TiPS - December 1991 [Vol. 121

Clinical future for Ca2+ antagonists looks more promising Molecular biological investi- gations of voltage-dependent Ca2+ channel function have now reached a stage where results from coexpression experiments allow unequivocal assignment of a func- tional role to each subunit. In addition another Ca** channel type ha; been cloned that most likely represents an N-type Ca*+ channel. New information has been gathered about the fast and stow modulation of Ca’+ channel expression and function in brain and heart. Tine oid claim of an anti-ischemic effect of Ca*+ antag- onists has been corroborated in latgel well-controlled clinical trials, and the way is paved for a variety of novel clinical cations of Ca*+ antagonists.

appli-

Gas+ channel molecular biology The structure of the voltage-

dependent Caz’ channel was re- ported at the last international symposium on Ca*+ antagonists two ycam ago. It was shown to be a hetero-oligomer consisting of a ___ _-._. large (-143475 kua) ai subunit carrying the receptor domains for the three main classes of Caz+ antagonist (dihydropyridines, phenytatkylamines and benzo- thiazepines) and believed to form the ion pore; a sli tly smaller a2 subunit (135-150 !FI a), which is heavily

I!! ycosylated; a very small

(24-29 k a) b subunit which is co- valently linked to the air subunit; a p subunit (-55 kDa) and a y sub- unit (-33 kDa) (see Ref. 1 for a recent review). A meeting this September+ highlighted extensive data, gathered over tlne past two years, on the electrophysiological properties of coexpressed cloned Cal+ channel subunits, allowing specific functional roles to be tentatively assigned to at least some of the L-channel subunits.

Although exciting, things are far from being settled; the only generally agreed point is that ex- pression of the al subunit alone

??5tJr Syn~posi~tr~t on Cd ’ Antqqonisls. Hosrtoa, Tms. USA. 25-28 Sepkrdwr 1991.

yields functionally active, di- hydropyridine-sensitive Ca*+ channels and that the co- expression of the al and g subunit increases the B,,, for dihydm- pyridine binding; changes in ac- tivation and inactivation rates as well as in channel amplitude upon addition of the f3 subunit seem to vary according to the type of cione used and the reporting laboratory.

Homologous coexpression of al and fi subunhs

Arthur Rmwn (Bayior Coiiige, Houston) presented evidence that the presence of the R subunit speeds L-channel activation up to lOO-fold while increasing the rate of deactivation only moderately. Brown’s group could not find any increase in flux amplitude al- though addition of the g subunit to the al increased the B,,, for dihydropyridines fivefold; all these experiments were, however, performed in the presence of 0.5 RM BayKW4. Using the same cell line (mouse LCa.11) for hom- oiogous coexpression of subunits also of skeletal muscle origin, Arnold Schwartz (University of Cincinnati) reported similar changes in kinetics, although after addition of the fi subunit his group found a decrease of the channel amplitude and a disrup tion of the stimulatory effect of BayRW4 (1 pi). Similar to the findings by Brown’s group, the B rnax for dihydropyridines was increased tenfold. Schwartz also reviewed the evidence obtained from other laboratories__ using L heteroiogous coexpressron tsuo- units expressed from clones ob- tained from different organs); an increase in the channel amplitude upon addition of the fi subunit was consistently found, whereas changes in kinetics could not be detected in most cases. The same uncertainty still governs the ex- tent and functional consequences of L-channel phosphorylation at the al and p subunits (Marlene Hosey, Northwestern University, Chicago).

439

Putative N-type Ca2+ channel clones have been obtained from the brain and electric lobe of the marine ray (Richard Tsien, Stan- ford University; see Ref. 2). Com- parisons of the derived amino acid sequences show that, com- pared with sequences for heart skeietai muscle Cai’ channel, both clones possess an additional positive charge (lysine) in seg- ment lllS4 (i.e. the fourth putative membrane-spanning region in the third transmembrane domain of the al subunit) and a slightly higher density of positive charges in segment IV%. This different clustering of positive charge in the putative voltage-sensor segments of the Ca2+ channel mieht rep- resent the structural basii~for the different voltage sensitivity of N channels versus L channels.

Modulation of N channels Tsien also presented evidence

that the neumtransmitter nor- adrenaline inhibits N-type Ca*+ channel-mediated Ca*+ infhrx into sympathetic varicosities by restricting the channels to a medium open-probability mode, a state that can also be assumed spontaneously by N channels after a few seconds of continuing voltage-step stimulation. In the medium open-probability mode, the POP,, is decreased and the cioseo time increased with respect to the fully activated channel. However, the current per opening is increased at the same time from 0.7 pA to 0.9 pA, and the overall effect on Ca*+ influx into the varicosities is not known.

As regards neural L-type Ca2+ channels, several findings from different research groups might help to explain the rather meav psychotropic effects of Ca-’ antagonists as well as the absence of CNS effects upon abrupt Ca2+ antagonist withdrawal: David VA__,_ ,CI_I_ tl;l..-z_:L;--~ L ,^_.. IrlggK &Ware unlvrrslry “I LVCW York, Buffalo) showed that after five days of exposure to the Caz+ channel blocker nifedipine (SO nhr), PC12 cells upregulated L-type Ca” channel density by roughly 30%. However, after five days of exposure to (d-Bay= (5OOmu), the B,, for drhydm- pyridine binding is decreased by about 25%. Accordingly, the five- day treatment with nifedipine ted to an increase in Wa2+ influx and Caz+ currents, whereas treatment

440 TiPS: - December 1991 [Vol. 121

with BayK8644 decreased both. Furthermore, in intact cerebellar granule cells, K’ depolarization (50 mu K+ from 5.8 mM K+) alone caused a fivefold decrease in B,,, that, at least partially, counter- acted the Weld depolarization- induced affinity increase. By con- trast, K+-induced depolarization of isolated myocytes from heart (where Ca2+ antagonists reliably produce distinct effects3) did not result in a change in B,,, for ( +)-13H]isradipine, whereas this Iigand’s affinity was increased slxtyfold. Furthermore, Kenneth P. Scholz (University of Chicago) reported converging evidence from several laboratories to suggest that - at least in some CNS preparations - both L and N channels (sensitive to dihydro- pyridines and u-conotoxin, re- epectively) together contribute to only 60-70% of the measured Cr+ influx. The most likely can- didates for the remaining 304% are w-agatoxin-sensitive Ca2’ channels, which cannot be accom- modated within the L, N, T and P channel classification. Further- more, in an elegant model using primary cultures of synapse- forming hippocampal cells, neuro- transmitter release from pre- synaptic terminals was only mildly sensitive to dihydropyri- dines and w-conotoxin.

It is becoming clear that Ca2’ channek exist on glial cells as well as neurons. L. Hertz (University of Saskatchewan, Saskatoon) re- ported that cultured astrocytes possess high-affinity [3H)nitren- dipine binding sites (I& 2 nM) that can be induced by treatment with dibutyryl-CAMP, and low-affinity sites (& 200 nr4 that are not affected by this treatment. A bi- phasic effect on intraglial Ca2+ concentration was observed for midazolam (ECm 5 nM, I&,, 50 nM) and diazepam (ECm 8On~, If& 600 nhr).

Ca’+ sensitivity of their contrac- tile apparatus from ions permeat- ing through L channels to Ca” stemming from the sarcoplasmic reticulum, by two mechanisms: an increased expression of ryanodine receptors and a redistribution of L channels to the junctional plas- malemma (day 3 versus day 30). In the neuronal PCl2 cell line, after 4-5 days of differentiation in- duced by nerve growth factor, there was a fivefold increase in N- channel density, whereas the very slight increase in L-channel den- sity was commensurate with the overall increase in cell surface (Harold Reuter, University of Bern).

In whole rat brain, the situation is less clear. Changes in density and’or affinity have been reported for both L and N channels; par- tially contrasting results (S. Gov- oni, University of Rome; Triggle) might be attributable to the different rat strains and age ranges studied. There is, however, consensus that aged neurons show more surface Ca2’ bindin

1z: an increased intracellular Ca load, prolonged Ca2+-dependent afterhyperpolarization (most likely resulting in decreased im- pulse velocities) but decreased sensitivity of Ca’+-dependent neurotransmitter release.

Research on the effects of Ca2+ antagonists in dementia seems to be very much focused on one drug, nimodipine. Clinical studies on primary degenerative dementia as well as multi-infarct-related deficits involving 1400 patients await analysis in mid-1992. Research in this field should be addressing a .variety of Ca2+ antagonists of diiferent chemical classes.

Protection of heatiand brain

Age-related Caz+ channel modulation

Apart from the Ca2+ antagonist- induced changes described above, a wealth of information has been gathered on age- related changes in Ca2+ chan- nel affinity and density in both brain and heart. Maurice Wibo WniversitB Catholique de Lou- vain, Brussels) reported that tat developing heart cells shift the racy __._._ _-_ _.-_

At the experimental level, more possible modes of action for the beneficial effects of Ca” antagon- ists in ischemic tissue have been described. For example, in is- chemia acylcamitines rise about threefold due to inhibition of B-oxidation (Mike Spedding, Syntex, Edinburgh); this causes a variety of deleterious effects, most notably a ryanodlne-sensitive Ca2+ release from the sarco- plasmic reticulum that can also be blocked by diphenylbutylpiperi- dine Cal+ antagonists such as flusnirilene or nimozide but not .

by dihydropy&$ne or phenyl- alkylamine

diastolic function, an effect also

antagonists. Camitine pal&toy1 transferase 1 (CIW inhibitors effectively lowered acylcamitine levek but results from clinical trials are not available yet. Another target structure for the anti-ischemic effects of certain Ca2+ antagonifts might be a specific, non-L- channel receptor described for dihydropyrfdfnes, phenylalkyl- amines and benzothiazepines that is located in the inner mito- chondrial membrane, associated with an anion channel (Gerald Zernig, University of Innsbruck). During oral therapy with nitren- dipine, up to an estimated 2B% of these mitochondrial receptors might be occupied by the drug’. By inhibiting this structure, CaZ* antagonists might prevent the Ca2+ overload of mitochondria from ischemically compromised tissues. This proposal, however, still awaits direct experimental validation.

There is considerable excite- ment about the fact that, after the first demonstration of the hen- eflcial effects of dlltirzem on re- infarctlon rate for non-Q-wave infarction in a double-blind, placebo-controlled and random- ized study enrolling 576 patients (Robert Roberts, Baylor College of Mediclne, Houston), another Caz’ antagonist has stood up to the clinical test. J. Fischer Hansen (Hvidovre Hospital, Hvidovre, Denmark) presented data fmm DAVIT II, the Danish Verqamil Infarction Trial (double-blind, placebo-controlled, r&on&ed multicentered and enrolling 1G patients), which demonstrated a significant decrease in mortality after treatment with l2Omg verapamil given three times a day - but only in patients who did not display signs of heart fail- ure in the coronary care unit and only If verapamil tmatment was delayed until one week after ad- mission. The venpamil-treated patients also showed a significant decrease in ischemic signs in the 24 h ECG and needed signifi- cantly less nitrate co-medication (both of these effects are most likely due to an anti-ischemic and/or anti-angina1 effect), as well as less diuretic co-medication. Reduced requirement for diuretics is most likely due to Increased

TYS - December 1991 /Vol. 121

demonstrated for the DEAANI trials by Paul Hugenholb (Socar, Givrins, Switzerland).

Similarly, an interesting meta- analysis of six studies (comprising more than 800 patients) on the effects of nimodipine on sub- arachnoid hemorrhage presented by Fredric Meyer (Mayo Clinic, Rochester), revealed that nimodi- pine clearly exerts an improve- ment in neurological deficits in patients without either demon- strating a clear effect on vaso- spasm induced by subarachnoid hemorrhage, increasing cerebral blood flow, decreasing the hemor- rhage itself, or decreasing mor- tality.

These studies highlight two main issues. First, the claim by basic scientists about a direct, energy-conserving, anti-ischemic effect of certain W+ antagonists (a claim stubbornly repeated in the face of accumulating contrast- ing clinical evidence) has now finally been confirmed in large, contn3Red dinical Mais. Secondly (and more importantly), it is clear that this anti-ischemfc effect can only be demonstrated after mrtch- ing the study design to the par- ticular path~hysiology of differ- ent organs affe&d by ischemia, and contmRing patient heterogen- eity. For example, DAVIT 1, the pmkcessor of DAVIT II, showed no improvement in death rates because, apparently, verapamil treatment was inittated too early. ConvemeIy, meta-analyses of the disappointing results of Ca*+ antagonists in the treatment of stroke was presented by M. Frontoni (University of Rome), and jan Gheuens (Janssen, Beer@ pointed out that, in the case of stroke intervention with Ca2+ antag&isb might have been started too late.

Gaatrohtatinale~ Caz’ antagonists with increased

selectivity for gastrointestinal smooth muscle cells are currently under investigation. Pinaverlum displays an absolute bioavail- ability of less than 0.5% and is therefore practfcaRy devoid of any systemic effects when given orally (Marie-Odik Christen, Labora- toires de Therapeuttque Modeme, Stuesnes). Although structuraEy different from the dihydro- pyrtdtnes, it acts as a competittve inhibitor of (+)-I’HJisradlpine in

both equilibrium and dissociation kinetic analyses. Six double-blind, placebo- or reference drug-con- trolled trials (totatting 237 patients) have demonstrated the effectiveness of pinaverium in irritable bowel syndrome and other gastrointestinal disorders.

In the canine colon, interstitial cells of Cajal are most likely of mesenchymal origin, and are l~ted at the submucosal border. These cells generate pacemaker action potenttats to which Ca*+ e$~te;roughbbot;;u;~d WE

Huizin&, h&Master University, Hamilton; Fig. 1). By contrast, smooth muscle cells in the circular and longitudinal muscle layers separated from the submucosal layers display oscillatory L-type channel aetivtty when challenged with BayKE644 (to open L chan- nets) and Ba*+ as a permeant ior+. Thus, to generate unequtvo- cal results, preparations of these gastrointestinal regions must be separated into different cell layers.

Ca’+ antagonists might be use- ful for treatfng esophageal con- tractfle disturbances (spasms, hypercontractllity or ‘nutcracker’ esophagus, achalaaia), non-uker- ogenic stomach motility disturb- ances, irritable bowel syndrome and other conditions (Richard M&Bum, University of Virgtnia, ChariottesvfRe). Interestfngly, the benefictal effect of ntfedipine given in angtnal pain might be attributable to the fact that ap proxtmately 50% of cheat pain is due to causes other than coronary vasoconstriction, particularly dis-

turbances in esophageal contrac- tility, which are blocked effectively by nifedipine.

Reversal Ofdmgre&tance in tumorltandrmlusl

It is now firmly established that a variety of Ca*+ antagonists interact with the multidrug resist- ance (mdr)-related P-glycoprotein (see Ref. 7 for a recent review), which consists of two repeats of a protein containing six putative transmembrane heltces and an Al-P-binding domain (recently reviewed in Refs 8 and 9). Lee Greenberger (LederIe Labora- tories, Pearl River) used photo- affinity labelling and sequence- directed antibody studies of the P-glycoprotein with [sH]azido- pine and I’ssIjiodoaryl-azido- prozosine to delimit the location of binding sites for these drugs to the putative transmembrane seg ments 5 and 6 of the second repeat. This finding points to similarities in the tertiary stntctum of the P-glycoprotein and the al subunit of the L-type Ca*’ chan- nel, where the lmtfon of the phenylalkylamine btndtng site could be delimited to the intra- cellular end of IVS6 (the putative sixth, and last, transmembrane helix of the fourth membrane- spanning domain of the al sub- unit) and the adjacent inbacelhrlar amino a&U”. The mdrl P-&o- protein, which according to Knox Van Dvke (West Virgina Dniver- sity, h&ga&wn) cG be re@ed as a non-stemoselective ATP- dependent pump transporting molecules with two positive

4-z

charges, does net display any stereoselectivity for Ca’+ antag- onists. Current clinical trials con- centrate on the distomers (bio- logically less active isomers) of the phenvlalkylamines [e.g. the (t )- enaniiomers of gallopamil and devapamil] with respect to the cardiovascular effects of this class of Ca.” antagonist. For the treatment of malaria, Van Dyke advocated the combined use of (+)-tetrandrine, a known L-chan- nel blocker (Ki 501~ for the benzothiazepine site in cardiac tissue”) that has been used in Chinese folk medicine for hun- dreds of years, with chloroquine (about 90% of the death toll from malaria is due to drug resistance). 1t1 ryitro experiments using chloro- quine-resistant strains of Pins- t&itttn fdciynrws had shown that tetrandrine given together with chloroquine at a molar ratio of 10~3 lowered the ICs, of chloro- quine for parasite growth from 186 nM to 24 nM (the ICW for tetrandrine is 80 mM; Ref. 12).

Ca*+ antagonists and the kidney Ex zliuo measurement of the

renal microvasculature using a videomicroscope proved that the beneficial effect of Ca2+ irntag- onists on glomerular filtration rate and renal blood flow is due to a dilatation of the vas afferens (Murray Epstein, University of Miami). Furthermore, it was found that one of the major ad- verse effects of ciclosporin A (which is used as an immuno- suppressive adjunct in renal transplantation) is in fact renal vasoconstriction, which can be blocked by Ca’+ antagonists; preliminary clinical trials have shown iheir beneficial effect on renal blood flow and graft re- jection (Ingemar Dawidson, South- western Medical Center, Dallas).

Novel and endogenous Ca” antagoniits

Finally, the magic word ‘en- dogenous’: Martin Morad (Uni- versity of Pennsylvania, Philadel- phia) presented further evidence for a peptide of approximately 900 Da that displaces the dihydro- pyridine nitrendipine from its binding sites, stimulates Ca’+ influx in the heart by increasing the mean open time and causes inhibition in neuronal tissue. .4n endothelium-derived hyperpolar-

ization factor has been described in vascular smooth muscle, the release of which is dependent on endothelial Ca” and calmodulin (Valerie Schini, Baylor College, Houston). There are, however, no data available on the structure or even chemical nature of the en- dothelium-derived hyperpolariz- ation factor. In cultured PC12 cell bodies, a bradykinin-sensitive Ca” permeation pathway exists that is regulated by [Ca2’]i and calmodulin (Harold Reuter); single-channel measurements, however, are not yet available for this system.

Acknowledgements G.Z. is supported by grant P7492-

MED. Austrian !3cience Foundation. GERALD ZERNIC

TiPS- Decentbcr 1997 IVol. 12j

References 1 Catterall, W. A.. Seagar. M. J. and

Takahashi, M. (19BB) 1. Biol. Chem. 363. 3535-3536

2 Tsien, R. W., Ellinor, P. T. and Home. W. A, (1991) Trends Phnnrrrof. Sri. 12. 349-354

3 Ferrante, J. and Triggle, D. I. (1990) Pharmacol. Rev. 42.29-44

4 Zemig, G. and Glossmann, H. WW Biochem. !.253,49-58

5 iknig, C. et nl. (1990) Mol. Phnrmacol. 38,362~369

6 Huizinga, J. D. Con. I. Physiol. Phanw- co/. (in +s)

7 Zemig, G. (1990) Trends Phnrmacof. Sri. 11,36-U

8 Gottesman. hf. M. and hstan, I. (1988) Trends Phnnacol. Sri. 9, M-58

9 Silver, S., Nucifera. G., Chu, L. and Misra, T. K. (1989) Treads Biochem. Sri. 14,76-B0

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12 Ye, 2.. Van Dyke, K. and Castranova, V. (19B9) EiocLem. Biophys. Res. Commun. 165,75&765

[ : -: : ,a , _. _’

EM

Role of adenosine in the trophic effects of sympathetic innervation In -the early 1960s it became apparent that adrenergic inner- vation exerts long-term trophic effects on effector organs12. Denervation supersensitivity in- cludes a postjunctional com- ponent, the mechanism of which has been at least partially clarified (see Ref. 3). Surgical or chemical sympathetic denervation also affects the removal and metab- olism of catecholamines more extensively than cocaine in- hibition of neumnal uptake of catecholamine8; this was assumed to be due to a denervation- induced impairment of the extra- neuronal corticostemid-sensitive 0-methylating systems. In paral- lel, morphological changes of a hypertmphic and hyperplastic nature were described in vascular smooth muscle cells and fibm- blasts, as well as in myocardial cells”. In agreement with in- vestigations on chicken expan- sor secundariorium’, rabbit aorta’”

and rabbit cerebral artery”, it was concluded that admnergic innervation exerts a repressive effect on the nuclear activity Of effector cells, thus denervation leads to hypertmphk-hyperpktic changes and de-differentiation.

Adenosineaealrophisfactor InitlalIy, noradrenaline ~8s

presumed to be responsible for these tmphic effects of the sym- pathetic nervous system. How- ever, vine-induced depletion of noradrenaline did not mimic the effects of denervation”, and continuous intisions Gf nor- adrenaline were ineffective in preventing the morphological and functional changes caused by denervationsJ. Co-transmitters were then considered as candi- dates. ATP is released from nor- adrenegic nerve varicosities and rapidly metabolized in the extra- neumnal space, so the effects of some adenosine receptor agonists