Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 274, No. 1, October, pp. 299-21X3,1989

Pertussis Toxin-Sensitive GTP-Binding Proteins May Regulate Phospholipase A2 in Response to Thrombin in Rabbit Platelets’ YASUO KAJIYAMA,

TOSHIHIKO MURAYAMA:

Department of Pharmmdogg, Faculty of Phu rmxeutical

AND

YASUYUKI NOMURA

Sciences, Hokkaido University, Sapporo 060,Japan

Received March 28,1989, and in revised form June 6,1989

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inosito1 trisphosphate (IP,) in [8H]inositol-labeled platelets, increases of [3H]arachidonic acid (TH]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase AZ activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5’-(3-O-thio)triphosphate (GTPrS), guanyld’-(@,y-iminio)triphosphate), or AlFi caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5’(20-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IPs formation, was diminished by 100 rig/ml of pertussis toxin, associated with ADP-ribosylation of membrane Il-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTPyS-stimulated release from permeabilized platelets were both markedly dependent on CaZ+.However, Ca2+addition could not enhance AA release without GTPrS even when Cazt was increased up to lop4 M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase AZ activity, not by phospholipase C activity, and that Ca2+is an important factor to the activation of phospholipase AZ but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase AZ. o 1989 Academic press, lnc. In platelets, thrombin can alter phospholipid metabolism through two pathactivation of ways: receptor-mediated phospholipase AZ and phospholipase C (1, 2). The activation of phospholipase A2 leads to release of AA3 and the subsequent 1 This work was supported by research grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture, Japan and from the Uehara Memorial Foundation, Japan. 2 To whom correspondence should be addressed. 3Abbreviations used: AA, arachidonic acid; PIs, phosphoinositides; IPs, inositol 1,4,5-trisphosphate; G protein, guanine nucleotide binding protein; G, and G,, guanine nucleotide regulatory proteins of adenyl0003-9861/89 $3.00 Copyright All rights

0 1989 hy Academic Press, Inc. of reprcduation in nny form resewed.

conversion to prostanoids such as prostaglandins and thromboxanes. It has been shown that the prostanoids are liberated into the extracellular fluids and bind to specific receptors on the platelets’ surface. The AA metabolites are able to modulate biological responses such as serotonin seate cyclase that mediate stimulation and inhibition, respectively; IAP, islet-activating protein (pertuss;S toxin); PAF. platelet-activating factor; Hepes, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GTPrS, guanosine 5’-(3-O-thio)triphosphate; GppNHp, guanyl+‘-(&y-imino)triphosphate; GDP@, guanosine 5’-(2-0-thio)diphosphatc; EGTA, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid.

!a0

PHOSPHOLIPASE

As REGULATION

cretion to certain drugs in platelets. Since the rate-limiting step in the synthesis of these metabolites appears to be the release of AA from phospholipids, it is important to clarify the mechanism of regulation of phospholipase AZ activity. Also, thrombin-stimulated activation of phospholipase C causes hydrolysis of PIs such as phosphatidylinositol 4,5-bisphosphate to inositol phosphates and diacylglycerol. The hydrolysis of PIs produces IP3 which increase intracellular concentrations of Ca2+by liberation of store Ca2’ (see Ref. (3) for review). Since Ca2+ is required for phospholipase A2 activity, these Ca2+mobilizations may also increase phospholipase A2 activity. The coupling of receptors to phospholipases is unclear. However, recent evidence suggests that G proteins are involved in activation of phospholipase C. In neutrophils (4) or mast cells (5), G protein, like Gi, which loses its function after being ADP-ribosylated by IAP (pertussis toxin), communicates between Ca2+-mobilizing receptors and phospholipase C. In 3T3 fibroblasts (6, 7) or hepatocytes (8), however, IAP exhibited no inhibition of receptor-mediated phospholipase C activation, suggesting that IAP substrate G proteins are not the sole mediators of Ca2+-mobilizing receptors. On the other hand, receptormediated AA release is abolished by IAP treatment in a variety of cells, including both cell types mentioned above (4-7). With this background, the present studies were designed to clarify the role of phospholipase A2 activity on serotonin secretion and to address two aspects of regulation of phospholipase A2 activity: (i) to determine whether the activity of phospholipase A2 is dependent on PI hydrolysis, and (ii) to determine whether thrombinstimulated AA release is, like adenylate cyclase or PI hydrolysis, mediated by G protein and, if so, to characterize that G protein. EXPERIMENTAL

PROCEDURES

Materials. IAP was purified from the 2-day culture supernatant of Bordetelkz pertussti (Tohama strain, phase I) according to the procedure described else-

BY GTP-BINDING

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201

where (9). [5,6,8,9,11,12,14,15-3H]AA (80-140 Ci/ mmol), 5-[3H]hydroxytryptamine (serotonin) (20-40 Ci/mmol), and myo-[2-3H]inositol (lo-20 Ci/mmol) were purchased from Amersham Corp. or New England Nuclear. Human a-thrombin, PAF, norepinephrine, GTP, mepacrine, and neomycin, were purchased from Sigma. Indomethacin was agift from Redary Corp. The sources of other materials were those described in previous papers (6,7,10,11). Isolation of rabbit platelets and prior radiolabeling. Blood (60 ml) was obtained from male Albino rabbits weighing 2-3 kg by heart puncture. Blood was mixed with 0.2 vol of ACD buffer (71 mM citric acid, 85 mM trisodium citrate, 111 mM dextrose, pH 5.5) to prevent coagulation. Platelet-rich plasma was obtained by centrifugation of the blood at 1509 for 10 min. Platelets were separated from platelet-rich plasma at 20°C by a lo-min centrifugation at 15009and washed twice with phosphate buffer solution (137 mM NaCl, 2.7 mM KCl, 8.0 mM Na,P04, 1.5 mM KHpP04, 5 mM dextrose, 0.1% bovine serum albumin (pH 7.4)). The cells were finally suspended in Hepes buffer (20 mM Hepes, 137 mM NaCl, 3.3 mM KHsPO,, 0.7 mM MgCls, 5 mM dextrose, 0.3% albumin (pH 7.4)), containing 10 PM indomethacin to inhibit cyclooxygenase, and were incubated for 2 h at 30°C with [3H]AA (5 &i/ml), [‘HIserotonin (5 &i/ml), or [3H]inositol (25 &i/ml). [‘HJAA release and [‘HJserotonin secretion from intact platelets in response to stimuli. The labeled platelets were suspended in the Hepes buffer after two washes with the same buffer and incubated for 7 min to assay for [‘H&A release and for 1 min to assay for [3H]serotonin secretion at 30°C with 1 mM CaClz and further additions shown in the tables and figures. In some experiments, these cells were first incubated with phospholipase inhibitors (in Fig. 1 and Table II) and digitonin (in Fig. 2) for 10 and 2 min, respectively. The reaction was terminated by addition of 1 ml of ice-cold Hepes buffer containing 2 mM EGTA and 10 mM EDTA followed by quick centrifugation at 8000~ for 2 min. The ‘H content of the supernatant was estimated. Digitonin treatment of the radiolabeled platelets to assay for [‘HjAA release. The labeled and washed platelet suspension was incubated with 15 pg/ml of digitonin for 7 min and the platelets were sedimented by centrifugation (15009 at 4”C, 10 min). In Table III, the incubation buffer was further supplemented with 100 rig/ml of the preactivated IAP, 100 PM NAD, 1 mM ATP, 10 mM thymidine, 10 mM dithiothreitol, for ADP-ribosylation of membrane proteins. This digitonin treatment was terminated by dilution with 10 ml of ice-cold Hepes buffer; the cell pellet was prepared and washed twice by centrifugation at 15OOgfor 10 min at 4°C. The reaction mixture for assay of [3H]AA release of this cell pellet consisted of the Hepes buffer supplemented with Ca2+and further ad-

KAJIYAMA,

MURAYAMA,

AND NOMURA

TABLE I EFFECTSOFTHROMBINONGENERATIONSOF IP, ANDRELEASEOF AA ANDSEROTONIN Additions None Thrombin PAF Norepinephrine PAF + norepinephrine

IP, generation (dpm/106 cells)

AA release (dpm/106 cells)

Serotonin secretion (dpm/lO’ cells)

51zk8 429 + 8” 159 k 8” 57k8 160 _+7”

40212 1600 f 54” 56k 3 432+- 51” 429k30”

74 f 14 2229 + 44” 100+ 3 93f14 359k21”

Note. [3H]Inositol-, [‘H&AA-, or [3H]serotonin-labeled platelets were incubated with stimulants for 10 s, 7 min, and 1 min, respectively, to measure the generation of IP3, the release of AA, and serotonin secretion as described under Experimental Procedures. The concentrations of additions are: thrombin, 1 unit/ml; PAF, 10 nM; norepinephrine, 10 PM. The data, means + SE, from three to four observations are shown. a Effects of stimulants were significant (P < 0.01).

ditions shown in the tables and figures. The released rH]AA was assayed as described above. Generation of [‘Hyp,. The [3H]inositol-labeled platelets were washed twice with the Hepes buffer before incubation in the same solution fortified with additions as shown in Table I. The temperature was maintained at 37°C during incubation. The incubation was terminated by the addition of 10% trichloroacetic acid, followed by extraction with five washes of 5 vol of water-saturated diethyl ether. The aqueous phase from ether-extracted samples was applied to Dowex AG l-X8 columns, for separation of IP3 as described elsewhere (1,5,7). Calcium &@r. Ca2+was buffered at concentrations between 10-r and 1O-4(pCa 7 to pCa 4) (pH 7.4) by the use of appropriate EGTA buffers which were prepared as previously described (12). Mg2+ was set at 1 mM. Data presentation All experiments were repeated under the same conditions. For single-point assays the mean + SE from three to four observations or means of duplicate observations which were reproducible and in agreement with each other within 10% error are presented. RESULTS

Roles of PI response and AA release on serotonin secretion. Radioactivities increased in the supernatant of the Hepes buffer when the rH]AA-labeled and [3H]serotonin-loaded platelets were incubated with thrombin (Table I and Fig. 1). Thrombin stimulated serotonin secretion and AA release in a dose-dependent manner. Thrombin also provoked rapid generation of IP3 when added to platelets within 10 s

as previously shown (13, 14). Thrombin-stimulated serotonin secretion was blocked by neomycin, an inhibitor of phospholipase C, and by mepacrine, an inhibitor of phospholipase AZ. In summary, stimulation of thrombin receptor resulted in (a) the generation of inositol phosphates, direct products of PI breakdown

Thrombln

(unltrlml)

Inhibitor

(-Log

MI

FIG. 1. Thrombin-stimulated serotonin secretion and effects of phospholipase inhibitors. [“HISerotonin-loaded platelets were incubated in the presence of phospholipase inhibitors for 10 min and further incubated for an additional 1 min with thrombin as described under Experimental Procedures. (A) The platelets were first incubated in the absence (0) or the presence of 10 PM mepacrine (0) or 1 PM neomycin (A) and further incubated with various concentrations of thrombin. (B) The platelets were first incubated in the presence of various concentrations of phospholipase inhibitors and further incubated with 1 unit/ml of thrombin.

PHOSPHOLIPASE

As REGULATION

BY GTP-BINDING

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mainly due to activation of phospholipase AZ but not to activation of phospholiEFFECTSOFPHOSPHOLIPASEINHIBITORS pase C. ONTHROMBIN-STIMULATEDAA Digitcnain treatment of platelet memRELEASEAND IPa FORMATION branes to permeabilixe GTPrS or fluoride. GTPrS is a hydrolysis-resistant analog of [‘H]AA lWIP, Additions release (%) formation (%) GTP that, in many systems, binds to and activates G proteins. GTPrS, however, Thrombin 100 100 which is not readily able to cross the 5.5 + 0.2” 86.3 z!z9.5 +lO pM Mepacrine plasma membranes of most cells, induced +lO PM Neomycin 88.8 f 6.3 15.8 f 7.1” no AA release in intact platelets. In the present experiments, platelets were “perNote. [3H]AA- or [3H]inositol-labeled platelets were meabilized” with increasing concentraincubated in the presence of phospholipase inhibitors tions of digitonin and mixed with GTPyS (10 PM) for 10 min and further incubated for an addiplatetional ‘7 min or 10 s in the presence of 1 unit/ml of (Fig. 2). In digitonin-permeabilized thrombin as described under Experimental Proce- lets, GTP$S evoked the release of [3H]AA. dures. The data are means + SE from three separate Basal AA release (without stimulants such experiments. [3H]AA release and rH]IP3 formation as thrombin or GTP+) was low regardless are indicated as a percentage of the thrombin-stimuof high Ca’+ concentration (Figs. 2 and 6). lated values without any inhibitors, which were 1200 The maximal increase was obtained with + 100 and 390 -I 20 dpm/106cells. lo-15 ~.LMdigitonin, which was used for the ’ Effect of inhibitor was significant (P < 0.01). subsequent experiments. Fluoride was found to stimulate AA release in permeabilized platelets (1094 via phospholipase C; (b) the release of AA dpm/106 cells for AlF; versus 243 for conand its metabolites, products of phospho- trol) but not in intact platelets. The welllipids hydrolysis via phospholipase Az; and known effect of the fluoride ion to stimu(c) the increase of serotonin secretion. late adenylate cyclase appears to be due to Effect (a), but neither (b) nor (c), was mimicked by PAF. Effect (b), but neither (a) nor (c), was mimicked by norepinephrine. PAF combined with norepinephrine induced three reactions, (a), (b), and (c), as did thrombin. These data suggest that the AA release by phospholipase A2 activation has an important role in platelet function such as serotonin secretion. Efects of phospholipase A, and C inhibitors on AA release. To determine whether AA release was an ultimate consequence of phospholipase C activation, the effect of thrombin on AA release was observed after inhibition of phospholipase C (Table II). Neomycin, which inhibits phospholipase C by binding PIs (15-1’7), blocked 0’ 8 3 5 3 ’ ’ thrombin-stimulated IP3 formation but 0 5 10 15 20 25 not AA release. Mepacrine significantly reDi@tonin ( )I Ml duced the AA release at a low concentraFIG. 2. GTPyS-stimulated AA release in digitonintion (1 PM) and blocked the release at 10 permeabilized platelets, [‘H]AA-labeled platelets PM. On the other hand, 10 PM mepacrine were incubated for 2 min in increasing concentrations did not reduce thrombin-stimulated IP3 of digitonin and incubated for an additional 7 min in formation. These findings suggest that the absence (0) or presence (0) of 10 PM GTPyS. The thrombin-stimulated AA liberation is data are means of duplicate observations. TABLE II

f 1500’

204

KAJIYAMA,

Ok+

\’

0.1

Thrombln

8

0.3

’

1

(unltalml)

I’#\

3

MURAYAMA,

AND NOMURA

.I QTP

(-Log

Ml

FIG. 3. GTP-dependent stimulation of AA release by thrombin and concentration dependency. The labeled platelets were treated with 10 pM digitonin and then assayed for AA release as described under Experimental Procedures. (A) Permeabilized platelets were incubated with increasing concentrations of thrombin in the presence (0) or absence (0) of 10 pM GTP. (B) Permeabilized platelets were incubated with increasing concentrations of GTP in the presence (0) or absence (0) of 1 unit/ml of thrombin. Each point is the mean of duplicate observations.

its activation of G,. Fluoride is also an effective activator of G protein(s) which can regulate phospholipase C-catalyzed PIs hydrolysis (18). These results suggest the possible involvement of G protein(s) in modulation of phospholipase A2 activity as well. G protein interactions with thrombin receptors and pho.spholipase A,. The response to different concentrations of thrombin or GTP was examined using permeabilized platelets (Fig. 3). Thrombin-stimulated [3H]AA release was not observed in the permeabilized platelets, in contrast with intact platelets. AA release was markedly stimulated by thrombin when the reaction mixture was supplemented with 10 PM GTP. The AA release attained its maximum value of SO-90% when the concentration of GTP was raised to lo-100 pM, a value comparable to the maximal value induced by thrombin in intact platelets. Similar experiments were repeated with increasing concentrations of GTP in the presence of 1 unit/ml of thrombin (Fig. 3B). Without receptor stimulation, GTP caused a little AA release. When hydrolysis-resistant GTP analogs, GTPrS or Gpp-

QTPVS

(-Log

QPDNNII

Ml

(-Log

Ml

FIG. 4. GTPrS- and GppNHp-stimulated AA release and its enhancement by thrombin addition. Permeabilized platelets were incubated with increasing concentrations of GTPrS (A) or GppNHp (B). The assay mixture was further supplemented with (0) or without (0) 1 unit/ml of thrombin.

NHp, replaced GTP, [3H]AA release was observed in its action on the phospholipase A2 even in the absence of thrombin (Fig. 4). This GTPrS- or GppNHp-induced AA release was enhanced by thrombin. In the studies shown in Fig. 5, the responses in permeabilized platelets stimulated with 10 pM GTP or GTPrS plus 1 unit/ml of thrombin were reduced by GDP/?S.The stable GDP analog GDP&S inhibited these rH]AA releases in a concen-

0’ t .‘. ’ 8

’ 7

5 6 GDPps

3 ‘s , 5 57-%+ (-Log

Ml

FIG. 5. Antagonism of thrombin-induced and GTP analog-stimulated AA release by GDPBS. Permeabilized platelets were incubated as described in the legend to Fig. 3. The assay mixture contained the indicated concentrations of GDP@ as well as no additions (0), 1 unit/ml of thrombin (A), 10 PM GTP analogs (0) (GTP in A, GTPrS in B), or thrombin plus GTP analogs (A).

PHOSPHOLIPASE

Az REGULATION

TABLE III IAP INHIBITS THROMBIN-INDUCED,GTP-DEPENDENT AA RELEASE

13HlIP~ Additions None Thrombin +GTP

IAP + +

rH]AA release (dpm/106 cells)

formation (dpm/106 cells)

80 251 76 259 840 (100) 415 (100) 492 (39.4 -t 2.2a) 414 (95.2 f 9.5a)

Note. [sH]AA- or [‘Hlinositol-labeled platelets were treated with 10 NM digitonin plus 100 PM NAD in the absence (IAP, -) or presence (IAP, +) of 100 rig/ml of preactivated IAP and then assayed for AA release as described under Experimental Procedures. The assay mixture was further supplemented with 10 MMGTP and 1 unit/ml of thrombin. The data are means of duplicate observations. The percentages of inhibition caused by IAP are shown in parentheses. The difference in release between the absence and the presence of thrombin plus GTP was indicated as 100%. The data in parentheses are the means of:SE from four separate experiments. a Effect of IAP was significant (P < 0.01).

tration-dependent manner. Half-maximal inhibition required 30 and 300 nM with GTP and GTP+, respectively. Similarly, the release by GTPrS without thrombin was antagonized by GDP&S addition. IAP-sensitive AA release by thrombin. Since the above experiments suggested the involvement of G proteins in AA release by phospholipase AZ, the effect of IAP was investigated. IAP catalyzed the ADP-ribosylation of certain G proteins, reducing their ability to couple receptors to effector proteins such as adenylate cyclase or phospholipase C (4-8,10,11). In our phospholipase AZ and C assay system using permeabilized platelets, IAP blocked thrombin-stimulated, GTP-dependent rH]AA release but not rH]IP3 formation (Table III). Lapetina et al. reported that the ADP-ribosylated protein from human platelets treated with IAP has the same apparent molecular mass as the 41-kDa ai subunit of Gi (19). Using rabbit platelets, 41-kDa proteins were ADP-ribosylated (data not shown). Cations requirements. Since it has been demonstrated that the activation of phos-

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PROTEIN

pholipase A2 requires extracellular Ca2’ and is modified by Mg2+ (20), the effects of cations were examined in digitoninpermeabilized platelets. Figure 6 shows that thrombin-stimulated, GTP-dependent rH]AA release was markedly dependent on free Ca2+.At the lowest Ca2’ levels, thrombin plus GTP did not induce AA release. This response was greatly enhanced by raising the free Ca2+ concentration to 0.1 mM. In the preceding experiments, the effects of stimulants was studied at 0.5-1.0 InM free Ca2+. It should be noted that higher Ca2’ concentrations could not activate phospholipase A2 in the absence of GTP+. Table IV shows the effects of Mg2+ on GTPyS-stimulated E3H]AA release of permeabilized platelets. GTPyS-stimulated response was completely dependent on Mg2+. This result is in agreement with previous reports that Mg2+ is a critical factor determining the activity of G, and Gi or other G proteins (21). DISCUSSION

A possible involvement sf AA release in serot-kin secretion. It was shown that ,

I””

0’

‘.f ’ 7

6

Free Ca2+

5

4

(-LOa M)

FIG. 6. Ca*+ concentration-response curve for AA release from permeabilized platelets in the presence of GTP-yS. Permeabiliaed platelets were incubated in the presence (0) or absence (0) of 100 FM GTPyS. The reaction mixture contained the indicated Ca”’ concentrations.

KAJIYAMA,

MURAYAMA,

AND NOMURA

to1 phosphates; (iii) Ca2+-induced phospholipase A2 activation leading to AA release. EFFECTOFMe ONAA RELEASEFROM To determine whether the activation of PERMEABILIZEDPLATELETS phospholipase C is necessary for the observed thrombin-stimulated AA release, pH]AA release (dpm/lo6 cells) we used neomycin to inhibit phospholipase Additions W C (15-17). Neomycin, which inhibited the 367 None PI hydrolysis in many cells including 360 + platelets at 10 PM, did not reduce throm333 GTP-yS bin-stimulated r3H]AA release (Table II). 879 + Mepacrine, a phospholipase A2 inhibitor, reduced the release at 1 l.cM and completely Note. Permeabilized platelets were incubated with at 10 PM. In human platelets, high concenor without 1 unit/ml of thrombin plus 10 PM GTP in ordinary Hepes buffer containing 0.7 mM MgC12 trations (>2 InM) of neomycin appear to exert some nonspecific effects (23,24) and (Me, +) or in Mp-omitted Hepes buffer (Me, -) as described under Experimental Procedures. The mepacrine inhibits both phospholipase A2 data are means of duplicate experiments. and phospholipase C (25). In rabbit platelets, however, low concentrations of the two inhibitors were selective (Table II). The results show that thrombin-stimustimulation of thrombin receptors re- lated AA release is mainly mediated by sulted in (a) increases in cellular IPB, (b) phospholipase A2, not by phospholipase C. AA release, and (c) serotonin secretion This idea is supported by another result in from platelets (Table I). Effect (c) of Table IV. In platelet membranes, Mg2+ was thrombin was abolished by prior treatan inhibitor of phosphatidylinositol 4,5ment of platelets with mepacrine or neo- bisphosphate hydrolysis stimulated by mycin (Fig. 1). PAF alone showed effect (a) guanine nucleotides (18). However, in our of thrombin but not (b) and (c). On the phospholipase A2 assay systems, GTPrSother hand, norepinephrine by itself stimulated [‘H]AA release was dependent showed effect (b) of thrombin but not (a) on the presence of Mg2+ (Table IV). The and (c). Interestingly, PAF combined with ability of agonists such as ADP and epinorepinephrine induced three effects of nephrine to cause AA release despite the thrombin, although the extents of any re- fact that they have little or no direct effect actions by PAF plus norepinephrine were on PI hydrolysis (2, 26), suggests that PI smaller than those by thrombin. These hydrolysis-independent pathways for AA data suggest that not only the hydrolysis release to agonist including thrombin do of PIs but also AA release is involved in exist. Only 3-69~ of the radioactive phosthrombin-induced serotonin secretion. phatidylcholine, but not phosphatidylinoAA release by phosplwlipase A, in Ca”sitol or diacylglycerol, was decreased durdependent manner without phospholipase C ing a short-term incubation of the labeled activatim A number of papers have re- platelets with thrombin (data not shown). cently been published which focus on a Also a diacylglycerol lipase inhibitor scheme of the reaction sequence responsi- (RHC 80267) did not affect the level of ble for signal transduction from Ca’+-mo- the released AA in thrombin-stimulated bilizing receptors to the eventual cell re- platelets (27). These data suggest that the sponses (see Ref. (22) for review). The gen- release is not catalized by the sequential eral scheme currently accepted is: (i) rapid actions of phospholipase C and diacylglycbreakdown of phosphatidylinositol4,5-biserol lipase. Indeed, phospholipase A2 has been rephosphate followed by accumulation of inositol phosphates and diacylglycerol as ported to require high concentrations of the initial step; (ii) Ca2’ mobilization from Ca2+ for its maximal activity. However, the extracellular or from cell-associated unexpectedly, increases in calculated Ca2+ stores as a result of the action of the inosi- to lop4 M had substantially no stimulatory TABLE IV

PHOSPHOLIPASE

A2 REGULATION

effects on AA release from permeabilized platelets, although GTPyS-stimulated AA release was dependent on Ca2’ (Fig. 6). This result may show that Ca2+ is an important but not sufficient factor for modulation of the phospholipase A2 activity of platelets. The involvement of G protein in receptormediated phospholipase A, activation. The following findings are in accordance with the idea that phospholipase A2 is coupled to thrombin receptors by G proteins in rabbit platelets: (i) [3H]AA-labeled and digitonin-permeabilized platelets were found to release [3H]AA or its metabolites by GTP+, GppNHp (Fig. 2), or AlF; addition. (ii) There was no liberation of AA by thrombin in the absence of guanine nucleotides, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate the release (Figs. 3 and 4). (iii) Thrombin-stimulated release observed in the presence of GTP or GTP-rS was fully antagonized by GDP@ addition (Fig. 5). (iv) The thrombin-induced, GTP-dependent release was much less in magnitude in IAP-treated platelets than in nontreated platelets (Table III); (v) GTPyS-stimulated AA release was dependent on Mg2+, which is a critical factor determining the association of CYsubunits with & subunits of G proteins (Table IV). In the adenylate cyclase system it has been proposed that GTP cannot persistently activate either G, or Gi because it is hydrolyzed to GDP by a GTPase associated with each G protein. The purpose of the agonist is to stimulate the receptor and enhance release of GDP from the associated G protein thereby allowing GTP access to the GTP-binding site (10, 11). GTP was able to cause a concentration-dependent enhancement of thrombin-induced AA release but had no effect when it was given alone (Fig. 3), suggesting that thrombin altered the efficacy of GTP. In contrast, the efficacy of GDP was unaltered by thrombin. GDP or its analog, GDP&S, did not evoke AA release and GDPfiS blocked thrombin-induced, GTP-dependent AA release by phospholipase A2 (Fig. 5). Similar findings regarding GTP analog-stimulated, IAP-sensitive phospholipase AZ ac-

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tivity have been reported previously in other cells including human platelets (2730). Additionally, light activation of phospholipase A2 has been shown to occur by a transducin (a GTP-binding protein that is an IAP substrate)-dependent mechanism in the rod out segments of bovine retina (31,32). There have been a number of G proteins described that comprise a family of regulatory proteins. Lapetina et al. reported that exposure of human platelet membranes to IAP resulted in the ADP-ribosylation of the M, 41,000 protein in membranes and treatment with thrombin inhibited the IAP-induced ADP-ribosylation of the protein (19). This IAP substrate is an a-subunit of Gi, ADP-ribosylation of which leads to complete loss of the Gi function to couple to receptors (33-35). These data suggest that thrombin receptors interact with IAP-sensitive G protein. Using rabbit platelets, 41-kDa protein was ADP-ribosylated and thrombin-induced inhibition of adenylate cyclase (data not shown) and increase of AA release (Table III) were diminished. It is likely, therefore, that Gi may play an important role in the phospholipase A2 system as well as in the adenylate cyclase system. This does not imply, however, that Gi and the putative G protein-coupled phospholipase A2 are the same protein. There may be more than one protein located within this band. Recently, receptors for Ca2+-mobilizing hormones have been proposed to be associated with a G protein that acts as a transducer between the receptor and phospholipase C which mediates hydrolysis of PIs in many cell types (22). In saponin-treated human platelets, thrombin-stimulated PI hydrolysis (35), but not thromboxane AZ analog (U46619)-stimulated (36) hydrolysis, was inhibited by IAP. In our experiments using digitonin-permeabilized rabbit platelets, thrombin-stimulated PI hydrolysis was unaffected by IAP (Table II). What is less clear is why PI hydrolysis should be affected by IAP in some tissues, but not in others. The differences seem to be receptor as well as tissue specific. Both events of receptor-mediated AA release and inhibition of adenylate cyclase were

208

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MURAYAMA,

markedly prevented by IAP in many cell types regardless of IAP sensitivity of PI hydrolysis (4-7,28). Whether activation of these different effector systems is a function of the same G protein or reflects action of different G proteins remains to be determined. The subunit specificity associated with phospholipase A2 activation in rabbit platelets is currently under investigation. REFERENCES 1. WATSON, P. S., RUGGIERO, M., ABRAHAMS, S. L., AND LAPETINA, E. G. (1986) J. BioL Chem 261, 5368-5372. 2. SWEAT’, J. D., BLAIR, I. A., CRAGOE, E. J., AND LIMBIRD, L. E. (1986) J. Bid Chem 261,86608666. 3. TAYLOR, C. W. (1987) Trends Phurm.acol Sci 8,

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