prostaglandin modification of membrane-bound enzyme activity: a possible mechanism of action?
TRANSCRIPT
PROSTAGLANDIN MODIFICATION OF MEMBRANE-BOUND ENZYME ACTIVITY: A POSSIBLE MECHANISM OF ACTION?
Malcolm Johnson* and Peter W. Ramwell*
Department of Physiology Stanford University Medical Center, Stanford, California
Present Address: Department of Physiology and Biophysics Georgetown UniversityMedical Center, Washington, D .C .
ABSTRACT
Prostaglandins El and E2 and their antagonist 7-oxaprostynoic acid modify Mg 2+-dependent Na+/K+zac tivated ATPase and adenylate kinase activities in human erythrocytes and platelets, rat liver mitochon- dria, and rabbit skeletal muscle. The kinetics of these interactions are suggestive of a possible allosteric mechanism. Specific differences in enzyme response to the different prostaglandins were observed and may be related to the membrane composition of the respective cell types. It is pro- posed that a common site for the wide variety of prostaglandin actions on different tissues may be a number of membrane-associated enzymes, and that the final cellular effect is the result of their individual responses.
ACKNOWLEDGMENTS
This work was supported by ONR grant #N00014-67A-0112-0055.
The prostaglandins were a gift from the Upjohn Company and the Alza Corporation. We wish to thank Dr. J . Fried of the Department of Chemistry, University of Chicago, for the gift of ‘I-oxaprostynoic acid. The authors express their gratitude to Mrs. M . Cornelson for technical assistance.
Accepted April 15
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INTRODUCTION
The most singular aspect of prostaglandin action is that few, if any, cell types appear to be unresponsive. This unusual and remarkable lack of cellular specificity (1)) which in complete contrast is associated with strict structural-activity requirements, has led us to conclude that prosta- glandins may exert their effects by a mechanism which is common to most cell types.
One possible cellular element, which was identified early was the adenylate cyclase system (2). for some of the effects of prostaglandins appear to be mediated via cyclic AMP. However, evidence has emerged that PGEl and PGE2 can have different effects on cyclic AMP accumulation and cellular response in the same cell type; as for example in platelets (3). Recently, we have drawn attention to the fact that a number of prosta- glandin effects are unlikely to be mediated via a cyclic AMP mechanism (1). For example, human red cells which are devoid of hormone-sensitive adenylate cyclase respond to PGEl and PGE2 at low concentrations (4). Also, the rat myometrial contraction elicited by PGF2g is not associated with changes in adenylate cyclase activity (5). In addition, cellular and tissue responses to prostaglandins show great species variation, which does not support a single and well defined obligatory role for cyclic AMP, However, the time course of prostaglandin action is such that the primary target site must be in the cell membrane. It is now becoming clear that prostaglandins modify membrane-associated enzymes other than adenylate cyclase , including adenylate kinase (6) and phosphodiesterase (7).
Prostaglandin effects on different cell types are as yet unpredictable, and therefore any analysis of prostaglandin action must be conducted on single cell populations. In the present communication we have used the human erythrocyte, the human platelet, and the rat liver mitochondrion as sources of accessible single membrane types, without resorting to the use of proteolytic enzymes.
MATERlALS AND METHODS
I. PLATELET LYSATE Platelet lysate was prepared by the method of Moake et al (8). The
final membrane suspension was homogenized to give a prote%Toncentration N 6 Omg/ml.
II. ERYTHROCYTELYSATE Erythrocyte ghosts were prepared from whole blood (Group 0, Rh+)
(9) and lysed in cold de-ionized water (4OC). The final precipitate was suspended in 5 x 10e4 - M histidine-imidazole buffer, pH 7.4 by homogeniza- tion to a protein concentration b 50 mg/ml .
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III. MITOCHONDRIAL LYSATE Intact rat liver mitochondria were fractionated in 0.3 M sucrose,
pH 7 (10) and resuspended in a 0.003 M K2HP04, pH 7. After centrifuga- tion, the pellet was resuspended to a protein concentration ~50 mg/ml.
Ail membrane preparations were divided into aliquots (1 ml) and stored (-20°C). Only once-thawed preparations were used for enzyme analyses. The preparations exhibited linearity between enzymatic activity and enzyme concentration in the protein ranges used.
Iv. ASSAY OF ATPase ACTIVITY Unless otherwise stated, the incubation mixture for the ATPase
reaction consisted of 115 mM NaCl, 10 mM KCl, 3 mM MgCl2, 20 mM Tris- HCl buffer and 3 mM ATP in a final volume of 2 ml at pH 7.4. Reactions were initiated by the addition of the cell lysate and carried out at 37OC for 20 minutes in polypropylene tubes. The test substances were included in the reaction mixture prior to addition of the enzyme and the quantity of cell lysate was adjusted so that less than 10% substrate ATP was hydro- lyzed in 20 minutes (8). Trichloroacetic acid (final concentration 5% w/v) was added to terminate the reaction. After centrifugation (12,500 g min) the phosphate in 1 ml of supernatant was measured by the method of Fiske and Subbarow , with correction by means of appropriate blanks for non-
. ~o?a;;J~s ,,,;,,,1s.
Protein was estimated by the method of Folin and dependent Na+/K+-activated ATPase activity is expressed
as umoles of phosphate released per hour per mg of protein in the presence of Mg 2++Na++K+.
V. ASSAY OF ADENYLATE KINASE Adenylate kinase was assayed in the direction of ATP synthesis in
a reaction mixture containing 50 mM Tris-HCl, 5 mM MgS04, 10 mM glucose, 9 units hexokinase , 10 units glucose 6-phosphate dehydrogenase and 0.2 mM NADP+ in a final volume of 1.4 ml. The reaction was initiated by the addition of ADP and the increase in absorbance at 340 nm was fol- lowed continuously in a Gilford Spectrophotometer . The test substances were added prior to addition of ADP . Adenylate kinase activity is ex- pressed as h OD/min/mg of protein.
RESULTS
I. PROSTAGLANDINS AND ATPases The observation that adenosine diphosphate (ADP) induces platelet
aggregation and concurrently inhibits Mg 2+-dependent Na+/K+-activated ATPase has been suggested as a possible mechanism in aggregation (8); ATPases , however, have previously been found to be unresponsive to prostaglandins (11). Owing to the importance of prostaglandins in this area, we have reinvestigated their effect over a range of concentrations
MAY 1973 VOL. 3 NO. 5 705
PROSTAGLANDINS
on the kinetics of ouabain-sensitive ATPase in a series of different mem- brane preparations.
1. Platelet The platelet enzyme responds to PGEl and PGE2 by exhibiting
anomalous kinetic orders with respect to substrate. The normal hyper- bolic curve of enzyme velocity (V) against substrate concentration (S) in the control response, becomes successively more sigmoid in the presence of increasina concentrations (0. l-l uM) of prostaglandins (Figure 1).
I I I I 1
I I I I
0.25 0.50 1.00 1.50
[ATP] mM
EFFECT OF PGEl AND PGE2 ON HUMAN PLATELET ATPase ACTIVITY. The figure illustrates the effect of varying the concentration of sub-
strate ATP (mM) on the enzyme activity expressed at phosphate (umoles) released per hour per mg of enzyme protein in the absence ( q ) and in the presenceofPGELatO.lpM (o), 1uM (o), andPGEZatO.luM (A), 1uM (A ) . Experimental conditions were as described in the text. Each point represents the mean of five estimations.
706 MAY 1973 VOL. 3 NO. 5
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In addition, PGEl and PGE2 elicit biphasic responses, low concen- trations (0.1 JIM), decreasing both the apparent Km (Michaelis constant) for ATP , and the corresponding Vmax in an uncompetitive manner. There is an apparent parallelism in the shift of double reciprocal plots. At higher concentrations ( 1 PM) both PGEl and PGE2 produce classical Michaelis Menton competitive inhibition by increasing Km, with no effect on Vm,. The order of potency in all these responses is such that PGEl a PGE2 (Figure 2). PGFId and PGF2& , which are ineffective in platelet aggregation (12)) were also tested for their ability to modify plate- let ATPase activity and at concentrations up to 1pM were inactive (13).
If one is to postulate a common membrane site for prostaglandin action, then their antagonists must also be active at the ssme site. The prosta- glandin antagonist ‘I-oxaprostynoic acid (14) was therefore investigated and at 15pM acts in a competitive manner to activate the enzyme by lowering the apparent Km (13); concentrations below this were found to be ineffect- ive. In our studies, we have found that much larger concentrations of this antagonist ( + 1000: 1 relative to prostaglandin) are essential for pharma- cological antagonist activity.
2. Mitochondria One obvious and potentially key site for prostaglandin action is in the
mitochondrial membrane. Lee (15) showed increased 32P incorporation into ATP by the addition of PGEI to mitochondria, and Polis (16) found that PGEl and PGBl restored respiratory control over oxidative phosphorylation in “aged” mitochondria exhibiting high ATPase activity. More recently, PGEl has been reported to facilitate the electrogenic exchange of Ca++ ions across the inner mitochondrial membrane (1’7).
In our studies, PGEl and PGE2 at 0.1 pM were found to have similar uncompetitive effects on liver mitochondrial ATPase to those observed in the platelet. The order of potency is, however, reversed in that PGE2 > PGEI. Increased concentration of PGE2 (1 FM) further lowers the apparent Km for ATP and decreases Vmax for the enzyme. PGE 1 at 1 pM exhibits a biphasic response and increases Km markedly (Figure 2). The site and mechanism of action of PGEI in the mitochondrial membrane appears dif- ferent from that for PGE2, and the mitochondrial membrane enzyme responds differently toward the prostaglandins than did the platelet enzyme. The antagonist 7-oxaprostynoic acid, at concentrations up to 30 pM , activates the enzyme by an effect on Km (13)) the effect being lost at higher concen- trations (60 @I) .
3. Erythrocyte The action of orostaglandins is of great interest if these compounds
are released following trauma. Weed (18) has suggested that there is a decrease in cell deformability when human erythrocyte ATP concentra- tions fall in vitro, and Allen and Rasmussen (4) have recently reported --
MAY 1.973 VOL. 3 NO. 5 707
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PROSTAGLANDINS
that PGE2 in low concentrations (0.1 nM) induces significant hardening of red cells, an event associated with changes in cellular ATP and calcium.
We have investigated the effect of prostaglandins on erythrocyte membrane ATPase. Low concentrations of PGEl and PGE2 (0.1 PM) activate ATPase by an effect on Km, the order of potency being El > E2. Higher concentrations (1 PM) produced inhibition by increasing the Km for ATP (Figure 2). This is a similar biphasic response to prostaglandins as that seen in the platelet. ‘I-oxaprostynoic acid (15-30 pM) activates erythro- syte ATPase by increasing Vmax (13).
II. PROSTAGLANDINS AND ADENYLATE KINASES Adenylate kinase catalyzes the reversible reaction ATP + AMP $ 2ADP.
Its function is to provide ATP in response to depletion of ATP pools, although it has been suggested that it controls ADP formation under normal conditions (19). Abdulla and MacFarlane (20) have recently shown that PGE2 and PGE3 modify the activity of this enzyme in skeletal muscle and blood platelets. In an attempt to correlate prostaglandin action to turnover or availability of ATP , we investigated the response of membrane-associated adenylate kinases to prostaglandins.
We have confirmed the earlier reports on the effect of PGE2 (1 PM) on this enzyme in muscle in stimulation of Vmax . We find, however, an associated increase in the apparent Km for ADP and a parallel reciprocal plot results. This kinetic phenomenon is similar to that observed in the ATPase studies. Furthermore, PGEl (FM) in contrast to PGE2 and PGE3 (20) was shown to inhibit muscle adenylate kinase by lowering Vmax, with little apparent effect on Km. PGFlc( and PGF2ti had no marked effect at the concentrations tested (0. l-l PM) (Figure 3).
The activity of the muscle enzyme was also increased by cyclic AMP (250 PM) and inhibited by ‘I-oxaprostynoic acid (20 PM), both effects being on Vmax .
As with the skeletal muscle enzyme, PGE 1 and PGE2 have opposing effects on platelet adenylate kinase activity. However, in contrast to the muscle enzyme, PGEl (1 pM) activates the platelet enzyme by lowering the apparent Km for ADP with little effect on Vmax. PGE2 (1 PM), on the other hand, exerts uncompetitive effects similar to those exhibited in muscle. A biphasic phenomenon was again apparent in this enzyme in response to lowering the concentration of both prostaglandins to 0.1 PM (Figure 4).
Erythrocyte adenylate kinase is also responsive to PGEl and PGE2 and their antagonist. They exhibit concentration-dependent uncompetitive effects, the order of potency being E2 > El (Figure 4) (13).
MAY 1973 VOL. 3 NO. 5 709
PROSTAGLANDINS
FIGURE 3
ACTION OF PROSTAGLANDINS AND OTHER SUBSTANCES ON MUSCLE ADENYLATE KINASE ACTIVITY.
Adenylate kinase was assayed as described in the text in the direction of ATP synthesis. The figure represents the mean linear regression Lineweaver-Burk plots from five estimations on each of two different enzyme preparations. The response of the enzyme to the prostaglandins and other substances at the following concentrations is compared to the control (C) enzyme activity: PGEl (1 PM), PGE2 (1 PM), PGFld (1 PM), PGF2d (lpM), cyclic AMP (250 PM), and 7-oxaprostynoic acid (20 PM). The velocity (V) of the enzyme reaction is expressed as A 0 .D . per min per mg enzyme protein and substrate ADP concentrations as mM-ADP .
710 MAY 1973 VOL. 3 NO. 5
PROSTAGLANDINS
KJ a.
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b.
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FIGURE 4
EFFECT OF PGEl AND PGE3 ON ADEN-YLATE KIFASE ACTIVITY. The prostaglandins were tested at 0.1 JJM @‘GE) and at 1 pM @‘GE)
on (a) human platelet and @) human erythrocyte adenylate kinase activity and compared to the respective controls (C) . Experimental conditions were as described in the text and in Figure 3.
MAY 1973 VOL. 3 NO. 5 711
PROSTAGLANDINS
DISCUSSION
It is apparent from these studies that in addition to adenylate cyclase, prostaglandins may modify other membrane-associated enzyme activity such as Mg2+-dependent Nat/K+-activated ATPase and adenylate kinase . In view of previous findings that prostaglandins modify adenylate cyclase in many tissues and also particulate phosphodiesterase in human platelets (21) it appears therefore that these compounds may exhibit general effects on a wide variety of membrane-enzyme activities.
Our findings, although obtained using broken cell preparations, may apparently be extrapolated to the pharmacological responses of the intact cell to prostaglandins . For example, at the respective concentrations active in platelet aggregation (22): PGEl (0.03 PM) activates platelet ATPase, an effect recently confirmed by other workers (23)) and decreases platelet ATP levels, a condition in which aggregation will not occur; PGE2 on the other hand, at 1 nM competitively inhibits enzyme. Also the prosta- glandin enzyme effects are observed in the range of normal physiological concentrations and at concentrations at which there is no detergency (24). In addition, such biphasic responses to prostaglandins are common in the literature and have been reported in platelet aggregation (25)) erythrocyte deformability (26) and lymphocyte mitosis (27).
A concurrent feature of the enzyme systems responsive to prostaglan- dins is a close association with ATP and with membrane components. There is therefore, strong evidence for a common membrane element in prostaglan- din action. The demonstration that prostaglandings may regulate a number of enzymes associated with ATP has implications with respect to their cellu- lar function. The opposing effects of prostaglandins on both ATPase and adenylate kinase may serve to regulate and maintain cellular ATP levels. ATP is a Ca++-chelating agent (28) and the complex formed is intimately in- volved in regulation of the physical state of membrane proteins (29). For example, the primary event in a cell, when ATP levels are depleted leading to an adverse ATP/Ca++ ratio is a twofold decrease in membrane deformabi- lity . This is followed by a classical disc to sphere transformation. Prosta- glandins and their antagonists have been demonstrated to be highly active during both phases (29, 30). In addition, ATP has recently been shown to be involved in the regulation of prostaglandin stimulation of adenylate cyclase in the platelet (31). It is therefore possible that prostaglandin effects on adenylate cyclase are attenuated by coincident effects on the ATP-regulating enzymes.
We would further suggest that the anomalous kinetic orders and biphasic responses exhibited by membrane enzymes in the presence of prostaglandins imply that an allosteric mechanism may be involved. Cyclic AMP has many effects similar to those of prostaglandins (13) and has been suggested previously as an allosteric effector (32).
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PROSTAGLANDINS
POSSIBLE BINDING SITES FOR PROSTAGLANDIN AS LIGANDS 1. Membrane phospholipids In view of the conformational possibilities and the amphipathic nature
of the molecule, we have previously suggested that prostaglandins may exert their actions by affecting phase changes in membrane components. Indeed, membrane type appears of prime importance in determining a spe- cific cellular enzyme response to prostaglandins. For example, the kinetic responses of ATPase and adenylate kinase in platelets and erythrocytes are similar but are different in mitochondria. The phospholipid spectra are widely different in the two membrane types. There is now an extensive list of membrane enzymes associated with two types of phospholipids: the basic type (phosphatidyl ethanolamine) which can be removed without affecting enzyme activity (33). and the acidic type which is critical for enzyme responsiveness (34).
Our data on platelet, erythrocyte and mitochondrial enzymes suggest that phosphatidyl serine and phosphatidyl inositol may be important in determining the order of potency and parity of responses in prostaglandin action. Phosphatidyl serine, which enhances ATPase activity (35, 36), represents w 13% of the total phospholipid in erythrocytes (37) and platelets (38) and phosphatidyl inositol, which specifically inhibits ATPase (39) represents y 1%. The order of potency of prostaglandins in these membrane types is such that PGEl > PGE2 but the effectiveness of the two prosta- glandins parallels each other at all concentrations. On the other hand, in the mitochondrial membrane which contains + 12% phosphatidyl inositol and b 3% phosphatidyl serine, PGE2 > PGEI in its effect on mitochondrial ATPase, and there is disparity between saturating concentrations of these prostaglandins. These data imply that different phospholipids may serve as discriminators for membrane enzymes responsive to prostaglandins.
It is interesting that the two apparently pertinent phospholipids with respect to prostaglandin-responsive membrane enzyme activity are both acidic and bind Ca++ (40). This ion inhibits adenylate cyclase (41), ATPase (42)) stimulates adenylate kinase (43) and binds ATP (44).
2. Membrane sulfhydryl groups It is unlikely, however, that membrane phospholipids alone could
satisfy the strict structural-activity requirements of prostaglandin action. In addition, there is mounting evidence for the involvement of functional protein groups in the prostaglandin “receptor .I’ Kuehl (45) has reported that the binding of prostaglandin to a specific adipocyte component is dependent on free sulfhydryl groups, and the four groups of enzymes affected by prostaglandins appear to have functional sulfhydryl groups (46, 47).
Membrane ATPases and adenylate kinase are characteristically de- pendent on two classes of SH groups which differ in their susceptibility
MAY 1973 VOL. 3 NO. 5 713
PROSTAGLANDINS
to complex formation (48). Prostaglandin modification of membrane ATPase kinetics is similar to that exhibited by sulfhydryl reagents (49, 50). Experi- mentation is now in progress on the effects of sulfhydryl reagents on prosta- glandin pharmacological responses. Indeed, prostaglandins are ineffective against U/V-induced platelet aggregation in which SH-SS interchange is stimulated (51) or in aggregating NEM-pretreated platelets (52).
We would suggest that classes of sulfhydryl groups with different reactivities could provide possible interactive sites for prostaglandin modi- fication of membrane enzyme activity. A similar membrane component has recently been proposed by Karlin (53) for the nicotinic acetylcholine re- ceptor . The ability of different cells to react in a characteristic manner to prostaglandins could then be based on the number, type, and more import- ant the relative availability of sulfhydryl groups, as they are modified by complexing phospholipids , in the respective membranes. Indeed, the sickling human erythrocyte, which is responsive to prostaglandins under conditions in which the normal erythrocyte is not (54)) differs from the healthy cell in the availability of its phospholipid reactive groups (55). These groups, especially those of phosphatidyl serine, are probably more tightly bound to protein in the abnormal cell.
A hypothesis has been proposed that the variety of prostaglandin effects noted in cells and tissues is due to an interaction with a common membrane component, possibly a protein sulfhydryl/phospholipid complex. Prostaglandins , because of their orientated polar groups, amphipathic qualities and structural flexibility may exert phase changes in this com- ponent , initiating a change in the activity of a number of membrane-bound enzymes, including adenylate cyclase , phosphodiesterase, adenylate kinase and ATPase. The final effect will then depend upon the individual res- ponsiveness of these enzymes to prostaglandin in that particular cell type. (Figure 5).
714 MAY 1973 VOL. 3 NO. 5
PROSTAGLANDIN
I MEMBRANE
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v
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I \I/
PHYSICAL EFFECTS 1
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b Ion Movqnents
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Figure V. HYPOTHETICAL MODEL OF PROSTAGLANDIN ACTION
PROSTAGLANDINS
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