fletcher 1993

Toxiean, Vol. 31, No. 6, PD. 669-695, 1993.

0041-0101/93 524.00 + .00Printed in ßrdt i3ritaio.

mon Pneu Ltd

REVIEW ARTICLE

POSSIBLE MECHANISMS OF ACTION OF COBRA SNAKEVENOM CARDIOTOXINS AND BEE VENOM MELITTIN

JAY E. FLSrcIIER'~ and Mnac-SIl1 JIANG'

Departments of'Anestheaiology, and ~13iochemistry, Hahnemann University, Philadelphia, PA 19102-1192,U.S.A .

(Received 10 September 1992 ; accepted 25 November 1992)

J. E. FLgrcIIEIt and M.-S. JIANG. Possible mechanisms ofaction ofcobra snakevenom cardiotoùns and bee venom melittin . Toxicon 31, 66995,1993.-Cobra snake venom cardiotoùns and bee venom melittin share anumber of pharmacological properties in intact tissues including hemolysis,cytolysis, contractures of muscle, membrane depolarization and activation oftissue phospholipase C and, to a far lesser extent, an arachidonic acid-associated phospholipase A2. The toùns have also been demonstrated to openthe Cat+ release channel (ryanodine receptor) and alter the activity of the Cat++ Mgt+-ATPase in isolated sarcoplasmic reticulum preparations derived fromcardiac or skeletal muscle . However, a relationship of these actions in isolatedorganelles to contracture induction has not yet been established. The toùnsalso bind to and, in some cases, alter the function of a number of otherproteins in disrupted tissues. The most difficult tasks in understanding themechanism of action of these toùns have been dissociating the primary fromsecondary effects and distinguishing between effects that only occur indisrupted tissues and those that occur in intact tissue . The use of cardiotoùnand melittin fractions contaminated with trace (`undetectable') amounts ofvenom-derived phospholipases A2 has continued to be common practice,despite the problems associated with the synergism between the toùns andenzymes and the availability of methods to overcome this problem. Withadequate precautions taken with regard to methodology and interpretation ofresults, the cobra venom cardiotoùns and bee venom melirttin mayprove to beuseful probes of a number of cell processes, including lipid metabolism andCat+ regulation in skeletal and cardiac muscle .

INTRODUCTION

SNAKE venom cardiotoùns (CTXs; 60-63 amino acids) derived primarily from variouscobra venoms and melittin (26 amino acids) from bee venom are small amphipathic basicpeptides that, although not identical, share a number of pharmacological properties.Several other venomcomponents also having similar actions have been grouped under theheading of cytolysic toùns (HARVEY, 1990) and these would also include toxins derivedfrom a diverse range of organisms, including plants (Pyrularia thionin; VEleNOx andROGERS, 1992), snakes (phospholipase A~; LEE et al., 1977; FL.ETCIIER et al., 1982),coelenterates (sea anemone, tenebrosin-C; GALE1-rIS and NOR~roN, 1990) and others

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J. E. FLETCHER and M.-S . JIANG

(HARVEY, 1990). These agents are all hemolytic and most have been demonstrated toinduce contractures in skeletal, cardiac or smooth muscle .

Several excellent reviews have appeared that cover many pharmacological aspects ofbee venom melittin (HABERMANN, 1972 ; DEMPSEY, 1990 ; VERNON and BELL, 1992) andsnake venom CTXs (CONDREA, 1974; CHANG, 1979 ; HARVEr, 1985, 1990, 1991 ; DUFTONand HroER, 1991 ; VERNON and BELL, 1992). Two recent reviews of CTXs (Dur-roN andHIRER, 1991 ; HARVEY, 1991) and one of melittin (DEMPSEY, 1990) are replete withreferences and are especially recommended to complement the present review .Additionally, an older, but comprehensive, review on melittin is highly recommended(HABERMANN, 1972) . There are fewer reviews regarding the pharmacology of melittinaction than CTX action and this reflects the greater emphasis on melittin as a tool tostudy the physical processes underlying protein-protein and protein-lipid interactionsthan as a pharmacological tool to understand cell function . The CTXs affect a widevariety of systems and have been alternatively termed direct lytic factors and membrane-active toxins (CorroREA, 1974, 1979). Structwe-activity relationships and interactions ofCTX (KINI and EVANS, 1989 ; GATINEAU et al ., 1990 ; MENEZ et al., 1990 ; DuFTON andHIRER, 1991 ; HARVEY, 1991) and melittin (DEMPSEY, 1990 ; IKURA et al., 1991 ; GROMOVA etal., 1992 ; WEAVER et al ., 1992) with membranes have been examined in detail and will notbe covered here . Instead the present review is primarily focussed on the actions of thetoxins on tissue-associated lipases [phospholipase A2 (PLAN, phospholipase C (PLC),triglyceride lipase], CaZ+ regulation in intact and disrupted preparations, the problems ofcontamination with venom PLAZ (new information on an old story) and new insights (orat least a new hypothesis) on the possible modes of action of these toxins in skeletalmuscle and red blood cells . Older studies are placed in perspective with recent findings andeven many of the newer data are reinterpreted.

Despite considerable research, the mechanisms) of action in excitable tissues or redblood cells of these toxins have continued to elude investigators . One barrier to identifyingprimary defects was the apparent tissue specific mechanisms (i .e . red blood cells vs .skeletal muscle ; HARVEY, 1985, 1991). This problem became more evident with the recentidentification of at least two independent primary mechanisms of action within the samecell type : release of membrane from the cells and activation of PLC (FLETCr->ER et al .,1991a) . Moreover, even within the cobra venom CTXs there are mechanisms of action notcommon to all members (e.g. PLAZindependent hemolytis ; FLEfCHIIt et al., 1991a;HARVEY, 1991) . An additional complicating factor is the activation of second messengersystems by these toxins, thereby indirectly affecting a broad array of cellular processes .Thus, simple cause-effect relationships are difficult to establish.The emphasis of this review is on cobra snake venom CTXs (primarily those from Naja

naja atra and Naja raja kaouthia venoms) and bee venom melittin (Apis mellifera), as theseare the agents that we are most experienced with . Hopefully, these principles can beapplied to some of the other CTX- and melittin-like agents under the broad heading ofcytolytic toxins .

Toward isolation ofpurified toxin-historical perspectiveCobra venom was identified as having cardiotoxic properties as early as 1905 (ELLIOT,

1905). However, it was not until 1942 that the venom was first fractionated (SARKAR et al.,1942) . Although a cardiotoxic fraction was not identified, SARKAR and coinvestigatorsspeculated that a cardiotoxic component existed that was not contained in the newotoxic

Mecéanisms of Cardiotoxin and Melittin

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or hemolytic fractions isolated . A crude cardiotoxic fraction was successfully isolated in1947 and was labeled CTX, based on its ability to arrest isolated hearts (SARKAR, 1947).More highly purified CTX fractions were isolated from several cobra snake venoms in theearly 1970s (see DUFTON and HroEIt, 1991) and details of the cardiac response to in vivoadministration of purified toxin have been reported (LEE et al., 1968 ; Suly and WnLxER,1986). These CTXs may play a more important role in the venom than was previouslyrecognized, as lethality of the Naja naja atra whole venom has been attributed tocardiotoxicity, not neurotoxicity (Sux and WnLxEtz, 1986). Also, CTX fractions causeextensive degeneration of skeletal muscle (D'ALSIS et al., 1988; CouTenux et al ., 1988).Even these purified CTX fractions still contained trace venom PLAZ activity contamina-tion (0.1-0.5%) that complicated at least some of the studies of the pharmacologicalproperties of the toxins, since CTX and even trace PLAZ contamination interact with amarked synergy in the red blood cell and some other systems, as discussed below. Whilethis synergism had been previously recognized (CoxnxEn et al., 19646; Coxnx>:n, 1974,1979), it was only with isolation of CTX virtually devoid of venom PLAZ contaminationthat the synergy with as little as 0.1 % became appreciated, as regards hemolysis(Lnxxlscx et al., 1971 ; Louw and VLS.SER, 1978 ; HroER and ICIInnEx, 1982 ; HnxvEY et al.,1983 ; HODGES et al., 1987 ; HARVEY, 1991) and this is discussed in greater detail below. Itshould be noted that some of the cobra snake venoms contain several isoforms of theCTXs that can be individually isolated (I~AIVEDA et al., 1977 ; Tax, 1982 ; Kn1~IE~n andHAYASHI, 1983; HARVEY, 1990; DuFrox and HroER, 1991).

Melittin comprises about 50% of the dry weight of bee venom (HABERMANN, 1972). Itwas first identified as a direct lytic factor (i .e . induced hemolysis in the absence of addedlecithin ; see HABERMANN, 1972) in 1953 (NEUMANIV et al ., 1953). The cause of death whenmelittin is administered in vivo is unclear, but is not due to hemolysis or neuromuscular organglionic blockade (HASERMAxIV, 1972), or a direct cardiotoxic action (MARSx andWHALER, 1980). Oddly, melittin does irreversibly paralyze isolated hearts, but these effectsare not apparent in vivo (MARSH and WHALER, 1980). As with CTX, melittin exhibits asynergism with its venom-derived PLAZ and the problem of copurification of traceamounts of enzyme with the melittin fraction is also well documented (MAULET et al.,1982 ; DuFOURCQ et al., 1986; DEMPSEY and WArrs, 1987; WILLE, 1989; FLETCi~R et al.,1990b) .

Synergism with venom PLAZ andsolutions for isolation of PLAZfree toxinTrace contamination of toxin fractions with venom-derived PLAZ has long been

identified as a serious problem in interpreting many CTX and melittin studies. While thistopic has been previously reviewed (HARVEY, 1985, 1991 ; DuFrox and HroER, 1991), it isimportant enough to be again emphasized . Also, methods are presented to circumventthese problems .For some poorly understood reason CTX and melittin exhibit a marked synergism with

their respective venom PLAIS . For CTX this synergy is most obvious in the red blood celland is hardly apparent in skeletal muscle (reviewed in HARVEY, 1985, 1990). A synergismis also observed in vivo with regard t0 LDS° VALUES (BOUGIS et al., 1987). In the red bloodcell this is really a mutual synergism in which the enzymatic activity of the venom PLAZ isincreased in addition to the lytic activity of CTX or melittin . Unsaturated fatty acids andlysophospholipids are the main products of PLAZ activity (Fig . 1A). Both of theseproducts have detergent actions and induce hemolysis (GuL and SMITH, 1974; FLETCFIIIt et

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J. E. FLETCHER and M:S. JIANG

al., 1987); however, it appears that unsaturated fatty acids (those primarily liberated bylipases) enhance, while saturated fatty acids do not affect the hemolytic action of CTXs(F~rct~R et al., 1990a) .While the most desirable starting point in a pharmacological study is a highly purified

toxin fraction devoid of venom PLAZ contamination (see Hmt;It et al., 1991, for methods),trace PLAZ contamination can be dealt with. Venom PLAZ activity is inhibited byp-bromophenacyl bromide (p-BPB) . This agent alkylates the histidine residue at thecatalytic site (VOLWERK et al., 1974) and abolishes both the enzymatic and pharmacolo-gical properties of the PLAZ enzyme (Coxnl~ et al., 1981 ; ROSENBERG, 1990). Thesequences of melittin (HABERMANN and J$xlscx, 1967) and a large number of CTXs(HARV~r, 1990 ; Dulrrox and HII)ER, 1991) are known. Melittin and many, but not all, ofthe CTXs do not contain the histidine residues which are highly reactive with p-BPB .Those fractions containing CTXs that lack a histidine residue (FLErcI-n=.R et al., 1991a) andfractions of melittin (FLSTCFISR et al., 1990b) when treated with p-BPB have the con-taminating venom PLAZ activity virtually eliminated without affecting the toxic propertiesofthe CTX or melittin molecules . It is important to remove most of the residual unreactedp-BPB before adding the toxin to preparations . We have found that a 2.5 ml SephadexG-25 column (PD-10 column ; Pharmacia LKB, Uppsala) is suitable, but this requires alarge amount (c. 2.5 mg) of toxin. We routinely employ p-BPB-treated CTX and melittinin our studies and recommend that they at least be tested as PLAZfree controls in allstudies with this group of toxins .A second and more costly approach to eliminating the problems associated with venom

PLAZ contamination is the use of synthetic toxin . Synthetic melittin (ScFntonnt et al.,1971) is commercially available (e.g . Peninsula Laboratories, Inc., Behnont, CA, U.S.A .and Merseyside, U.K.) . Although biologically active CTXs have been synthesized (Woxcet al., 1978; Du et al., 1986), to our knowledge these are not readily available. We haveobtained similar results in studies of PLC activation with p-BPB-treated (Fi.ETCt~x et al .,1990b) and synthetic (FLETCHHt et al ., 1991a) melittin .

Binding and internalizationThe specific cellular binding sites for CTXs and melittin have not been conclusively

identified . Both protein and lipid have been suggested as potential primary binding sitesand there is evidence supporting either hypothesis (reviewed in Dur-rox and HII)ER, 1991 ;HnxvEV, 1991) . A few interesting points regarding CTX and melittin binding deserveattention . CTXs bind by a mechanism that is displaced by high levels of Caz+ (CxnxG etal., 1972 ; EAxL and EXCEr.L, 1972 ; LuJ SHIAU et al., 1975, 1976 ; VirrcEtvT et al., 1976 ;HARVEY et al., 1982; JIANG et al., 1989a,b) . Melittin binding is apparently not displaced byhigh levels of Caz+ in skeletal muscle (LIx SfuAU et al., 1975) ; however, the hemolyticactivity of melittin is antagonized by high Caz+ levels (Bnsi->FORn et al., 1989) . Negativelycharged phospholipids, especially phosphatidylserine, have been suggested as CTXbinding sites (DuFOUxcQ and FAUOOx, 1978; VINCENT et al., 1978 ; BOUGI3 et al., 1981,1983), but specific binding to neutral phospholipids can be observed depending on thespecific CTX, phospholipid and surface pressure or packing state of phospholipids(BouGIS et al ., 1981, 1983) . In contrast to CTXs, the interactions between melittin andphospholipids are less specific (Boucrs et al., 1981), but melittin may still have a moresubtle preference for phosphatidylserine over neutral phospholipids (GROMOVA et al.,1992) . CTXs and melittin have not been suggested to be internalized and it would seem

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673

unlikely that this wouldbe required for their action . However, there have been insufficientdata gathered regarding such a mechanism and there is insufficient evidence to support orrefute this possibility .

E,B`ects on isolated tissue preparationsExcitable cells. The name CTX for the cobra venomderived toxins was originally based

on their efficacy in arresting the isolated heart (S~txwx, 1947) and this is the primaryaction resulting in death following in vivo administration of these toxins (L~ et al., 1968;Sux and Wncx~, 1986). These toùns are believed to be responsible for the ca,rdiotoùcproperties of whole cobra venom (ELLIOT, 1905; Sux and Wnix~, 1986). In addition totheir effects on the heart, CTXs affect awide variety of excitable and inexcitable cells. Dueto its relative stability, ease of preparation, suitability for electrophysiological studies,relatively well-defined excitation-contraction mechanism and large mass for biochemicalstudies, skeletal muscle has been used extensively to elucidate the mechanism of action ofthe CTXs in excitable tissues in general. The toxins depolarize (Ms.nxuht, 1965; L~ et al.,1968 ; Chang et al., 1972 ; Earl and Excet.L, 1972; Ho et al., 1975; Hnxv~r et al., 1982,1983; Surf and Wni.KBt, 1986; HOnoFS et al., 1987) and induce contractures (Snxxnx,1951 ; L~ et al., 1968 ; Chang et al., 1972 ; Lin Shiau et al., 1976; Hwxv~r et al., 1982, 1983;FLgrct~tt and Lizzo, 1987) in skeletal and cardiac muscle . The mechanisms underlyingcontracture induction and depolarization are not known, nor is the cause~ffect relation-ship of depolarization to contracture induction in skeletal muscle well understood . Thesetoùns also induce contractures in smooth muscle (L~ et al., 1968 ; Hmat and HARVEY,1982; HII)ER and ICHADER, 1982; Luv-SHIAU et al., 1986) and depolarize nerves (CxArrc etal., 1972). The CTXs are cytolytic to a variety of cell cultures (BRAGANCA et al., 1967 ;ZAiiEER et al., 1975 ; LEUNG et al ., 1976; HARVEY et al., 1983 ; KAIVEDA et al ., 1985 ; HixMAxet al., 1987 ; TZENG and Cm?rr, 1988; CxAn~-Mn~rYAS et al., 1991), with cultures of skeletalmuscle being readily lysed after depolarization (HARVEY et al ., 1983).

There are actions of the CTXs in skeletal muscle and red blood cells with eitherimmediate or delayed onset and some that are transient and others that are sustained(Fr urcm?tt et al., 19916) . It is uncertain how these apparently distinct actions are relatedto cardiotoùcity and hemolysis (FLETCI-IER et al., 1991b) . Immediate effects includemembrane depolarization, activation of PLC and contracture induction in intact tissueand Caz+ release from isolated terminal cisternae-containing preparations (Fr.ETC~t etal., 1991c) . Contractures in skeletal muscle usually have a latency to onset of about10-180 sec (HARVEY et al., 1982 ; Fi.Erc~R and Lizzo, 1987) and reach the maùmumpeak after about 0.5- 0min (HARVEY et al., 1982; FLETCHER and Lizzo, 1987 ; Fr..src~Ret al., 1991b), depending on the muscle preparation and the specific toùn and concentra-tion employed . The latency to depolarization has a similar variable time course (secondsto minutes) for onset, also depending on the muscle preparation and the specific toùn andconcentration employed (LEE et al., 1968 ; CHAxa et al ., 1972; EARL and EXCEi.L, 1972 ; Hoet al., 1975 ; HARVEY et al., 1982 ; HoncES et al., 1987). Actions with a delayed onsetinclude inhibition of Na+ currents and hemolysis (Fi src~t et al ., 19916) . Transienteffects include contractures and sustained effects include activation of PLC (FLErCFIIIt etal., 19916).As regards skeletal muscle, melittin, like CTX, induces contractures (IIABERMANN, 1972;

Lnv SFUAU et al., 1975 ; HARVEY, 1990; FLETCFISIt et al ., 1992) and depolarization(HABERMANN, 1972). Melittin is cardiotoxic in vitro, causing arrest of the rat heart, but

674


only induces a slight hypertension in vivo (MwRSH and WHALER . 1980). Melittin is alsocytotoxic (HABERMANN, 1972; LEE and Hwrr, 1985; Bwst->FOxv et al ., 1989 ; SHARMw, 1992).

The red blood cell. An additional model system used extensively to understand themechanisms of action of both melittin and CTX is the red blood cell . While there is somequestion as to whether this is an appropriate model to study the cardiotoxic action of theCTXs (HARVEY, 1985, 1991), it has recently become more apparent that the CTXs possessa number of actions, and at least one primary action may be examined for a subgroup ofCTX with direct hemolytic activity (FLETCi->Etlt et al., 1991a; HARVEY, 19~ 1) . The majorproblem with the red blood cell model, as opposed to skeletal muscle, is the markedsynergy observed with trace PLAZ contamination. Indeed, most of the CTXs appear to benonhemolytic on their own and require some contamination with PLAZ in order to inducehemolysis (HARVEY et al., 1982, 1983; HIDER and HARVEY, 1982; HARVEY, 1985). Thisobviously means that the effects of the PLAZ must be dissociated from the effects of theCTX-an impossible task considering the synergy that occurs . In some cases hemolysiscanbe completely PLAZindependent (WoxG et al., 1978 ; FLETCHER et al., 1991a; HARVEY,1991). The effects of PLAZ contamination on the hemolytic activity of CTX are mostevident at concentrations of the Naja naja kaouthia and Naja raja atra venom CTXsgreater than about 5~M (JIANG et al., 1989a) . Assuming a contamination of about 0.1 %,this would be roughly 5 nM PLAZ for a 5~M concentration of CTX. In contrast, inskeletal muscle a much higher concentration of PLAZ (c . 1 ~M) is required for aninteraction with comparable concentrations of CTX and the increase in depolarization(CwwxG et al., 1972) or contractures (FLETCi-~R and Llzzo, 1987) is not as dramatic asthat for hemolysis in the red blood cell . While the reason for the tissue-specific synergismis not understood, it has been suggested that this may relate to differences in basal lipaseactivities in the various tissues and the consequent levels and fluxes of free fatty acids(FLETCHER and Llzzo, 1987). For example, the levels of unsaturated fatty acids in human(c . 1200 pmole/mg protein; FLETCHER et al., 1989) and porcine (c. 400 pmole/mg protein;FLETCHER et al., 1988) skeletal muscle are much higher than in red blood cells fromhumans, horses, sheep and goats (30-130 pmole/mg protein; FLETCHER et al., 1990a) .Therefore, low levels of PLAZ activity that would significantly increase the free fatty acidlevels in red blood cells, would be masked by the already higher levels of free fatty acids inskeletal muscle. Also, the enzymes involved in processing and removing these fatty acidsmay be more active in skeletal muscle since metabolism of fatty acids through ß-oxidationis the preferred energy source at rest. Melittin also induces hemolysis by a mechanismindependent of PLAZ contamination that is markedly enhanced by venom PLAZ(FLETCHER et al., 1990b) . Overall, melittin is more potent and efficacious than any of thecobra CTXs in inducing hemolysis in the absence of PLAZ contamination .The red blood cell allows a number of highly detailed studies to be conducted that

would be impossible or extremely diflïcult in other cell types. The red blood cell is aunique model system since practically all of the lipid is contained in the plasma membrane(vwN DEENAN and DE Gmt, 1974). Furthermore, there is considerable variation in the lipidcomposition of the red blood cells from different species (NELSON, 1967a,b; FLETCHER etal., 1990a) and the red blood cells from different species have different susceptibilities tocobra venoms and their direct lytic components, the CTXs (TURNER, 1957; CONDREA etal., 1964a; CONDREA, 1974, 1979; FLETCHER et al., 1990a), and melittin (OSORIO E CASTROet al., 1990). Early studies suggested that the amount of phosphatidylcholine (lecithin) inthe red blood cell determined the susceptibility to lysis by cobra venom (TURNER, 1957) .Although there are some exceptions to the rule (e .g . camel erythrocytes), the phospha-


675

tidylcholine to sphingomyelin ratio predicts the relative susceptibility of red blood cells toCTXs (Coxnxi?n, 1979). When a source of phosphatidylcholine is added to red blood cellpreparations, the production of lysophosphatidylcholine and unsaturated fatty acids byvenom PLAZ accounts for the indirect hymolysis by many snake venoms (CoxntzEn, 1979).There also seems to be an inverse relationship between the amount of saturated fatty acidin the red cell and the susceptibility to lysis by CTX (FLETCHER et al., 1990a) . This latterobservation is consistent with the inactivity of saturated fatty acids on CTX-inducedhymolysis and the enhancement of CTX-induced hymolysis by unsaturated fatty acids(Fr sTCi-n?tt et al., 1990a) . The free fatty acid pool in a cell normally is predominant insaturated fatty acids. Activation of lipases enriches this pool with unsaturated fatty acidsmaking these more available to interact with CTX.

Activation of tissue lipasesGeneral. Both CTX and melittin activate cellular lipases. Since the products of lipase

activity have become recognized as important second messenger systems, it is necessary tounderstand the enzymes activated and the subsequent metabolism of the products tounderstand the mechanisms of action of CTX and melittin (Fig. 1). The major recognizedlipid-derived second messengers are inositol 1,4,5-trisphosphate (IP3), diacylglycerol(diglyceride) and free fatty acids. Phosphatidylinositol 4,5-bisphosphate, the source of IP3generated by PLC, is a subclass of phosphatidylinositol, which is a minor component ofthe membrane phospholipids. Diacylglycerol can be derived from PLC activity, tri-glyceride hydrolysis, or PLD activity with subsequent phosphohydrolase activity . Thebulk of the acylated (fatty acid containing) lipid is phospholipid and triglyceride.Depending on the cell type and state of the cell the relative amount oftriglyceride can varywidely from about 5-60% of the total lipid. Free fatty acids can be liberated by PLA�PLAZ, triglyceride hydrolysis and PLC activity with subsequent deacylation of thediacylglycerol . The effects of IP, on CaZ+ metabolism (HENZI and MncD>;xMOTT, 1992;MErrxrri et al., 1992) and diacylglycerol on protein kinase C activation (BELL, 1986;Rnxno, 1988; Ct-i.~uxnx et al., 1990) have been reviewed. Fatty acids alter the function ofa number of ion channels (OxnwnY et al., 1991), including Na+ (Lirrosty andRouTTExssxc, 1989; W~r nrro et al., 1992), K+ (KiM and CLAPIiAM, 1989; OxnwnY et al.,1989), CaZ+ (LnvnEx and RouTTExsma, 1989; Glow et al., 1990; FLETCIiER et al., 1990c)and Cl - (Axni?xsox and WFS.sx, 1990; HwnxG et al., 1990). Fatty acids are known tobind covalently to proteins through an enzymatically mediated process termed acylation(Towr.~t et al., 1988 ; SALTIEL et al., 1991). A further complication in studying the effectsof CTX and melittin is that the enzymes involved in cellular lipid metabolism are wellknown to be up- and down-regulated in response to hormonal factors and the state ofcelldifferentiation .

Phospholipase A2 (PLA?) . Originally, CTX (Sxmt, 1979) and melittin (MOLLAY et al.,1976; Si-m:x, 1979) were both thought to specifically activate tissue PLAZ activity . Thetoxins clearly enhance the activity of venom PLAZ activity (MOLLAY and Kttntr., 1974;Yuxas et al., 1977) with some specificity as to the enzymes stimulated by the toxins(Coxxicons and Ocxs, 1989) and the state of the substrate (YUNES et al., 1977) and it wasa natural extension of these observations to suggest that tissue PLAZ was specificallyactivated by the toxins . These early studies had noted that when CTX and melittinfractions having no detectable venom PLAZ contamination on artificial substrates wereadded to cell cultures they produced high levels of lysophosphatidylcholine (Si-mix, 1979)and free fatty acids, both of which are major products of PLAZ activity (Fig . lA).

67 6


PLAZA . PE-'LPE + FA

B. PLCPE ---~ DG

GP + FA

TGaseC . TG

HSL

3FA + G

P-Eth

D. IPg

Ca s+

® `~ Ca2+-ATPasePLAZ ..~

FA

~ Ca~+releasechannel

Na+, Ca~, K+, CI-channels

Toxln

FIG. 1 . ENZYA~SS OF LIPII) I~rABOLISM AND 17~ AGTIVAI'ION OF PLC BY CTX OR MELITI'IN.A. Activation of PLAZ results in the production of free fatty acids and lysophospholipid. In thiscase the substrate is phosphatidylethanolamine (PE) and the products are lysophosphatidyl-ethanolamine (LPE) and fatty acid (FA) . LPE can also be further degraded to glycerol phosphate(GP) and frce FA . The fatty acid attached to the ~ 2 position of the phospholipid that is removedby PLAZ is usually unsaturated (one or more double bonds) . B. Activation of PLC results in theproduction of diacylglycerol (DG) and phosphorylated base, in this case phosphcethanohunine (P-Eth). P-Eth can be dephosphorylated to ethanolamine (Eth), which is also a direct product of PLDactivity. DG can be further deacylated by lipases, yielding free FAs and glycerol (G), orphosphorylated to phosphatidic acid (PA), which is also a direct product of PLD activity. C.Activation of triglyceride (TG) breakdown. At least two separate enzymes catalyze the hydolysis ofthe triglyceride-associated fatty esters, triglyceride lipase (TGase) and honmone-sensitive lipase(HSL). D. The toxins appear to activate PLC. However, it is also likely that the toxins affect anumber of proteins indirectly through the products of PLC activity . PLC activity onphosphatidylinositol 4,5-bisphosphate yields inositol 1,4,5-trisphosphate (IPA which elevatesintracellular CaZ+ levels . CaZ+-dependent PLAZ could subsequently be activated by elevation ofcytoplasmic CaZ+ levels. Both PLAZ activity and the deacylation of DG would yield free FAs,

which affect a variety of ion channels and other proteins .

However, since phosphatidylcholine is primarily located on the outer leaflet of themembrane bilayer (ZWML et al., 1975 ; Dsvnux, 1991), lysophosphatidylcholine shouldnot be detected to a significant extent by activation of a PLAZ acting on the inner leaflet .We have observed lysophosphatidylcholine production by venom PLAIS, which primarilyattack the outer leaflet of the membrane bilayer (Fr.E~rCr->ER et al., 1990b, 1991a) .Originally we had proposed that the primary source of lysophosphatidylcholine formelittin (FLSrct-mt et al., 19906) and CTX (FLETCt~It et al ., 1991a) fractions washydrolysis by venom PLAZ contamination in the fraction, not activation of tissue PLAZ.The preparations of CT'X and melittin used in most studies contain trace amounts ofvenom PLAZ, but the enzymatic activity cannot be detected with the assay conditions


677

commonly used . Most of these assay systems are based on artificial substrates, which arenot always as good a substrate matrix for the enzyme as a biological membrane . Also,snake venom CTXs (Fr.ErcHHt et al., 1991a) and bee venom melittin (FI.srcHBt et al.,1990b) greatly enhance the enzymatic activity of the PLAIS derived from their respectivevenoms when detenmined on cell cultures . Consequently, the activity of the venom PLAZand the synergistic increase in that activity in the presence of CTX explain about 75% ofthe fatty acid release and all of the lysophosphatidylcholine observed in cell culturesystems treated with melittin (FLgrcH~x et al., 1990b) or CTX (Fi.srcHax et al., 1991a)fractions . However, it has subsequently become apparent that the situation . is morecomplex than we had originally supposed .The original studies examining PLAZ activation by CTXs and melittin radiolabeled the

lipid fractions with low concentrations of ['H]arachidonic acid . One consistent feature ofthe PLAZ activation using ['H]arachidonic acid is a 10-20 min latency to onset (SHmt,1982 ; CHOt et al., 1992). This latency is not observed when using either higher concentra-tions (10 pM) of ["C]linoleic acid to radiolabel the fatty esters in the phospholipid pools(Fig . 3A~), or monitoring the release of tritiated inositol phosphates (CHOt et al., 1992) .The shorter labeling time (overnight) used in the arachidonic acid (20 : 4; 20 carbons and 4double bonds) studies and the unique metabolism of arachidonic acid, including a muchmore selective and unusual labeling of the phospholipid pools (phosphatidylinositol >phosphatidylethanolamine > phosphatidylcholine » phosphatidylserine; C~-roi et al.,1992) and processing through the cyclooxygenase and lipoxygenase pathways mayaccount for the apparent discrepancies in these seemingly similar, but very differentapproaches. It is important to note that the distribution of labeled arachidonic acid in thephospholipids is discordant with the relative distribution of arachidonic acid-containingmolecular species of phospholipids in a wide variety of tissues (ANSELL and SPANNER,1982), cultures of skeletal muscle ($AIffH and FINCH, 1979) and even in the phospholipid(TRAxtvoR et al., 1982 ; AiuGA et al., 1988), or fatty ester (TxAYrrox er al., 1982)distribution in PC12 cells, which were used in the melittin studies with radiolabeledarachidonic acid . Other investigators have not found that melittin induces insulin releaseby mechanisms consistent with PLAZ activation (METZ, 1986) . Linoleic acid (18 : 2) is apredominant fatty ester in skeletal muscle, comprising about 25% of the total phospho-lipid fatty ester (arachidonic acid is about 15%) and about 15% of the triglyceride fattyesters (arachidonic acid is about 0.2%) . Linoleic acid is an essential fatty acid and is aprecursor of arachidonic acid . The distribution of radiolabeled linoleic acid into skeletalmuscle phospholipids using a prolonged labeling period and a 10 pM concentration offatty acid (Fig.2 and FLE'rCHER et al., 1991a) is similar to the natural distribution ofphospholipids in skeletal muscle (SsurH and FINCH, 1979 ; ANSELL and SPANNER, 1982 ;FLEfCHER et al., 1988) and the expected distribution of fatty esters into the molecularspecies of phospholipid in muscle cell cultures (SMrrH and FirrcH, 1979); that is,phosphatidylcholine » phosphatidylethanolamine » phosphatidylinositol =phosphatidyLserine . Using ["C]linoleic acid, or even simultaneous labeling with['°C]choline, we have never obtained evidence for activation of tissue PLAZ activity byp-BPB-treated CTJC or melittin, or synthetic melittin (Fi.srCHER et al., 19906, 1991a ;J.E.F ., unpublished observations) . The concentration of radiolabeled fatty acid usedgreatly affects the distribution into phospholipids (Bi,ANx et al., 1992; SNYDER et al.,1992) . A recent study using synthetic melittin demonstrated activation of tissue PLAZ,supporting this as truely an action of the toxin and not due to venom PLAZ contamination(CHOI et al., 1992) . Therefore, the activation of PLAZ activity observed using

67 8


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t d

.

,;J rr~l ~t.___I~.

Determine radioactivityassociated with each

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FIG. Z . METHODOLOGY EMPLOYED TO EXAMINE PLC AC'ITVAT70N BY CTXs AND MELITI'IN IN PRIMARYCULTIJRFS OF HUMAN AND EQUINE SREI~TAL MUSCLE .

A. Cell cultures are plated into 35 mm dishes and, after allowing 2 weeks for growth and division,the cells are radiolabeled for 3 days. For some experiments ["C]choline is substituted for["Clethanolamine . All radiolabels are present in 10 ~M concentrations . The prelabeling andpostlabeling times are varied, depending on the growth characteristics ofthe cell type. The cells arewashed to remove tmincorporated radiolabel and then exposed to toxins in a HEPES-bufferedmodified Krebs solution with no BSA present (FLETCHER et al., 1990b), the supernatant removedand the radioactivity counted. The lipids are extracted from the cells, separated by 1-D TLC andthe radioactivity in each lipid spot quantitated by a radioactivity scanner . H. The totalradioactivity released into the bathing medium is determined as well as the total cell-associatedradioactivity and the radioactivity in the cellular aqueous phase (polar head groups) and the

organic phase (phospholipids and neutral lipids).

[3H]arachidonic acid may relate to a specific, and apparently minor, aspect of arachidonicacid metabolism with a prolonged onset following toxin addition that is unrelated tocontracture induction or depolarization . In the case of the CTXs, the PLAZ activitystimulated has been suggested to be lysosomal (SH>St, 1982).

Phospholipase C (PLC) . One shared action of the CTXs and melittin in intact tissue isthe activation of PLC (FLETCHER et al., 1991a; FLETCHm and JiArtG, 1992). This activityhas been demonstrated by the elevation of inositol phosphates (JAM>ESON and VTLLEREAL,1987 ; GusovsxY et al ., 1991 ; ZEITLER et al., 1991 ; CFTOT et al., 1992), phosphoethanolamine(FLETCFIIIt and JIANG, 1992 and Fig.3C) and diacylglycerol (FLETC:HER et al., 1990b,


679

1991a,b; FLETI.HER and hwrtG, 1992) . We first identified this action as PLC activation bythe detection of diacylglycerol production (FLETCFIIIt et al., 1991 a) . PLC activation occurswithin 1 min whether the release of inositol phosphates (Cxot et al., 1992),phosphcethanolamine (Fig. 3C), or diacylglycerol (Fig. 3B) is monitored . Since bothinositol phosphate and phosphcethanolamine are released and since IP 3 is not elevated toa greater extent than other inositol phosphates, this is not due to the activation ofphosphatidylinositol-specific PLC, but rather a PLC or group of PLCs with a less limitedsubstrate preference . It is unclear whether the toxins directly interact with the PLC, orwhether this action takes place through signal transduction, or physical perturbation ofthe membrane . It would seem unlikely that the action is direct, as the toxin should not beable to penetrate the membrane bilayer. Melittin inhibits the activity of a bacterial PLCon several types of artificial bilayers of phosphatidylcholine (Rno, 1992) and stimulatesthe activity of a rat liver PLC on detergent mixed micelles (Hnam et al ., 1991), suggestingan interaction with the lipid phase could be involved . The majority of the PLC activityactivated by melittin is Cat+-independent (Fig . 3D-E) and is not antagonized by pertussistoxin (Cot et al., 1992 ; M.-S .J ., unpublished observations) or cholera toxin (M.-S.J .,unpublished observations). Pertussis and cholera toxin also did not antagonize the PLCactivity stimulated by CTX (M.-S.J ., unpublished observations). It is improbable thatactivation of PLC by CTXs or melittin is involved in hemolytis, since the Naja naja atravenom CTX has no hemolytic activity independent of PLAZ activity, yet this toxin and theNaja naja kaouthia venom CTX both activate PLC (FLErCHIIt et al., 1991a) .

Phospholipase D (PLD). The phosphorylated base (e.g. phosphcethanolamine)produced directly by PLC activity could be indirectly produced by PLD activity andsubsequent phosphorylation of the base (e.g. ethanolamine ; Fig. 1B) . Conversely, ethanol-amine could be indirectly produced from phosphcethanolamine by PLC and subsequentphosphatase activities (Fig . 1B) . At times we have observed significant toxin-inducedincreases in ethanolamine in cells in which phosphatidylethanolamine has been radio-labeled (FLETCHIIt and JtnxG, 1992) . However, at other times the toxin-induced increasesin phosphcethanolamine release are considerably greater than ethanolamine to the extentthat ethanolamine levels are almost undetectable (Fig . 3C). Therefore, we must considertwo possibilities. Either the toxins activate PLD activity (but only when the PLD proteinis expressed in the tissue), or the presence of phosphcethanolamine phosphatase isvariable and the expression of this enzyme (not necessarily a site of CTX or melittinaction) in the tissue converts the phosphcethanolamine to ethanolamine (Fig. 1 B) .

Triglyceride hydrolysis. At a high concentration (10 ~M) melittin (but not CTX)activates triglyceride hydrolysis (FLETCiiIIt et al., 1991a) . We have not determinedwhether this action is mediated through a lipoprotein lipase-like triglyceride lipase, orwhether hormone-sensitive lipase is involved (Fig . 1C). The time course has also not beendetermined . The activation of triglyceride breakdown appears to be dependent on thelevels of triglyceride existing before addition of the toxin (FLETCf~R et al., 1991a) . Thehigher concentrations of melittin causing triglyceride breakdown (Fr..srcmut et al., 1991a)are in the range in which the release of radioactivity into the medium is decreased (St-uER,1979 ; FLerc~x et al., 1991a ; Cxoi et al., 1992) .

Modulation of CaZ+General. The elevation of myoplasmic CaZ+ levels is an important signal for contrac-

tures of skeletal and cardiac muscle . Since the CTXs and melittin induce contractures inskeletal muscle, it is most likely that they increase CaZ+ levels . CTX (Tzstvc and C~v,

680


A .ôö

vwiiK

ÂObéea

Time (mln)

D .

x x0.0vw

1 S.00

Diglyceride

Free Fotty Acid

Reease to Supematont

FIG . 3 . TIME COURSE AND C8~ + DEPENDENCE OF MELITTIN-INDUCED PLC ACIiVATION AND uF, n~aF OFRADIOACIMTY IN70 THE BATHING MEDIUM IN PRIMARY CULTURES OF HUMAN SKEIECAL MUSCLE.

The time course (Panels Ate) of response at 37°C was determined in cells radiolabeledsimultaneously with ['~Cjethanolamine and ['~C]linoleic acid . A. Release of radioactivity into thebathing medium. B. Production of diacylglycerol (open circles) and free fatty acids (filled circles)within the cells . C. Production of phosphcethanolamine (open circles; product of PLC) andethanolamine (filled circles ; product of PLD) within the cells, separated by TLC as previouslypublished (FLETCHER and JIANG, 1992). The mean of the values for time zero were subtracted fromeach point for Panels Ate. The Cat+ dependence (Panels D and E) was determined in cellsradiolabeled with ["C]linoleic acid incubated with melittin (2 alai) for 2 hr at 37°C. Cells wereincubated with EDTA (10mM) to prevent Cat+ influx and ruthenium red (10pM) to prevent Ca=+release from intracellular stores . D. Release of radioactivity to the bathing medium for control(open bar and diagonal bar) and melittin-treated (filled bar and cross-hatched bar) preparations innormal bathing medium (Ca=+; open bar and filled bar) or medium containing EDTA andruthenium red (-Ca2+; diagonal bar and cross-hatched bar) . E. Production of diacylglyoerol andfrce fatty acids (symbols identical to Panel D). The levels of diacylglyceride and free fatty acid werereduced (P < 0.0~ in the -Cap+ medium ; however, the bulk ofthe PLC activity was still evidentunder these conditions. The presence of EDTA induced release of radioactivity into the medium(P < 0.01) . In Panels A-E melittin induced increases in lysophosphatidylcholine were notdetectable and each symbol represents the mean with associated SD bar (when larger than the

symbol) for three determinations .

1988) and melittin (Mnc et al., 1984 ; Cxoi et al., 1992) do increase CaZ+ levels in other celltypes. Considerable insight has been gained as to the mechanisms underlying Cap+

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681

regulation in skeletal muscle (Fig.4A). Skeletal muscle, unlike cardiac muscle, has nodependence on extracellular CaZ+ for contractions or contractures . That is, the Cat+

stored in the sarcoplasmic reticulum is sufficient to sustain muscle contraction forextended periods in a CaZ+-depleted medium . Possible mechanisms by which the toxinscould increase myoplasmic CaZ+ include: (1) depolarization of the membrane, whichwould indirectly increase CaZ+ through the normal process of excitation-contractioncoupling; (2) opening pores in themembrane to allow extracelluiar CaZ+ to move down itschemical gradient into the cell ; (3) directly acting on the dihydropyridine receptors toactivate excitation-contraction coupling; (4) activation of a signal transduction process(e.g . PLC); (5) entering the cell and either directly stimulating CaZ+ release or blockingCaZ+ uptake . The toxins do depolarize excitable cells (discussed above), form pores inmembranes (discussed below), activate PLC (discussed above), and they interact with theCaZ+ +MgZ+ATPase and CaZ+ release channel in isolated organelles (discussed below) .Contractures induced by CTX in skeletal muscle are partially antagonized by dantrolene(FLETCFIIIt and Lizzo, 1987), suggesting some role of intracellular CaZ+ stores. However,membrane depolarization and CaZ+ influx to trigger CaZ+-induced CaZ+ release from thesarcoplasmic reticulum could account for the action of dantrolene.

CaZ+ +Mgt+-ATPase/Ca Z+ pump. Melittin inhibits (MAHANEY and TxoMns, 1991; Vosset al., 1991) and CTX has no effect on (FOURIE et al., 1983) the CaZ+ +MgZ+-ATPase inskeletal muscle sarcoplasmic reticulum. The effects of melittin are in part attributed to ahydrophobic interaction with the membrane (Voss et al., 1991). The CaZ+ pumpingactivity in skeletal muscle sarcoplasmic reticulum is also reduced by melittin (Fr~rci~x etal., 1992) and CTX (Foam: et al., 1983). In cardiac sarcoplasmic reticulum an increase inthe CaZ+ +MgZ+-ATPase activity and a decrease of CaZ+ uptake is observed (TRUMBLE etal., 1992b) . In cardiac sarcolemmal vesicles the CaZ+ +MgZ+-ATPase and CaZ+ pumpingactivity are both stimulated by CTX (Hunxc and T1tUMHLE, 1991). In contrast to that inskeletal and cardiac muscle, the erythrocyte CaZ+ +MgZ+-ATPase is inhibited by CTX(Four et al., 1983).

CaZ+ release. CTXs (Fr.>rrcl~t et al., 1991b, 1993) and melittin (Fr.>?rci->at et al., 1992)induce CaZ+ release from terminal cisternae-containing preparations from skeletal muscle .CTX also induces CaZ+ release from cardiac sarcoplasmic reticulum (Txu~r~ et al.,1992b) . This action is specific for CaZ+ release through the CaZ+ release channel (orryanodine receptor) as the CaZ+ release by CTX (FLETCFIIIt et al., 1993; Txu~r.E et al.,1992b) and melittin (Fr grCFmt et al., 1992) is blocked by ruthenium red, ruling out anonspecific membrane action of the toxins, causing leakage of CaZ+ . Most assaysmonitoring CaZ+ release (Fig. 4B) are actually detecting the net flux of CaZ+ release andCaZ+ uptake and the toxins may only appear to cause CaZ + release by blocking CaZ+

uptake through the ATPase-driven pump . Either of these actions would raise extravesi-cular CaZ+ . Different experimental approaches (e.g . asCaZ+, spectrophotometric CaZ+analysis) all require dissociating an apparent decrease in uptake from release. Forexample, using asCaZ+, what initially appeared to be a decrease in net CaZ+ uptake incardiac sarcoplasmic reticulum was subsequently interpreted using ruthenium red as anincreased release of CaZ+ by CTX (Tku~L$ et al., 1992b) .

It is unclear whether the effects of CTX and melittin are mediated by a direct andspecific action on the CaZ+ release channel, or whether they involve an indirect actionmodulating CaZ+ release, such as production of fatty acids by activating PLC. Ryanodinebinding is enhanced by agents opening the CaZ+ release channel, as this plant alkaloidbinds to the open channél pore (P>?ss~x et al., 1985). Although radiolabeled CTXbinds to

68 2


A.

FIG . 4. CaZ* REGULATION AND MONTrORING OF Ca t* RFt FecF AND UPTAKE BIOCnEbIICALLY INSKELETAL MUSCLE.

A. The action potential generated at the endplate region of the neuromuscular junction (l) iscoupled through the t-tubules to the dihydropyridine receptors shown as small circles (2) . Thedihydropyridine receptors in skeletal muscle function as voltage sensors and are coupled to theCaZ* release channels, or ryanodine receptors (3), which are shown as large circles . Opening of theCaZ* release channels results in release of the terminal cisternae (4) CaZ* stores and elevation ofmyoplasmic Ca'* . The Cat* then interacts with the troponin-tropomyosin complex associatedwith actin and allows interaction of actin with myosin for mechanical movement (5) . The Cat*signal is terminated by pumping the CaZ* into the longitudinal sarcoplasmic reticulum by anATP-driven process (6) . B. Fractions containing resealed terminal cistemae regions are isolatedand CaZ* concentrations can be monitored using a metalochrome indicator, such as arsenazo IIIor antipyrylazo III (AIII) . CaZ* fluxes can also be monitored with "CaZ* . Usually an ATPregenerating system (CK plus CP) is used to prevent ATP depletion. The CaZ* flux actuallymeasured is the net result of uptake and release from the vesicles . Ruthenium red (c. l ~M) can beused to distinguish uptake from release, as it blocks CaZ* release through the CaZ* release channel

(filled circles).

a protein believed to be the ryanodine receptor (TRUMBLE et al ., 1992a), neither melittin(FLETCHER et al ., 1992) nor CTX (FLETCHER et al., 1993) enhances the binding ofradiolabeled ryanodine to the CaZ+ release channel under standard binding conditions .Indeed, CTX slightly inhibits ryanodine binding (FLETCHER et al., 1993) . However,caffeine releases CaZ+ from the sarcoplasmic reticulum through the CaZ+ release channeland has been found to interact with a modulatory site on the CaZ+ release channel (PESSAHet al., 198 and this would not be detected under the same binding conditions previouslyused with CTX or melittin . Caffeine overrides the MgZ+-induced inhibition of ryanodinebinding. Similar modulatory sites on the CaZ+ release channel may be involved in theactions of CTX and melittin .The inhibition of the CaZ+ pumping activity in skeletal muscle sarcoplasmic reticulum

preparations (about 10%) does not seem to play a major role relative to the effects of


683

melittin on CaZ+ release at low concentrations (0 .1 ~M) of melittin (Ft.srcr-~R et al.,1992). However, it is possible that the inhibition of the CaZ+ +Mgt+-ATPase and pumpplays a much more important role at higher (1 ~M) concentrations of melittin (Fi.ErcFmtet al., 1992).

CaZ+ antagonists and divalent cation substitution. One possible mechanism of increasingintracellular CaZ+ is by opening pores or CaZ+-specific ion channels in the sarcolemma.Therefore, the effects of CaZ+ channel antagonists and divalent cation substitution are ofinterest in understanding the mechanisms of these toxins. Verapamil, an organic CaZ+

channel antagonist, was only slightly effective in antagonizing CTX-induced contractuuesin the chick biventer cervicis (HARVEY et al., 1982). Melittin-induced CaZ+ influx in PC12cells was slightly inhibited by organic CaZ+ channel antagonists (nifedipine, verapamil,diltiazam) ; whereas, inositol phosphate formation and arachidonic acid release wereunaffected (CI-IOi et al., 1992). La3+ (1 mM), an inorganic blocker of CaZ+ influx, had noeffect on CTX contractures (HARVEY et al., 1982). One basic principle must be understoodbefore examining individual divalent canons on CTX action . A high (10mM) concentra-tion of BaZ+ and SrZ+ has the same inhibitory effect as the same concentration of CaZ+ andthis may simply relate to antagonism of CTX binding (JiArvo et al., 1989b). Therefore,there is no specificity to divalent cation action at concentrations around 4mM and higher .An exception to this divalent cation rule is that a 10 mM concentration of MgZ+ enhancesCTX-induced contractuues (Lix SHIAU et al., 1976) and a 3 mM concentration increasesthe cytolytic activity (LEUxc et al ., 1976). At a concentration of 2 mM, both SrZ+ andBaZ+ decrease hemolysis (JiAxG et al., 1989a) and release of deoxyglucose-6-phosphate(Jinxc et al., 1989a) from red blood cells relative to that with the same concentration ofCaZ+ at concentration of CTX of 10ItM . In contrast, at a lower (3 ~M) concentration ofCTX CaZ+, SrZ+ and BaZ+ are equivalent in supporting hemolysis (JiAxa et al., 1989a) .The antagonistic effects of SrZ+ and BaZ+ at high concentrations of CTX may be related toantagonism of the enzymatic activity of trace PLAZ contamination in the CTX prepara-tions (JtArrc et al., 1989a). MnZ+ has a much more dramatic effect in inhibitingCTX-induced hemolysis at all concentrations of CTX (JiAxc et al., 1989a) . MnZ+ (1 mM)also completely reverses many of the ca.rdiotoxic effects in the rat atrium (membranedepolarization, action potential blockade, decreased contractile activity) of a basic PLAZfrom Naja nigricollis snake venom (Fi.ETCi~R et al., 1982). These anticardiotoxic effects ofMnZ+ on the Naja nigricollis PLAZ could not be attributed to an antagonism of toxinbinding (Fi.ETI:HER et al., 1982). Similar reversal studies have not been conducted withregard to the cobra venom CTXs or melittin . MnZ+ does not antagonize caffeine-inducedcontractures in skeletal muscle (Lix SmAU et al., 1976), or effects on electrical andcontractile activity of rat atria (FLSrci-tt~ et al ., 1982), suggesting a specificity to theaction of MnZ+ forCTXs. In the case of the Naja nigricollis venom PLAZ with cardiotoxicproperties, the hydrolysis of phosphatidylserine was specifically antagonized by MnZ+(Fi,ETCt-1Ex et al., 1982). SrZ+ adequately substitutes for CaZ+ in contracture induction byCTX (Fi,ETCi~R and Lizzo, 1987). Three inorganic CaZ+ antagonists (CoZ+ , MnZ+ , CdZ+)

at a 10mM concentration greatly antagonize melittin-induced CaZ+ influx into PC12 cellsand partially antagonize arachidonic acid release (Cxoi et al., 1992). While CdZ + andMnZ+ completely antagonize melittin-elicited inositol phosphate formation, CoZ+ has noeffect on this action (Gioi et al ., 1992). In the absence of CaZ+ (actually low CaZ+ , as noEGTA was added) CTX-induced contractures occur in rat diaphragm, but are greatlyattenuated in human vastus lateralis skeletal muscle (Fi.ETCi~R and Lizzo, 1987). SinceCaZ+ was not required for CTX contractures, at least in the rat diaphragm preparation,

684

J. E. FLETCHER and M:S . JIANG

then influx across the sarcolemma is not essential for increasing intracellular CaZ+ levels inall preparations. Indeed, CTX-induced contractures of the chick biventer cervicis (LiNSHIAU et al., 1976) and hemolysis of human red blood cells (JIANG et al., 1989a,b) areincreased with lower than physiological concentrations of Cat+ . Also, the ineffectivenessof verapamil and La3+ would suggest that extracelular CaZ+ is not essential for contrac-ture induction by CTX in skeletal muscle . However, the effectiveness of EGTA inblocking CTX-induced contractures in chick biventer cervicis (Lity Si-nAU et al., 1976 ;HARVEY et al ., 1982) and a Cat+-free medium in antagonizing contractures in humanskeletal muscle (FLETCHIIt and LIZZO, 1987) add some complications to this simpleinterpretation . It seems clear that sarcolemmal CaZ+ channels blocked by the organic CaZ+antagonists do not play a specific role in the action of CTX or melittin . Inorganic CaZ+antagonists, especially MnZ+ and CoZ+, may be important tools for dissecting outindividual actions of the CTXs and melittin .

E~`ects involving other ionsFormation of pores. Melittin has been demonstrated to form pores in artificial

membranes (planar bilayers) by an aggregation of monomers (T06TESON and TosrFSON,1981 ; T06TESON et al., 1980 ; STANicowsxi et al ., 1991). These pores are more permeable toanions than to canons (T03TBSOIV and T05TESON, 1981). Pore formation does not seem tocorrelate with membrane disruption, including hemolysis (STANKOWSKi et al ., 1991). Therelationship of these pores in artificial membranes to ion conductance in biologicalmembranes remains to be established .Na+ channels . CTX has been suggested to inhibit Na+ channels in whole cell patch-

clamp of skeletal muscle myoballs (Fi.ETCi~R et al., 1991 b) . Also, melittin inhibits two ofthree slow Na+ currents in embryonic chick hearts that have properties similar to adultCaZ+ channels (BxAU,Y et al., 1988). These slow Na+ currents are not blocked by MnZ+ or10-sM tetrodotoxin . However, the contribution of venom PLAZ contamination has notbeen sufficiently ruled out in either of these studies since neither p-BPB-treated norsynthetic melittin were used (see above). Since the addition of fatty acids to the externalleaflet of muscle membranes inhibits Na+ currents (WiELAND et al., 1992), PLAZ con-tamination, consequent fatty acid production and the synergism with the toxins could stillaccount for this action.[Na+,K +]-ATPase . Melittin and the CTXs have been suggested to be potent inhibitors

of the [Na+ ,K+]-ATPase (Zniii~R et al., 1975 ; Curt and Lnv-Stunu, 1985; CUPPOLETTIand AssoTT, 1990; RAYNOR et al., 1991). However, inhibition of the [Na+,K+]-ATPasedoes not mimic (LAxiciscii et al., 1972 ; Fr erci~t and Llzzo, 1987) or antagonize(HARVEY et al ., 1982; Fi.ETCiiER and Lizzo, 1987) CTX action and at least one CTX (Najaraja kaouthia) does not inhibit [Na+,K+]-ATPase activity in cardiac muscle (HunxG andTRUMBLE, 1991). Furthermore, Boutais and coinvestigators have proposed that the inhibi-tion of [Na+ ,K+]-ATPase activity by CTX fractions is due to PLAZ contamination(Boutais et al., 1989). Therefore, inhibition of [Na+,K+]-ATPase activity does not appearto play a role in the mechanisms of action of the CTXs and melittin and a direct effect ofthe toxins on the enzyme independent of PLAZ contamination remains to bedemonstrated .

E,~ects on other cell proteinsA number of studies, usually conducted in disrupted cell systems, have demonstrated

that the toxins bind to and/or alter the function of several proteins . Melittin (Kn'i'ox et al.,1982 ; O'BRiAN andWARn, 1989; RAYNOR et al., 1991) and CTX (Juo er al., 1983 ; RAYxoR


685

et al ., 1991) inhibit protein kinase C. Melittin binds with high affinity to calmodulin(Cones et al., 1983 ; KnTeoxe et al., 1989). Melittin stimulates the guanylate (Len andS~tt, 1979) and adenylate (Len and Sit, 1980) cylases. Melittin stimulates the GTPaseactivity of Go and G;, which are two G proteins ADP-ribosylated and inhibited byperfusais toxin (HtcesFUnnse et al., 1990). However, since perfusais toxin and choleratoxin, an inhibitor of G� have no effect on melittin or CTX action (see above), it isunlikely that effects on GTPase occur in intact tissue .

Membrane disruptionThe formation of blebs on the cell membrane by CTX (Tzsrtc and C~rr, 1988) or the

release of membrane fragments by melittin (Kezsv et al., 1988, 1989) and CTX (Gur..nc-Kxznvrciu et al., 1981) suggests a gross disruption of the membrane that may relate tothe release of radioactivity into the medium (S~, 1979 ; FLgrcFmt et al., 1991x),especially since the lipid distribution of radioactivity released into the incubation mediumin the absence of BSAresembles that of the cell membrane lipid composition (F~rcFmt etal., 1991x) . Such a disruption of the membrane, most likely by a nonspecific physicalinteraction, may expose new substrates to the toxins (VERNON and B~., 1992), such asphosphatidylserine, which is normally on the inner leaflet of the bilayer. Thus, these toxinsmay be membrane perturbing probes of lipid metabolism and the results obtained withtheir use, while potentially valuable, should be interpreted with caution. It is important tounderstand that assays run in the presence of BSA will extract large amounts of free fattyacids that may mask the release of membrane . This may be why the levels of phospholipidare only 23-30% of the total released radiolabel in the presence of BSA (Cxot et al.,1992), instead of higher levels in the absence of BSA (FLE"rc~t et al., 1991x) . With thelimitations of membrane disruption in mind, the toxins may still prove to be extremelyuseful probes of lipid metabolism .

Variability in toxin actionCTX and melittin have been shown to have actions that are variable depending on the

cell type, state of the cell and the species examined. Certain types of cells are moresusceptible than others to CTX (BxnGexce et al., 1967; Kexsne et al., 1985; HnvMex etal ., 1987) or melittin (CxnnK-Mez~res et al., 1991 ; SHARMA, 1992). In the case of melittinthere appears to be a greater susceptibility in those cells expressing high levels of the rasoncogene (SHAAMA ~ 1992) and in the case of CTX chick embryonal fibroblasts trans-formed with the Rous sarcoma virus are more sensitive than untransformed cells (Kexmeet al., 1985). As mentioned above, there is a species difference in hemolysis induced by theCTXs (TuitxEx, 1957 ; Coxnaua et al., 1964x; F~TCm tt et al., 1990x) and melittin(Osoxto s Ces~rxo et al., 1990). Additionally, red blood cells from humans become moresusceptible to lysis when they are `aged' (Ct-~x et al., 1984; JiexG et al ., 1989b) . As regardsthe action of CTX in skeletal muscle, the Cat+-dependence of contracture induction(FLETCiIIIt and Lizzo, 1987) and the lowering of the threshold of CaZ +-induced CaZ+release (Ft,srcFmt et al., 1993) are examples in which there are species differences. There isalso considerable variability among individuals from the same species in the effects ofmelittin (Fr.ETC~t et al., 1992) and CTX (FLETCHER et al ., 1993) on Cat+ release . Wehave observed considerable variability in the activation of PLC by the toxins in primarycultures of human skeletal muscle with no detectable effects at times (unpublished

686


1 P3 --"~

Release ofradioactivity

DG~FA `~

Contracture

.FA

to medium____

ce s*AIPam

TOXIN

M~mbranodlvuplion

RESPONSE

TARGET

F1G . S . P06SIBLE PRIMARY AND 3ECONDARY ACTIONS OF Ttfl: TOXINS.We present three potential primary responses (activation of PLC, activation of triglyceridehydrolysis and membrane disruption). Two other actions (indicated by ?), CaZ+ release from thesarcoplasmic reticulum and inhibition or stimulation of CaZ++MgZ*-ATPase, could be primaryactions, or could be a secondary consequence of elevating fatty acid (FA) levels, or in the case of

CaZ+ release, elevating IP 3 levels .

observations) . We believe that the variability may relate to the state of differentiation ofthe cultures and the degree of expression of various enzymes involved in lipid metabolism,although this has not been proven. An alternate explanation is an up- and down-regulation of toxin receptors. Despite the variability in activation of PLC, we consistentlyobserve the release of radioactivity into the incubation medium and would attribute thisto a physical interaction with the membrane independent of either specific binding sites oractivation of lipases .

Primary vs . secondary effectsA major problem in this field is identifying which toxin actions are primary (direct

target sites) and which are secondary (indirect consequence of a primary action). Anexample of a secondary action would be the production of fatty acids by a direct action onan enzyme (e.g . PLA2 or PLC) . These fatty acids could, as a secondary effect, alter thefunction of several other proteins, as shown in Fig. 5. The functions of a number of ionchannels are altered by fatty acids, as described above. Also, a number of proteinscovalently bind fatty acids through a process called acylation (palmitoylation andmyristoylation) and this could alter their function, although the actual consequences ofacylation on protein function are poorly understood (SALTIEL et al., 1991). The CTXs andmelittin may themselves be acylated, as covalent attachment of fatty acids to Lys residueson a hemolytic toxin from Cerebratulus lacteus has been reported (Lru and BLUMIIV'THAL,1991).

Biochemical artifactsSince this review is concerned with mechanisms of action of the toxins, it is important to

differentiate between actions in intact and disrupted tissues. In some cases the toxinswould not normally gain access to substrates presented in disrupted systems. Also, if thetoxins act through proteins or cofactors that are lost during the isolation of organelles,some of their actions may not be detectable in disrupted preparations . This does not


687

negate the utility of the toxins as probes to examine function in disrupted systems, or theuse of isolated organelles to understand the toxin action . However, it does mean that if theintention of the study is to determine what action is causing an effect in intact tissue, thenefforts must be taken to verify that the effects examined in isolated organelles are relevantto intact tissue . The CTXs and melittin depolarize cells, cause membrane release (celldisruption) and activate PLC and $n arachidonic acid-associated PLAZ. All of theseactions occur in intact tissue . While the toxins cause CaZ+ release and interact with anumber of proteins in isolated sarcoplasmic reticulum preparations, the relevance of thesefindings to inducing contracture in intact tissue has not been determined . Dantrolene doesafford partial antagonism of CTX-induced contractures in skeletal muscle (intact tissue),supporting Cat+ release from internal stores (Fr src~t and Lizzo, 1987). However,activation of PLC could cause CaZ+ release indirectly through IP, (HENZI andMncDmMOZ-r, 1992 ; MErrxt~ et al., 1992) and fatty acid formation (FLETCHER et al.,1990c), or depolarization and CaZ+-induced CaZ+ release could be involved .

Differences between CTX and melittinAlthough the bulk of this review has focused on similarities in mechanisms between the

two toxins, it is important to note that there are some differences . Structurally, CTXconsists of three loops, while melittin has more of a cylindrical shape that is bent in themiddle separating two a-helical stretches by 60° (Haxvt:x, 1990). While CTX bindspreferentially to negatively charged phospholipids, such as phosphatidylserine, melittinstrongly interacts with neutral phospholipids, although there may be a more subtlespecificity of melittin for phosphatidylserine (see above) . Also, high CaZ+ concentrationsinhibit the binding of CTX to membranes and thereby antagonize all of the actions of thetoxin. In contrast, high CaZ+ concentrations have no effect on contracture induction bymelittin (see above) . Additionally, the release of radioactivity from cells treated withmelittin is decreased at higher toxin concentrations (c . 10 ~M) and this is not observedwith CTX (see above) . Also, hemolysis induced by CTX is slow, whereas that induced bymelittin is fast (Ln~ Sxtnu et al., 1975). Melittin (FLfiTCHER et al ., 1992) is about 100 timesmore potent in inducing CaZ+ release than CTX (FLSTCF~R et al ., 1991b, 1993).Melittin is far less cytotoxic to HL-60 cells than CTX (Rnrnrox et al., 1991). Furthermore,melittin inhibited ~Rb uptake into and differentiation of these cells, while CTX could notinduce these effects at subcytolytic concentrations (RAYNOR et al., 1991). The CaZ+dependence of PLAZ activation is different for CTX and melittin (Smut, 1982). Weconclude that the actions of these toxins are similar and may involve the same bindingsites ; however, the exact mechanisms are not identical.

Future directionsDespite some very interesting findings regarding the pharmacological mechanisms of

the cobra venom CTXs and bee venom melittin, it seems that we are still at the beginningof our understanding the actions of these agents. Areas for future research that seemespecially important in better understanding these toxins include: (1) the relationshipbetween depolarization and contracture induction ; (2) the relationship, if any, betweencontracture induction and activation of PLC; (3) the interaction of the toxins with PLAZ,PLC, and triglyceride metabolizing enzymes and the best approaches for examining theseinteractions (radiolabeling cells with linoleic acid, arachidonic acid, etc.); (4) the toxin

68 8

J. E. FLETCHER and M.-S. JIANG

Fic. 6. Sos~ rossrei,e rwROEts of CI'X, s~arrnv wxn x~tx x~r~ve vexo~ PLA~s uv uv~rwcrstcII.E-rwi. Mvscr~.

The PLCand PLAZ activated by the toxins are apparently acting on different substrates . The PLCappears to be located intracellularly and definitely hydrolyzes phosphatidylethanohunine (shownin figure) and phosphatidylinositol, but hydrolyzes phosphatidylcholine to a lesser extent, if at all .The PLAz appears to hydrolyze a minor pool of phoapholipid not associated with linoleic acidmetabolism and specific for arachidonic acid that may include phosphatidylcholine, but this hasnot clearly been established. The toxins cause Cap+ release from isolated termiaal cisternaevesicles, but it is unclear (~ whether this occurs in intact tissue. The toxins also release membranefragments through a physical interaction with the membrane. Venom PLA2s primarily hydrolyzethe phoapholipid in the outer leaflet of the membrane bilayer, and this is primarily

phosphatidylcholine.

binding sites) in intact tissue and those modulating Caz+ release in the isolated sarco-plasmic reticulum; (5) whether internalization of the toxin occurs; (6) the role of CaZ+release from internal Cap+ stores in contractures ; (~ the relationship between thehemolytic action of a subpopulation of the CTXs and effects in other tissues; (8) theinteractions between fatty acids and the toxins ; (9) why high concentrations of melittinstimulate triglyceride breakdown and inhibit the release of radioactivity (membranefragments) from the cells; and (10) why there is considerable tissue and species variation inresponse to the toxins in some systems. Some of the tools that may prove useful are theinorganic CaZ+ antagonists (especially Coy+ and Mn2+), the species and individualdifferences in toxin action and the wide variety of mechanisms and CTXs that can be usedfor correlation studies .


689

ConclusionsWhile our understanding of some of the actions of the cobra venom CTXs and melittin

has become more clarified, other actions have become more obscure. These toxins affectmany different proteins in disrupted systems and it is unclear which, if any, of theseactions are relevant to mechanisms in intact tissues. It is also unclear as to which primarytarget is responsible for triggering the cascade of secondary events . The production ofsecond messengers with widespread actions, such as IP3 and free fatty acids, greatlycomplicates the dissociation of primary from secondary actions. Two actions of thetoxins, PLC activation and membrane depolarization, are relatively rapid and these eventsmust be dissociated to better understand the mechanisms of toxin action . The majorappeal of these latter two mechanisms as potentially responsible for contracture inductionis that they occur in intact tissue and follow a time course similar to contracture induction .In contrast to activation of PLC, the activation of PLAZ by the toxins does not relate tothe immediate actions of the toxin and the PLAZ activity activated appears highly specificfor arachidonic acid . Understanding toxin action is further confused by the physicalremoval of membrane by the toxins . Although an understanding of the mechanisms of thetoxins in excitable tissue is one interesting endpoint, these toxins will most probably notbe useful probes in intact tissue to examine processes like contractures due to theirmultiplicity of actions . While of limited utility as specific pharmacological probes in intacttissue, these toxins may still be valuable probes of lipid metabolism and CaZ+ regulation,the former in intact and isolated systems and the latter in isolated organelles .Additionally, melittin has been an especially useful probe of protein-protein and protein-lipid interactions . However, in all studies of melittin and CTX, it is essential to eliminatethe confounding contribution of venom PLAZ contamination .

In closing, this review is intended not to act as a compendium of all the facts related tomelittin and CTX action, but to act as the starting point for stimulating future research onthe mechanisms of action of these toxins. Presented are testable hypotheses that will haveto be reevaluated as contradictory data are uncovered .

Acknowledgements-Funding was provided by the U.S . Army Medical Research and Development Command,Contract No . DAMD17-90-C-0134 and the Hahnemann Anesthesia Research Foundation . Opinions, interpreta-tions, conclusions and recommendations are those of the authors and are not necessarily endorsed by the. U.S .Army .

REFERENCES

D'At.acs, A., COUTEAUX, R., Jexsim~, C., Router, A. and MIRA, J.-C. (1988) Regeneration after cardiotoxininjury of innervated and denervated slow and fast muscles of mammals. Myosin isoform analysis. Eur. 1.Biachem . 174, 103-110.

ArmERSOx, M. P. and WEI~I, M. J. (1990) Fatty acids inhibit apical membrane chloride channels in airwayepithelia. Proc. natn . Acad. Sci. 87, 7337338.

Al~sel-L, G. B. and SrAxrmt, S. (1982) Phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine .In: Phospholipids, pp . 1~9 (HAwTxa~RrIE, J. N. and Arlsal,l ., G. B., Eda) . Amsterdam: Elsevier Biomedical .

ARIGA, T., MACALA, L. J., $AI1'O, M., MARaolas, R. K., GRemvE, L. A., MAROOLL4,R. U. and Yv, RK. (1988)Lipid composition of PC12 pheochromocytoma cells: characterization of globoside as a major neutralglyeoplipid. Biochemistry 27, 52-58.

BAS~oRn, C. L., RODRIOUE3, L. and PASTERxAx, C. A. (1989) Protection of cells against membrane damage byhaemolytic agents: divalent cations and protons act at the extracellular side ofthe plasma membrane. Biochim .biophys. Acta 983, 56-ßr4 .

BELL, R. M. (1986) Protein kinase C activation by diacylglycerol second messengers. Cell45, 631-632.

690


Btutt.v, G., JwCQuFS, D., YAMAMaI'O, T., SCUt.rrotsEwrru, A. and Pwvgr, M. D. (1988) Three types of slowinward currents as distinguished by melittin in 3~ay-old embryonic heart. Can. J. Phyriol. Pharmac. 66,1017-1022.

Bt-wtvs, M. L., SsarH, Z. L. and Sxvn>at, F. (1992) Contributing factors in the trafficking of [tH]arachidonatebetween phospholipids . Biochim . biophys . Acta 1124, 262-272.

Boucts, P., Rocxwr, H., PtFxoxt, G. and Vtatcett, R. (1981) Penetration of phospholipid monolayers bycardiotozins . Biochemistry 20, 4915-4920.

Boucts, P., Tt~x, M., Vwx Rmtsceo~trtv, J., Rocxwr, H., FwucoN, J. F. and DusouxCQ, J. (1983) Areinteractions with phospholipids responsible for pharmacological activities ofcardiotoxins . Mol. Cell. Biochem.SS, 49fi4.

Boucts, P. E., MARCHOT, P. and RocHwr, H. (1987) In viva synergy of cardiotoxin and phospholipase AZ fromthe elapid snake Naja mossambica mossambica . Toxicon 25, 427-431.

Boucts, P. E., KHEWF, A. and Rocxwr,H. (1989) On the inhibition of [Na*,K*]-ATPases by the components ofNaja mossambica mossambica venom: evidence for two distinct rat brain [Na+,K*}ATPase activities .Biochemistry 28, 3037-3043.

BRAGANCA, B. M., PA1EL, N. T. and BwnxtxwTt~t, P. G. (1967) Isolation and properties of a cobra venom factorselectively cytotoxic to Yoshida sarcoma cells . Biochim . biophys . Acta 136, 508-520.

CHAIM-MATYAS, A., Botttcow, G. andOvwOU, M. (1991) Isolation and characterization of a cytotoxin P4 fromthe venom of Naja nigricollis nigricollis preferentially active on tumor cells . Biochem . Int. 24, 415-421.

CiiwNG, C. C. (1979) The action of snake venoms on nerve and muscle . In : Snake Venoms, pp . 309-376 (Lg,C. Y., Ed .) . Berlin: Springer.

CttwxG, C. C., CHUANG, S:T., Lam, C. Y. and Wet, J. W. (1972) Role of cardiotoxin and phospholipase A in theblockade of nerve wnduction and depolarization of skeletal muscle induced by cobra venom. Br . J. Pharmac.44,752-764.

C~twurux, V. P. S., CHwuxwx, A., DtatnrtutcH, D. S. and Bxoctcrxxo~, H. (1990) Lipid activators of proteinkinase C. Ltfè Sci. 47, 981-986.

Carat, C.-C. and Ltx-St-nwu, S.-Y. (1985) Mode of inhibitory action of melittin on Na'-K*-ATPase activity ofthe rat synaptic membrane . Biochem . Pharmac. 34, 2335-2341.

Cttt~t, Y.-H., Hu, C. T. and YwNC, J. T. (1984) Membrane disintegration and hemolytis of human erythrocytesby snake venom cardiotoxin (a membrane-disruptive polypeptide). Biochem . Int. 8, 329-338.

CHOI, O. H., Pwnctrrr, W. L. and Dwt,av, J. W. (1992) Effects of the amphipathic peptides melittin andmastoparan on calcium influx, phosphoinositide breakdown and arachidonic acid release in ratpheochromocytoma PC12 cells . J. Pharmac. exp . Ther . 260, 369-375.

CHOW, S. C., ANSO~t~ecut, I. J. and Joxnwt., M. (1990) Inhibition of receptor-mediated calcium influx in T-cellsby unsaturated non-esterified fatty acids. Biochem. J. 267, 727-732.

CoM~, M., Mwut .er, Y. and Coz, J. A. (1983) CaZ*dependent high affinity complex formation betweencalmodulin and melittin . Biochem. J. 209, 269-272.

Corrottew, E. (1974) Membrane-active polypeptides from snake venom: cardiotoxins and haemocytotoxins.Experientia 30, 121-129.

Corrote>:w, E. (1979) Hemolytic effects of snake venoms. In : Snake Venoms, pp . 44879(Lta:, C. Y., Ed .) . Berlin :Springer.

Coxottrw, E., MAMMON, Z., At .OOt=, S. and DE Vtuzs, A. (1964a) Susceptibility of erythrocytes of various animalspecies to the hemolytic and phospholipid splitting action of snake venom. Biochim . biophys. Acta 84, 365-375.

Coxnttrw, E., DE Mars, A. and Mwcex, J. (1964b) Hemolytis and splitting ofhuman erythrocyte phospholipidsby snake venoms. Biochim. biophys. Acta 84,6073.

Corrottaw, E., FLEI'CI~R, J. E., RAPUANO, B. E., Ywxc, C.-C. and RoseNSettc, P. (1981) Effect ofmodification ofone histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase Az from Najanigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja naja atrasnake venoms . Toxicon 19, 61-71 .

CONRICODE, K. M. and OOHS, R. S. (1989) Mechanism for the inhibitory and stimulatory actions of proteins onthe activity ofphospholipase Ar. Bicehim . biophys . Acta 1003, 363.

Cou~rewux, R., Mtttw, J.-C. and D'At,tus, A. (1988) Regeneration of muscles after cardiotoxin injury . I .Cytological aspects . Biol . Cell 62, 171-182.

Gbrt>otErrt, J. and Asttau, A. J. (1990) Interaction of melittin with the (Na*-K*)ATPase : evidence for amelittin-induced conformational change. Archs Biochem . Biophys. 283, 249-257.

DEMI~SEY, C. E. (1990) The actions of melittin on membranes. Biochim . biophys . Acta 1031, 143-161.DeMt~t,C. E. and WwTTS, A. (1987) A deuterium and phosphorus-31 nuclear magnetic resonance study ofthe

interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of wntaminatingphospholipase A2. Biochemistry 26, 5803-5811.

DEVwUX, P. F. (1991) Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30, 1163-1173.Du,Y. C., SHEN, J. H., Wu,W. Y., Wu, C. R., Wwxc, K. Z., Gu, B. X. and Fu,P. (1986) Chemical synthesis of

India cobra cytotoxin II by solid-phase condensation of fragments. Biopolymers 25, 58596.Dut=outtCQ, J. and FwucoN, J.-F. (1978) Specific binding of a cardiotozin from Naja mossambica mossambica to

charged phospholipids detected by intrinsic fluorescence . Biochemistry 17, 1170-1176.


69 1

DutouttcQ, J., FAUCON, J.-F., Fouxct~, G., D~ux, J.-L., Mwtxi:, M. L. and GULne-KtezYwtctu, T. (1986)Morphological changes ofphosphatidylcholine bilayers induced by melittin : vesiculari7ation, fusion, discoidalparticles . Biochim . biophys. Acta 859, 338.

DuFrox, M. J. and HtnEtt, R. C. (1991) The structure and pharmacology of elapid cytotoxins . In : Snake Toxins,pp. 259-302 (H~xvt:v, A. L., Ed .) . New York: Pergamon Press.

Enxt� J. E. and Exct=.t.t., B. J. (1972) The effects of toxic components of Naja nivea (Cape cobra) venom onneuromuscular transmission and muscle membrane permeability . Comp. Biochem . Physiol. 41A, 59715.

Et.uoT, R. H. (1905) A contribution to the study of the action of Indian cobra venom. Phi. T Roy . B . 197,361-~05.

Ft .Erct-mix, J. E. and Jtwrrc, M.-S. (1992) Activation of tissue phospholipases C and D by a snake venomcardiotoxin and bee venom melittin . In: Recent Advances in Toxinology Research, Vol.2, pp.215-221(Gor~t.ntcxtst~tNetcor>E, P. and Tax, C. K., Eds). Singapore: Venom & Toxin Research Groupa, NationalUniversity of Singapore.

Ft .src~x, J. E. and Ltzzo, F. H. (1987) Contracture induction by snake venom cardiotoxin in skeletal musclefrom htunans and rats . Toxicon 25, 1003-1010.

Ft,erct~x, J. E., Yarra, C. C. and Rasetvaz:ac, P. (1982) Basic phospholipase AZ from Naja nigricollis snakevenom: phospholipid hydrolysis and effects on electrical and contractile activity ofthe rat heart. Toxicol. appl.Pharmac . 66, 39-54.

Ft,erct~x, J. E., Ktsrt.ea, P., Raserteexa, H. and Mtcttwux, K. (1987) Dantrolene and mepacrine antagonize thehydolysis of human red blood cells by halothane and bee venom phospholipase Ar Toxicol. appl. Pharmac.90, 41019.

Ft .erct~at, J. E., Rost:rrnEtta, H., MICHAUX, K., Cl~tt, K. S. and CI~AFt, A. M. (1988) Lipid analysis ofskeletalmuscle from pigs susceptible to malignant hyperthermia. Biochem . Cell. Biol. 66, 917-921.

Ft .srcttex, J. E., Rosßvanto, H., MtCHAUX, K., Tturot,tTts, L. and Ltzzo, F. H. (1989) Triglycerides, notphospholipids, are the source of elevated free fatty acids in muscle from patients susceptible to malignanthyperthermia. Eur. J. Anaesth . 6, 355-362.

Ft.ercrrFtt, J. E., h~tvo, M.-S., Twrot.trts, L., SMn'H, L. A. and Bt:ECx, J. (1990a) interactions in red blood cellsbetween fatty acids and either snake venom cardiotoxin or halothane. Toxicon 28, 65767.

F~ .Erct-mix, J. E., M~cttwux, K. and JuNC, M.-S. (1990b) Contribution of bee venom phospholipase AZcontamination in melittin fractions to presumed activation of tissue phospholipase Az. Toxicon 28, 647-656.

Ft.Erctmtt, J. E., TRIt~LITl3, L., Exwtx, K., Hetvsort, S., RosErtHetto, H., Coin, P. A. and Bgcx, J. (1990c)Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulumfractions: implications for malignant hyperthermia. Biochem . Cell. Biol. 68, 1195-1201.

Ft,grct~x, J. E., J»a, M.-S., Goxo,Q.-H. and San~rtt, L. A. (1991a) Snake venom cardiotoxins and bee venommelittin activate phospholipase C activity in primary cultures of skeletal muscle . Biochem . Cell . Biol. 69,274-281.

FI,EI'CIiCA, J. E., Jt~xo, M-S., Goxo, Q-H., YUDKOWSKY, M. L. and W>EUxn, S. J. (1991b) Effects of acardiotoxin from Naja naja kaouthia venom on skeletal muscle : involvement of calcium-induced calciumrelease, sodium ion currents and phospholipases A2 and C. Toxicon 29, 1489-1500.

FLSrct~te, J. E., Mwt~t:aot~t, S., Txtrot.rng, L., YUDKOWSICY, M. and RasParaeao, H. (1991c) Fatty acidsmarkedly lower the threshold for halothane-induced calcium release from the terminal cisternae in human andporcine normal and malignant hyperthermia suceptible skeletal muscle . Life Sci. 49, 1651-1657.

FLE1CIi©t, J. E., Txtrot .tns, L. and BEecx, J. (1992) Bee venom melittin is a potent toxin for reducing thethreshold for calcium-induced calcium release in human and equine skeletal muscle. Life Sci. 51, 1731-1738.

Ft.srcttett, J. E., TRtI~I,Trts, L. and Beecft, J. (1993) Species difference in modulation ofcalcium release by Najanaja kaouthia snake venom cardiotoxin in terminal disternae from human and equine skeletal muscle. Toxicon31, 45-51 .

Four, A. M., MEt.TZea, S., B~t~tv, M. C. and Louw, A. I . (1983) The effect of cardiotoxin on (Ca'+ +Mgz+)-ATPase of the erythrocyte and sareoplasmic reticulum. Biochem . Int . 6, 581-591.

Get.errts, P. and Nox~rox, R. S. (1990) Biochemical and pharmacological studies of the mechanism of action oftenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone, Actinic tenebrosa. Toxicon28, 695-706.

GATTNEAU, E., T~ctn, M., Bout=r, F., MANSUELLE, P., ROCt~T, H., H~xvt:v, A. L., MorrtExnY-Gnxasrtex, T.and MErtEZ,A. (1990) Delineation of the functional site ofa snake venom cardiotoxin : preparation, structure,and function of monoacetylated derivatives. Biochemistry 29, 6480489.

GROMOVA, I. A., Mowtcovstcv, J. G. and BettoEtsox, L. D. (1992) Anthrylvinyl-labeled phospholipids asfluorescent membrane probes. The action ofmelittin on multilipid systems. Chem. Physics Lipids 60, 235-246.

Gut,, S. and St~uxtt, A. D. (1974) Haemolysis of intact human erythrocytes by purified cobra venomphospholipase A2 in the presence of albumin and Ca'+ . Biochim . biophys . Acta 367, 271-281 .

Gut.ttc-Ktszrwtcxt, T., B~t~xx~, M., VtNCt:rrr, J.-P. and Lnznutvsta, M. (1981) Freeze-fracture study ofcardiotoxin action on axonal membrane and axonal membrane lipid vesicles . Biochim . biophys . Acta 643,101-114.

Gusovsxv, F., Sat?ttota., D. G. and D~t.v, J. W. (1991) Effects of hastoparan and related peptides onphosphoinositide breakdown in HL-60 cells and cell-free preparations . Eur. J. Pharmac . 206, 309-314.

692


HAaea, M. T., Fus:ut, T ., Lt~owtrz, M. S . and Lowexs'tEtx, J . M . (1991) Activation of phosphoinositide-specific phosphoGpase Cgamma from rat liver by polyamines and basic proteins. Archs Biochem. Biophys . Z88,243-249 .

Hwa~estwnx, E . (1972) Bee and wasp venons. Science 177, 314-322 .HwaeßsrwxN, E . and Jstv'rsctr, J. (1967) Sequen~analyse des melittins ~us den tryptischen anti peptischen

spaltshtcken . Happe-Seyler's Z. Physiol. Chem . 348, 37-50 .HARVEY, A . L. (1985) Cardiotoxins from cobra venons: possible mechanisms ofaction . J. Toxicol. Toxin Rev. 4,41fi9.

HwRVEV, A. L . (1990) Cytolytic toxins. In : Handbook ojToxinology, pp. 1~6 (St~tt, W. T. and MEas, D ., Eds) .New York: Marcel Dekker.

HwRVEY, A. L . (1991) Cardiotoxins from cobra venons . In : Reptile Venons and Toxins, pp . 85-106 (Tu, A . T.,Ed.) . New York : Marcel Dekker.

HARVEY, A. L ., M "RSHAr L, R . J. and KARLS90N, E. (1982) Effects of purified cardiotoxins from the Thailandcobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations. Taxiton 20, 379-396 .

HwRVEY, A. L ., Hrnnt, R . C . and KHAnER, F . (1983) Effects of phospholipase A on actions of cobra venomcardiotoxins on erythrocytes and skeletal muscle . Biochim. biophys. Acts 728, 215-221 .

HExz~, V . and Mwcnetu~o'c-r, A. B. (1992) Characteristics and function of Ca'+- and inositol 1,4,5-trisphosphate-releasable stores of Ca'+ in neurons. Neuroscience 46, 251-273 .

Hn~ER, R. C . and HwxvEY, A . L. (1982) The structure and actions of cardiotoxins isolated from cobra venons .S. Afr. J. Sci. 78, 350-356 .

Hu>ER, R. C. and KNADER, F. (1982) Biochemical and pharmacological properties ofcardiotoxins isolated fromcobra venom . Toxicon 20, 175-179 .

H~nER, R. C ., KwRrssox, E. and NAtruRwxtwx, S . (1991) Separation and purification of toxins from snakevenons . In : Snake Toxins, pp . 1-34 (IiwRVeY, A. L ., Ed.) . New York: Pergamon Press .

H~cwsFnrnaw, T., Buat~trFrt, J. and Ross, E . M . (1990) Regulation of G; and Go by mastoparan, relatedamphiphilic peptides, and hydrophobic avines . Mechanism and structural determinants of activity. J . biol.Chem. 266, 14,176-14,186 .

HtrrMwx, C . L ., LErrs~ro, E., S~veNS, R., Morrrco~tY, N., RAUCt~t, H . C . and Htrosox, R . A . (1987) Effects ofcardiotoxin D frov Naja naja siamerrsis snake venom upon marine splenic lymphocytes . Toxicon 25,1011-1014 .

Ho, C.-L ., LEE, C. Y . and Lu, H. H . (1975) Electrophysiological effects of cobra cardiotoxin on rabbit heartcells . Toxicon 13, 43746 .

HODGES, S. l ., AGHAJI, A . S ., HARVEY, A. L . and HmER, R . C. (1987) Cobra cardiotoxins . Purification, effects onskeletal vuacle and structure/activity relationships . Eur. J. Biochem. 165, 373-383 .

Huwxo, J.-L . and TRUMBIE, W. R . (1991) Cardiotoxin from cobra venom affects the Ca-Mg-ATPase of cardiacsareolemmal membrane vesicles . Toxicon 29, 31-41 .

HWANG, T.-C ., Guccnvo, S. E . and Guccnvo, W. B . (1990) Direct modulation ofsecretary chloride channels byarachidonic and other cis unsaturated fatty acids. Proc . natn. Acad. Sci . 87, 5706-5709 .

IICURA, T ., Go, N. and INAOAICI, F. (1991) Refined structure of melittin bound to perdeuterateddodecylphosphocholine micelles as studied by 2D-NMR and distance geonetry calculation. Prat. Struct . Func.Genet . 9, 81-89.

Jw~sox, G . A . JR and Vrr r Fop " r , M. L . (1987) Mitogen-stimulated release of inositol phosphates in humanfibroblasts . Archs Biochem. Biophys. 252, 478-~86 .

Jwvo, M:S., Fr.nrct~tt, J . E . and SMI7H, L. A . (19ß9a) Effects of divalent cations on snake venom cardiotoxin-induood hemolyais and ~I-I-deoxyglucose-6-phosphate release from human red blood cells. Taxiton 27,1297-1305 .

Jwvt;, M.-S ., FIETCfflat, J . E . and Ssvrx, L . A. (19ß9b) Factors influencing the hemolysis of human erythrocytesby cardiotoxins from Naja naja kaouthia and Naja naja atra venons and a phospholipase A Z with cardiotoxin-like activities from Bungarus jasciatus venom. Toxicon 27, 247-257 .

Kwr~w, N . and HwYA3HI, K. (1983) Separation ofcardiotoxins (cytotoxins) from the venons of Naja naja andNaja naja atra by reversed-phase high-performance liquid chromatography . J. Chromat. 281, 389-392.

KAN®w, N., Swswtci, T . and HwYwstn, K. (1977) Primary structures ofcardiotoxin analogues II and IV from thevenom of Naja naja arra . Biochim . biophys . Acts 491, 536.

KAN®A, N., HAMAaucttt, M., KoJ~tw, K ., KwxES~w, H . and HwYwsrn, K . (1985) Action of cobra venomcardiotoxin on chick embryonal fibroblasts transformed with a temperature-sensitive mutant ofRous sarcomavirus . FEBS I,ett . 192, 313-316 .

Kwrwoaw, M., HEwn, J . F., SewTON, B. A . and Exoa.Mwx, D . M . (1989) Melittin binding causes a largecalcium-dependent conformational change in calmodulin . Proc . nain. Acad. Sci. 86, 6944-6948 .

ICArorr, N ., RwYxoR, R. L., WISE, B . C., ScxwrzMwrr, R . C ., TuRriat, R . S ., Her.~wx, D . M., Fw~x, J . N. andKuo, J:F. (1982) Inhibition by melittin of phosphoGpid-sensitive and calmodulin-sensitive Ca'+-dependentprotein kinases. Biochem . J. 202, 217-224 .

Kw~ISU, T ., Nnvo~rw, C ., Kuxolco, M., KoswYASxi, H ., HmoTw, T . and Fulrrw, Y . (1988) Action ofamphipathic peptides gramicidin S and melittin on erythrocyte membrane . Biochim . biophys. Acts 939, 573.


693

ICe~tstr, T., Kuxo~o, M., MORIICAWA, T ., Swxct~e, K., FunTe, Y., Yexeestrtu, H. and Une, M. (1989)Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin . Biochim.biophys . Acts 983, 135-141.

Knt, D. and G.erHe~, D. E. (1989) Potassium channels in cardiac cells activated by arachidonic acid andphospholipids. Science 244, 1174-1176 .

Knot, R. M. and Evexs, H. J. (1989) A common cytolytic region is myotoxias, hemolysins, cardiotoxins andantibacterial peptides . Int. J. Peptide Prot. Res. 34, 277-286.

Kuo, J. F., Renvox, R. L., Mwz~, G. J., Scxerz~ux, R. C., Tuxr~t, R. S. and KBes, W. R. (1983) Cobrapolypeptide cytotoxin I and marine worm polypcptide cytotoxin A-IV are potent and selective inhibitors ofphospholipid-sensitive Cat*-dependent protein kinase . FEBS Lett . 153, 183-186.

Len, P. J. and Smut, W. T. (1979) Activation of microsomal guanylate cychtse by a cytotoxic polypcptide :melittin. Biochem. biophys. Res. Commun. 89, 315-321 .

Len, P. J. and Stagy, W. T. (1980) Effect of melittin-induced membrane alterations oa rat heart adenylatecyclase activity . Archs Biochem. Biophys . 204, 41824.

Lextuscx, P. G., LAB, L., Ot~tcs, H. D. and VocT, W. (1971) Binding of phospholipase A to the direct lyticfactor revealed by the interaction of Cap* with the haemolytic effect . Biochim. biophys. Acts 239, 267-272.

Let~t, P. G., Scxonmt, K., Sctiot~, W., Kuwze, H., Hotav, E. and Vocr, W. (1972) Inhibition of Na*- andK*-activated ATPase by the direct lytic factor of cobra venom (Naja naja) . Biochim. blophys. Acts 266,133-134.

LBE, C . Y ., Ctiexc, C. C., C84u, T. H., Ctau, P. J. S., TsEtve, T. C. and LBE, S. Y. (1968) Pharmacologicalproperties of cardiotoxin isolated from Formosan cobra venom. Nawryn-Schmiedebergs Arch. Pharmac. 259,360-374.

LEE, C. Y., Ho, C. L. and Ee~t, D. (1977) Cardiotoxin-like action of a basic phospholipase AZ isolated fromNaja nigricoUis venom. Toxicon 15, 355-356.

LBE, G. L. and Herr, W. N. (1985) Inhibition ofgrowth of C6 astrocytomacells by inhibitors ofcalmodtilin . LifeScè. 36, 347-354.

LEUNC, W. W., Ksuxe, W. M. and Koxe, Y. C. (1976) The cytolytic effect of cobra cardiotoxin on Ehrlichascites tumor cells and its inhibition by Cap* . Naunyn-Schmiedebugs Arch. Pharmak. 292, 193-198 .

LINDEN, D. J. and RovrtwaEae,A. (1989) cis-Fatty acids, which activate protein kinase C, attenuate Na* andCaz* currents in mouse neuroblastoma cells. J. Physiol. 419, 95-119 .

LtN Staeu, S. Y., Huexe, M. C., Tswe, W. C. and LEE, C. Y. (1975) Comparative studies on the biologicalactivities of cardiotoxin, melittin and prymnesin. Nawiyn-Schmiedebergs Arch. Pharmak. 287, 349-358.

Lnv Staeu, S. Y., Huexc,M. C. and LBE, C. Y. (1976) Mechanism of action ofcobra cardiotoxin in the skeletalmuscle . J. Pharmac. exp. Ther. 1%, 758-770.

Lnv-St~aeu, S. Y., HueNC, H. C. and Lam, C. Y. (1986) Acomparison of the actions of cobra cardiotoxin andscorpion toxin II on the guinea-pig taenia coli . Toxicon 24, 131-139.

Ltu, J. and Bute~vrttet, K. M. (1991) Identification of oleic acid binding sites in cytolysin A-III from theheteronemertine Cuebratuha lacteus. Toxicon 29, 13-20.

Louw, A. I . and VIS3Elt, L. (1978) The synergism of cardiotoxin and phospholipese Az in hemolysis . Biochim.biophys . Acts 512, 163-171 .

MAHANEY, J. E. and Tt~DSUS, D. D. (1991) Effects of melittin on molecular dynamics and Ca-ATPase activity insarcoplasmic reticuhun membranes: electron paramagnetic resonance . Biochemistry 30, 7171-7180 .

MwRSx, N. A. and WHAT-Ety B. C. (1980) The effects of honey bee (Apis mellijua L.) venom and two of itsconstituents, melittin and phospholipase A2, on the cardiovascular system of the rat. Toxicon 18, 42735.

Meutsr, Y., BxoDeBCtc, U. and Ftn.rtus, B. W. (1982) Purification from bee venom of melittin devoid ofphospholipase Az contamination . Analyt. Biochem. 127, 617.

MBt.Dxues, B. S. (1965) Actions of whole and fractionated Indian cobra (Naja naja) venom on skeletal muscle .Br . J. Pharmac. 25, 197-205 .

MENEZ, A., Ge~tu~eu, E., Rou~sreNn, C., HARVEY, A. L., Mouewen, L., Ga.Qtmv, B. and Tote, F. (1990)Do cardiotoxins possess a functional site? Structural and chemical modification studies reveal the functionalsite of the cardiotoxin from Naja nigricollis. Biochimie 72, 575-588.

MBNNIII, F. S., Bata, G. S. J., GtBNNON, M. C., Oaa; J. F., Rossa~rt, M. F. and Ptrrno?v, J. W. JR (1992) Inositolpolyphosphates and cak:ium signaling. Mol. CeU. Neurosci. 3, 1-10 .

MB~tz, S. A. (1986) Lack of specificity of melittin as a probe for insulin release mediated by endogenousphospholipase Az or lipoxygenase . Biochem. Pharnrac. 3S, 3371-3381 .

MDC, L. L., DINHts1PdN, R. J. and Vn ~ uup~_r ., M. L. (1984) Mitogens and melittin stimuLite an increase inintracellular free calcium concentration in human fibrobhtsts . Biochem. biophys. Res. Common. 119, 69-75.

MOLLAY, C. and KRBa., G. (1974) Enhancement of bee venom phospholipase AZ activity by melittin, direct lyticfactor from cobra venom and polymyxin B. FEBS Lett . 46, 141-144.

MOLLAY, C., KREa., G. and BHtcER, H. (1976) Action of phospholipases on the cytoplasmic membrane ofEscherichia coli . Stimulation by melittin . Biochim. biophys . Acts 426, 317-324.

NBtsoN, G. J. (1967a) Composition of neutral lipids from erythrocytes of common mammals. J. Lipid Res. 8,374-379.

694


Nrasox, G. J. (1967b) Lipid composition of erythrocytes in various mammalian species . Biochim. biophys. Actu144,221-232.

NEUMANN, W., HAat:xMAxx, E. and HANSex, H. (1953) Differentiation of two hemolytic factors in bee venom.Nawyn-Schmiedebergs Arch . exp. Path . Pharmak. 217, 130-143.

O'BatAx, C. A. and WAxo, N. E. (1989) ATP-sensitive binding of melittin to the catalytic domain of proteinkinase C. Mol. Pharmac. 36, 355-359.

ORUWAY, R. W., WALSH, J. V. and StxcrJrt, J. J. (1989) Arachidonic acid and other fatty acids directly activatepotassium channels in smooth muscle cells. Science 244, 1171x1179.

ORDWAY, R. W., Stxcea, J. J. and WAtsH, J. V. (1991) Direct regulation of ion channels by fatty acids. Tr .Neurosci . 14, 91x100 .

Osotuo E CASTRO, V. R., Asrrwoou, E. R., Woos, S. G. and VeaxoN, L. P. (1990) Hemolysis of erythrocytesand fluorescence polarization changes elicited by peptide toxins, aliphatic alcohols, related glycols andbenzylidene derivatives . Biochim. biophys. Acta 1029, 252-258.

PFSSAH, I. N., WAIERHOUSE, A. L. and CASIDA, J . E. (1985) The calcium-ryanodine receptor complex of skeletaland cardiac muscle . Biochem. biophys. Res. Common . 128, 44956.

PessAH, I. N., $TAMBUK, R. A, and CASrnA, l. E. (198

Caz+-activated ryanodine binding: mechanisms ofsensitivity and intensity modulation by Mgz+, caffeine, and adenine nucleotides. Mol . Pharmac. 31, 232-238.

RAxno, R. R. (1988) Regulation of protein kinase C activity by lipids. FASEBJ. 2, 2348-2355.RAO, N. M. (1992) Differential susceptibility of phosphatidylcholine small unilamellar vesicles to phospholipases

A~, C and D in the presence of membrane active peptides. Biochem. biophys. Res. Common . 182, 682-688.RAVrtox, R. L., Zt->Exa, B. and Kuo, J. F. (1991) Membrane interactions of amphiphilic polypeptides

mastoparan, melittin, polymyxin B, and cardiotoxin . Differential inhibition of protein kinase C,Cap+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na` pumpand differentiation . J. biol. Chem. 266, 2753-2758.

Rosexneac, P. (1990) Phospholipases . In : Handbook of Toxinology, pp. 67-277 ($litFa, W. T. and Mt:as, D.,Eds) . New York: Marcel Dekker.

SALTIEL, A. R., IL+verctr, J. and ADEREM, A. A. (1991) Functional consequences of lipid-mediated protein-membrane interactions . Biochem. Pharmac. 42, 1-1 l .

SARICAR, N. K. (1951) Action mechanism of cobra venom, cardiotoxin and allied substances on musclecontraction . Proc . Soc. exp. Biol . 78, 46971.

SARKAR, B. B., MArraA, S. R. and GIiOSH, B. N. (1942) The effect ofneurotoxin, haemolysin and choline esteraseisolated from cobra venom on heart, blood pressure and respiration . Ind. J. med. Res. 30, 45366.

SARICAR, N. K. (1947) Isolation ofcardiotoxin from cobra venom (Naja tripudians, monocellate variety) . J. lnd.them . Soc. 24, 227-232.

Set~ueonm, E., LuatcE, K., L>~ArtN, M. and BeErz, I . (1971) Haemolytic activity and action on the surfacetension of aqueous solutions of synthetic melittins and their derivatives. Experientia 27, 764-765.

SHARMA, S. V. (1992) Melittin resistance : a counterselection for ras transformation . Oncogene 7, 193-201.Stm:a, W. T. (1979) Activation of high levels ofendogenous phospholipase A~ in cultured cells . Proc. natn . Acud.

Sci. 76, 195-199.St~e, W. T. (1982) Cytolytic mechanisms: self~estruction of mammalian cells by activation of endogenous

hydrolytic enzymes. J. Toxicol. Toxin Rev. 1, 1-32 .SMrrtt, P. B. and FtxcFr, R. A. (1979) Alterations in lipid metabolism of developing muscle cells in culture .

Biochim. biophys. Acta 572, 139-145.SxvnFJe, F., Lse, T.-C. and Buxrc, M. L. (1992) The role oftransacylases in the metabolism ofarachidonate and

platelet activating factor. Prog. Lipid Res. 31, 65-86.STANKOWSICI, S., PAWLAK, M., KAISF~VA, E., ROBERT, C. H. and SCHWARZ, G. (1991) A combined study of

aggregation, membrane affinity and pore activity of natural and modified melittin . Biochim. biophys. Acta1069, 77-86.

S~x, J. J. and WALICEa, J. A. (1986) Actions ofcardiotoxins from the Southern Chinese cobra (Naja raja atra) onrat cardiac tissue . Toxicon 24, 233-245.

TAx, N.-H. (1982) Cardiotoxins from the venom of Malayan cobra (Naja naja sputatrix) . Archs Biochim.Biophys. 218, 51-58.

Tos~sox, M. T. and Tos~sox, D. C. (1981) The sting. Melittin forms channels in lipid bilayers . Biophys. J. 36,109-116.

Tosresox, M. T., ALVAREZ, O., HuBaet,t., W., BrECAxsrct, R. M., ATIENHACH, C., CAronat.FS, L. H., Levy, J. J.,Nu~rr, R. F., R08ENBLATT, M. and Tosrnsox, D. C. (1990) Primary structure of peptides and ion channels .Role of amino acid side chains in voltage gating of melittin channels . Biophys. J. 58, 1367-1375.

Towr-ete, D. A., Gotu~ox, J. L, ADAMS, S. P . and Gtasnt, L. (1988) The biology and enzymology of eukaryoticprotein acylation. A. Rev. Biochim. 57, 69-99.

TRAYNOR, A. E., Sct~Rr, D. and Ar.t.ax, W. R. (1982) Alterations of lipid metabolism in response to nervegrowth factor. J. Neurochem. 39, 1677-1683.

TxuMaLe, W., HE, Z., Wassox, C., HUANG, M., HuAxo, J.-L. and TauMat.e, M. (1992a) Cobra cardiotoxininduces Ca =+ release from cardiac sarcoplasmic reticulum membrane vesicle . Biophys. J. 61, A433 .


695

Txustet.i;, W. R., Huarrc, J.-L., Txuatst.n, M. J., W~soN, C., Huaxc, M. and He,Z. (1992b) Cobra cardiotoxinmediates calcium release from muscle sarcoplasmic reticulum vesicles and binds to specific membrane proteins.In: Recent Advances in Toxinology Research, Vol. 1, pp . 65769 (GOPALArcRIC tüanKONe, P. and Tax, C. K.,Eds) . Singapore: Venom 8c Toxin Research Group, National University of Singapore.

Tuter~x, J. C. (1957) Absence oflecithin from the stromata of the red cells of certain animals (ruminants) and itsrelation to venom hemolysis. J. exp. Med. 105, 189-193.

T7~fiG, W.-F. and Ct~v, Y:H. (1988) Suppression of snake-venom cardiotoxin-induced cardiomyocytedegeneration by blockage of Cap+ influx or inhibition of non-lysosomal proteinases. Biochem. J. 256, 89-95.

VaNDt~xax, L. L. M. and ue Gv?te, J. (1974) Lipids of the red cell membrane . In : The Red Blood Cell, pp . 147-21 l (SUROENOR, D. M., Ed.) . New York : Academic Press.

VERNON, L. P. and BELL, J. D. (1992) Membrane structure, toxins and phospholipase AZ activity . Pharmac. Ther .54,269-295 .

VERNON, L. P. and RocExs, A. (1992) Binding properties ofPyrularia thionin and Naja naja kaouthia cardiotoxinto human and animal erythrocytes and to murine P388 cells . Tozicon 30, 711-721 .

Vrrtc~tr, J.-P., ScxwErrz, H., CFUCt~pox~rtc~, R., Foss~r, M., BaLEßNa, M., LExouz, M. C. and LaznuxsxyM. (1976) Molecular mechanisms of cardiotoxin action on axonal membranes. Biochemistry 15, 3171-3175.

Vnvcexr, J.-P., BALERNA, M. and Laznuxstu, M. (1978) Properties of association of cardiotoxin with lipidvesicles and natural membranes. Afluorescence study. FEBS I,ett . 85, 103-108.

VOLWERK, J. J., PtLrt~ttsox, W. A. and DE Haas, G. H. (1974) Histidine at the active site of phospholipase AZ.Biochemistry 13, 1446-1454.

Voss, J., BIRMACHIJ, W., HussEV, D. M. and Txoutas, D. D. (1991) Effects of melittin on molecular dynamicsand Ca-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy . Biochemistry30, 7498-7506.

WEavert, A. J., KE~trLE, M. D., BxauNEa, J. W., MENDELSOHN, R. and PxExnEacasr, F. G. (1992) Fluorescence,CD, attenuated total reflectance (ATR) FTIR, and ~'C NMRcharacterization of the structure and dynamicsof synthetic melittin and melittin analogues in lipid environments . Biochemistry 31, 1301-1313.

Wm.arro, S. J., FLETCHEIt, J. E. and GONG, Q:H. (1992) Differential modulation of a sodium conductance inskeletal muscle by intracellular and extracellular fatty acids. Am . J. Physiol. 263, C308~312 .

W~LLE, B. (1989) A preparation of melittin depleted of phospholipase A, by ion exchange chromatography indenaturing solvents . Analyt . Biochem. 178, 118-120.

Woxo, C. H., Cr~N, S. T., Ho, C. L. and Waxo, K. T. (1978) Synthesis of a fully active snake venomcardiotoxin by fragment condensation on solid polymer. Biochim. biophys. Acta 536, 376-389.

YUNFS, R., GOLDHAMAIER, A. R., GARNER, W. K. and CoanES, E. H. (1977) Phospholipases: melittin facilitationof bce venom phospholipase A,-catalyzed hydrolysis of unsonicated lecithin liposomes. Archs Biochem.Biophys. 183, 105-112.

ZAtmeR, A., NoxoNt~tA, S. H., HOSPATTANKAR, A. V. and BRaaANCa, B. M. (1975) Inactivation of(Na++K+}-stimulated ATPase by a cytotoxic protein from cobra venom in relation to its lytic effects on cells .Biochim. biophys. Acta 394, 293-303.

7FrrLER, P., Wu, Y. Q. and HANDWFRGER, S. (1991) Melittin stimulates phosphoinositide hydrolysis andplacental lactogen release : arachidonic acid as a link between phospholipase AZ and phospholipase Csignal-transduction pathways . I,iJe Sci. 48, 2089-2095.

ZwAaL, R. F. A., Roet .oFSarr, B., CoMEURnts, P. and VANDEENEN, L. L. M. (1975) Organization ofphospholipidsin human red cell membranes as detected by the action of various purified phospholipases . Biochim. biophys.Acta 406, 83-96.

fletcher 1993

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