Platelets From Spontaneously Hypertensive Rats Exhibit Decreased Expression of Inhibitory Guanine Nucleotide Regulatory Protein

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1 1032 Platelets From Spontaneously Hypertensive Rats Exhibit Decreased Expression of Inhibitory Guanine Nucleotide Regulatory Protein Relation With Adenylyl Cyclase Activity Madhu B. Anand-Srivastava We have recently demonstrated an enhanced expression of inhibitory guanine nucleotide regulatory protein (Gi) in the heart and aorta from spontaneously hypertensive rats () as compared with control Wistar-Kyoto () rats; this enhanced G, expression was associated with an increased inhibition of adenylyl cyclase by inhibitory hormones and decreased stimulation of adenylyl cyclase by stimulatory hormones. In the present studies, we have determined the levels of stimulatory and inhibitory guanine nucleotide regulatory proteins (G, and Gi, respectively) in platelets from by cholera toxin- and pertussis toxin-catalyzed ADP-ribosylations, respectively, as well as by immunoblotting techniques using specific antibodies for G, and Gi. Cholera toxin catalyzed the ADP-ribosylation of a single protein of M, in rat platelets from and rats, and the labeling of this band was not altered in as compared with rats. Pertussis toxin, on the other hand, catalyzed the ADP-ribosylation of a single protein band of Mr in platelets from and rats, and unlike the response in heart and aorta, the labeling of this band was significantly decreased in as compared with rats. Furthermore, immunoblotting experiments using AS/7 antibody, which is specific for G1al and Git,.2 showed a decrease in Gi12 in platelets from as compared with rats. In addition, the inhibitory effects of angiotensin II and atrial natriuretic factor on adenylyl cyclase and camp levels were completely abolished in platelets, whereas the stimulatory effects of GTP, N-ethylcarboxamide adenosine, prostaglandin E1, and forskolin on adenylyl cyclase and camp levels were enhanced. These results suggest that altered responsiveness of hormones to stimulate or inhibit adenylyl cyclase and camp levels in platelets from may partly be attributed to the decreased expression of Gi levels and not to the overexpression of Gs protein. (Circ Res. 1993;73: ) KEY WoRDs * adenylyl cyclase activity * G proteins * spontaneously hypertensive rats * platelets T he adenylyl cyclase/camp system is one of the signal transduction systems implicated in the regulation of cardiovascular functions including arterial tone and reactivity. Several abnormalities in the adenylyl cyclase activities and camp levels in cardiovascular tissues of spontaneously hypertensive rats () have been implicated in the pathogenesis of hypertension.1-3 The adenylyl cyclase system is composed of three components: extracellular receptor, catalytic subunit, and guanine nucleotide regulatory proteins (G proteins). The hormonal stimulation and inhibition of adenylyl cyclase are mediated via two different G proteins: stimulatory (GQ) and inhibitory (G), respectively.4 G proteins are heterotrimeric and composed of three distinct subunits: a, l3, and y.5 The a-subunit (Gsa) binds and hydrolyzes GTP and confers specificity in receptor and effector interactions.6 Two different forms of the Gsa protein with a molecular size of 45 and 52 kd have been characterized.7 However, a third form of the Gsa Received October 28, 1992; accepted August 11, From the Department of Physiology, Faculty of Medicine, University of Montreal, Quebec, Canada. Correspondence to Dr Madhu B. Anand-Srivastava, PhD, Department of Physiology, Faculty of Medicine, University of Montreal, C.P. 6128, Succursale A, Montreal, Quebec, Canada H3C 3J7. protein having a molecular size of 47 kd has also been detected in heart.8 These different forms of Gsa arise from several species of mrna that appear to be the products of alternative splicing of a common precursor.9 10 On the other hand, three distinct forms of GQ, (Gia, Ga,-2, and Gia 3) have been identified and characterized and have been shown to be the products of different genes The G proteins are also targets of bacterial toxins that are useful probes for defining the interaction of the regulatory proteins with other components of the adenylyl cyclase system. The bacterial toxins such as cholera toxin (CT) and pertussis toxin (PT) catalyze a reaction in which an ADP-ribose is transferred from NAD to specific amino acid acceptors on Gs, or Gi, and a G protein of unknown function (G,), respectively, and thereby modify the characteristics of these proteins CT irreversibly activates the Gs proteins, which mediate the stimulation of adenylyl cyclase, whereas PT, in addition to GO, acts on the Gi protein, which regulates inhibition and attenuates the GTP-dependent and receptor-mediated inhibition of adenylyl cyclase.17 The functions of Go are not yet known, but it may interact with enzymes associated with Ca2' mobilization and not with adenylyl cyclase.

2 Anand-Srivastava Adenylyl Cyclase/cAMP Signal Transduction in Hypertension 1033 The alterations in the expression of G, or Gi have been demonstrated in various pathophysiological conditions, such as heart failure, diabetes, and hypothyroidism We have recently shown an enhanced expression of Gja2 at protein and mrna levels and its relation with the adenylyl cyclase inhibition in heart and aorta from and deoxycorticosterone acetate-salt hypertensive rats, whereas the levels of Gsa and GQa mrna were not altered Several abnormalities in platelet functions have been reported in hypertensive patients as well as in ; these abnormalities include an abnormal, augmented, or decreased aggregation in response to various stimuli such as ADP, epinephrine, thrombin, prostaglandins, and Ca21 ionophore A Since the adenylyl cyclase/camp system has been implicated in platelet aggregation, the observed abnormalities in platelet functions in hypertension may be attributed to the impaired adenylyl cyclase/camp system, including G proteins, which play a key role in the regulation of camp levels. The present studies were therefore undertaken to investigate the levels of G proteins (Gi and GQ) and their relation with adenylyl cyclase inhibition and stimulation in platelets from. Materials and Methods ATP, camp, and isoproterenol were purchased from Sigma Chemical Co, St Louis, Mo; creatine kinase (EC ), myokinase (EC ), and GTPyS were from Boehringer Mannheim, Montreal, Quebec, Canada. [ad2]atp was from Amersham Corp Ontario, Canada, and [a-32p]nad was from Dupont Canada, Mississauga, Ontario, Canada. The electrophoresis chemicals were obtained from Bio-Rad, Mississauga, Ontario, Canada. Atrial natriuretic factor-(99-126) (ANF) was acquired from Peninsula Laboratories, Inc, Belmont, Calif. PT was from List Biochemicals, Campbell, Calif, and CT was from Sigma. AS/7 and RM/1 antibodies were from Dupont Canada. Preparation of Platelet Membranes Platelet membranes were prepared as described previously.28 Blood was drawn from femoral arteries of anesthetized rats into citrate anticoagulant containing (mmol/l) sodium citrate, 87; dextrose, 105; citric acid, 11; and Na3P04, 7. A 1-mL portion of anticoagulant was used for each 10 ml of blood. After centrifugation of whole blood at 350g for 10 minutes, platelet-enriched plasma was collected and further centrifuged at 1000g for 10 minutes. The pellet was washed twice with a buffer containing 10 mmol/l Tris and 1 mmol/l EDTA, ph 7.5. Platelet suspensions in 10 mmol/l Tris and 1 mmol/l EDTA were frozen in liquid N2 and stored at -70 C. Before the assay, the frozen platelet suspension was thawed and homogenized, and this freeze thawing was repeated twice. Adenylyl Cyclase Activity Determination Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [a-32p]atp, as described previously.21'28 The assay medium contained 50 mmol/l glycylglycine (ph 7.5), 0.5 mmol/l Mg-ATP, 1 to 1.5 x 106 cpm [a-'2p]atp, 5 mmol/l MgCl2 (in excess of the ATP concentration), 100 mmol/l NaCl, 0.5 mmol/l camp, 1 mmol/l 3-isobutyl-1-methylxanthine (or oth- erwise as indicated), 0.1 mmol/l EGTA, 10 limol/l GTPyS (or otherwise as indicated), and an ATP-regenerating system consisting of 2 mmol/l creatine phosphate, 0.1 mg creatine kinase per milliliter, and 0.1 mg myokinase per milliliter in a final volume of 200,uL. Incubations were initiated by the addition of the membrane preparation (20 to 70 jig protein) to the reaction mixture, which had been thermally equilibrated for 2 minutes at 37 C. The reactions, conducted in triplicate for 10 minutes at 37 C, were terminated by the addition of 0.6 ml of 120 mmol/l zinc acetate containing 0.5 mmol/l unlabeled camp. camp was purified by coprecipitation of other nucleotides with ZnCO3 and by the addition of 0.5 ml of 144 mmol/l Na2CO3 and subsequent chromatography by the double-column system, as described by Salomon et al.29 The unlabeled camp served to monitor the recovery of the [32P]cAMP by measuring absorbance at 259 nmol/l. Under the assay conditions used, adenylyl cyclase activity was linear with respect to protein concentration and time of incubation. Protein was determined essentially as described by Lowry et al,30 with crystalline bovine serum albumin (BSA) as standard. Determination of camp Levels camp levels in platelets were determined by radioimmunoassay as described previously31'32 by using a radioimmunoassay kit from Dupont Canada. ADP-Ribosylation by PT and CT ADP-ribosylation of platelet membranes by PT or CT was performed as described earlier.21'33 The membranes from age-matched control and rats were incubated in 25 mmol/l glycylglycine buffer, ph 7.5, containing 15 gumol/l [a-32p]nad (20 gtci/ml), 0.4 mmol/l ATP, 0.4 mmol/l GTP, 15 mmol/l thymidine, 10 mmol/l dithiothreitol, and ovalbumin (0.1 mg/ml) with and without PT (5,ug/mL) for 30 minutes at 37 C in a total volume of 100 4L. The reaction was terminated by the addition of 20,uL of a "stop mix" containing 5% sodium dodecyl sulfate (SDS) and 50%,3-mercaptoethanol, followed by heating for 10 minutes in a boiling water bath. The labeled proteins were analyzed by subjecting the aliquots to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli34 with 12% SDS-polyacrylamide gels. After electrophoresis, the gels were fixed, stained, destained, dried, and autoradiographed by exposure to Kodak XAR-5 film, as described previously.33 They were calibrated by using molecular weight standards (Pharmacia): phosphorylase b (Mr, ), albumin (Mr, ), ovalbumin (Mr, ; carbonic anhydrase (Mr, ), trypsin inhibitor (Mr, ), and P-lactalbumin (Mr, ). CT-Catalyzed ADP-Ribosylation CT-catalyzed ADP-ribosylation of the membranes from age-matched and rats was performed in a manner similar to that catalyzed by PT except that the CT (500,ug/mL) was preactivated for 20 minutes at 37 C in a mixture containing 20 mmol/l dithiothreitol, 1,g/mL BSA, and 25 mmol/l KH2PO4 (ph 8.0). For studying the effect of CT on adenylate cyclase activity, the platelet membranes were pretreated with and without CT for 30 minutes at 30 C in the same reaction mixture as described above for ADP-ribosylation except

3 1034 Circulation Research Vol 73, No 6 December 1993 F- 0 C Wj 0 QE &,- -0- > C) LL uj cn C) U- z LUl.'E G) E n < a 500 -, BASAL A II ANF NECA PGE 1 CD llj Or.0 cz : TI- \ GTP (M) FIG 1. Graph showing the effect of GTP on adenylyl cyclase activity in platelet membranes from 12-week-old Wistar-Kyoto () rats and age-matched spontaneously hypertensive rats (). Adenylyl cyclase activity was determined in the absence or presence of various concentrations of GTP as described in "Materials and Methods." Values are mean ±SEM of six separate experiments. Basal enzyme activity in rats and was 154.8±15.3 and 205.7±30.0 pmol camp per milligram protein for 10 minutes, respectively. Six animals were used in each experiment. 1 mmol/l NAD instead of [a-32p]nad was used. The platelets were washed twice with buffer containing 10 mmol/l Tris and 1 mmol/l EDTA (ph 7.5) and finally suspended in the same buffer for adenylyl cyclase activity determination. Immunoblotting After SDS-PAGE, the separated proteins were electrophoretically transferred to nitrocellulose paper (Schleicher & Schuell) using a minitransfer apparatus (Bio-Rad) at 100 V for 1 hour or a semidry transblot apparatus (Bio-Rad) at 15 V for 45 minutes as described previously.21 After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% BSA at room temperature for 2 hours. The blots were then incubated with antisera against G proteins in PBS containing 1% BSA and 0.1% Tween 20 at room temperature for 2 hours. The antigen-antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio- Rad) conjugated with horseradish peroxidase for 2 hours at room temperature. The blots were washed three times with PBS before reacting with diaminobenzidine and hydrogen peroxide as described by Hsu and Soban.35 The autoradiograms and immunoblots were quantified by densitometric scanning using an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Quebec, Canada) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was one dimensional and 0- FIG 2. Bar graph showing the effect of hormones on adenylyl cyclase activity in platelet membranes from 12-week-old Wistar-Kyoto () rats and age-matched spontaneously hypertensive rats (). Adenylyl cyclase activity was determined in the absence of drugs (basal) and in the presence of 10 gtmol/l angiotensin il (A II), 0.1,umol/L atrial natriuretic factor-(99-126) (ANF), 10,umol/L N-ethylcarboxamide adenosine (NECA), and 1.0 gmol/l prostaglandin E1 (PGE1) as described in "Materials and Methods." 3-lsobutyl-1-methylxanthine was replaced by Ro (Roche). Values are mean+sem of six separate experiments. Six animals were used in each experiment. Statistical analysis was performed with Student's t test for comparison between and rats: for A 11, ANF, and PGE1, P<.05; for NECA, P<.01. scanned the entire area of protein band in autoradiograms and immunoblots. Results Effect of GTP on Adenylyl Cyclase Activity Guanine nucleotides stimulate or inhibit the adenylyl cyclase activity by interacting with G proteins. We have recently reported a significant decrease in the sensitivity of guanine nucleotides in the stimulation of adenylyl cyclase activity in heart and aorta from as compared with age-matched normotensive rats.3221 To investigate if such an impaired response of guanine nucleotides to adenylyl cyclase stimulation also exists in platelets from, we studied the effect of GTP on adenylyl cyclase activity, and the results are shown in Fig 1. GTP stimulated the adenylyl cyclase activity in platelets from both and rats in a concentration-dependent manner; however, unlike the response in heart and aorta, the extent of stimulation was greater in than rats. For example, at 10,umol/L, GTP stimulated the adenylyl cyclase activity in rats by -20%, whereas -::75% stimulation was observed in. These results indicate that the G, protein may be hypersensitive or overexpressed in. Hormonal Responsiveness ofadenylyl Cyclase If G, is hypersensitive or overexpressed in platelets, the GQ-mediated hormonal effects on adenylyl cyclase should also be augmented. To investigate this possibility, we examined the effects of prostaglandin E, (PGEL) and N-ethylcarboxamide adenosine (NECA), which have been shown to inhibit platelet aggregation, on the adenylyl cyclase activity in platelets from and rats. As shown in Fig 2, NECA and PGE,

4 Anand-Srivastava Adenylyl Cyclase/cAMP Signal Transduction in Hypertension 1035 oe.f ~- W.'r >--J cm Z0CL E W-5 CO o o ANF (M) FIG 3. Graph showing the effect of various concentrations of atrial natriuretic factor-(99-126) (ANF) on adenylyl cyclase activity in platelet membranes from 12-week-old Wistar-Kyoto () rats and age-matched spontaneously hypertensive rats (). The enzyme activity was determined as described in "Materials and Methods." Values are mean±sem of six separate experiments. Six animals were used in each experiment. stimulated the enzyme activity to various degrees in and rats; however, the extent of stimulation was higher in as compared with rats. On the other hand, angiotensin II (Ang II) and ANF, which inhibit adenylyl cyclase through Gi, inhibited the enzyme activity by -:'30% and 50%, respectively, in rats, whereas the inhibition was almost completely attenuated in. Fig 3 shows the effect of various concentrations of ANF on adenylyl cyclase in and platelets. ANF, as reported earlier,28 inhibited the adenylyl cyclase activity in platelets from rats in a concentration-dependent manner; the maximal inhibition observed was '=70% with an apparent Ki between 0.1 and 1 nmol/l. However, in, the ANF-mediated inhibition was completely attenuated. Effect of Hormones on camp Levels Table 1 shows the effect of some hormones and forskolin (FSK) on camp levels in platelets from and rats. PGE1, NECA, and FSK increased the camp levels in platelets from and rats to various degrees; however, the increases were greater in. For example, NECA and PGE1 increased the camp levels by :z-25% and 520%, respectively, in rats and ':"150% and 1240%, respectively, in. Similarly, FSK-stimulated camp levels were '-3-fold augmented in as compared with rats (:==23- fold increase in as compared with 7-fold increase in rats). On the other hand, inhibitory hormones such as Ang LI and ANF decreased the camp levels by '-40% in platelets from rats; however, this inhibitory effect was completely abolished in platelets from. These results indicate that the inhibitory G protein (Gi,), which TABLE 1. Effect of Various Agonists on camp Levels in Platelets From Spontaneously Hypertensive Rats and Wistar-Kyoto Rats camp Levels, pmol/mg protein Addition None ±2.5 PGE1, 1,umol/L * ADA, 5 U/mL ADA+NECA, 10 gmol/l ± ±5.9* FSK, 50,umol/L * Ang II, 10 1 mol/l * GG, 50 mmol/l GG+ANF, 0.1,tmol/L 74.9± * indicates Wistar-Kyoto rats;, spontaneously hypertensive rats; PGE1, prostaglandin E1; ADA, adenosine deaminase; NECA, N-ethylcarboxamide adenosine; FSK, forskolin; Ang 11, angiotensin 11; GG, glycylglycine; and ANF, atrial natriuretic factor-(99-126). Values are mean+±sem of six experiments. Platelets from and rats were preincubated with 1 mmol/l Ro (Roche) for 5 minutes at 370C. Various agonists were then added to the cells and were further incubated for 10 minutes. The reaction was stopped by the addition of 6% trichloroacetic acid, and camp levels were determined in the supernatant by radioimmunoassay using a kit from Dupont. *P<.001 vs by Student's t test. mediates the inhibitory effects of hormones on adenylyl cyclase, may be impaired in platelets from. To investigate whether the hypersensitivity of GQmediated hormonal stimulations were due to the enhanced expression of Gsa, we determined the levels of G, protein by CT-catalyzed ADP-ribosylation and immunoblotting technique using specific antibodies against G, (RM/1). As shown in Fig 4, CT in the presence of [32P]NAD catalyzed the ADP-ribosylation of a single protein band of 45 kd in both and platelets; however, the labeling of this protein band was not different in as compared with rats ( rats, arbitrary units;, arbitrary units; n=5) (Fig 4, left). Similar results were also observed by using specific antibodies against G5, (Fig 4, right) that recognized a protein with a molecular mass of 45 kd in both and platelets, with no alterations in the relative amount of immunodetectable Gsa in as compared with rats ( rats, 1.1±0.10 arbitrary units;, arbitrary units; n=5). These results indicate that the Gs, protein is not altered in platelets from. To corroborate these results with the functions of GQa protein, the effect of CT treatment on the GTP-sensitive adenylyl cyclase was performed in platelets from and rats, and the results are shown in Table 2. CT treatment augmented the GTPsensitive adenylyl cyclase activity in both and platelets; however, the extent of stimulation was not different in as compared with rats. We also determined the levels of Gia in platelets from and rats by PT-catalyzed ADP-ribosylation and immunoblotting techniques (Fig 5) to investigate whether the attenuation of inhibitory responses of ANF and Ang II on adenylyl cyclase and camp levels were due to the attenuated levels of Gia in. As shown in

5 1036 Circulation Research Vol 73, No 6 December 1993 PLATELETS CT PLATELET 94 - (v) x F- I LL ax s > _a 31 - _i -Ji _3w) FIG 4. Left, Autoradiograph illustrates the cholera toxin (CT)-catalyzed ADP-ribosylation of platelet membranes from 12-week-old Wistar-Kyoto () rats and spontaneously hypertensive rats (). Platelet membrane protein (40 jig) from rats (lanes 1 and 2) and (lanes 3 and 4) were incubated with [ a-32p]nad in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 100 gg/ml CT at 300C for 30 minutes. The 32P-labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described in "Materials and Methods." The autoradiograms shown are representative of four separate experiments. Right, Immunoblots are from platelet membranes of 12-week-old rats and age-matched. The membrane proteins (40 jig) from rats and were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using RM/1 antibody as described in "Materials and Methods." The detection of the stimulatory G protein a-subunit (cs45) was performed by using diaminobenzidine and hydrogen peroxide technique as described in "Materials and Methods." The autoradiograms shown are representative of four or five separate experiments. Fig 5, left, PT in the presence of [a-32pinad catalyzed the ADP-ribosylation of a single protein band of 40/41 kd in both and platelets; however, the labeling of this band was significantly lower (55% to 62%, 60±4%, n=4) in as compared with platelets. Similarly, AS/7 antibodies, which recognize Gia and Ga2,36 recognized a single protein having the molecular mass of 40 kd (Gja 2) in both and platelets, and the relative amount of immunodetectable Gia2 was significantly decreased (40% to 50%, 44+5%, n =4) in as compared with rats (Fig 5, right). These results indicate that the expression of Gia2 implicated in adenylyl cyclase inhibition36 is decreased in platelets from. Discussion We3,21,37 and others38,39 have previously reported a decreased sensitivity of various hormones to adenylyl cyclase stimulation in several tissues including heart and aorta from different models of hypertension; this de- TABLE 2. Effect of Cholera Toxin Treatment on Adenylyl Cyclase Activity in Platelets From Wistar-Kyoto and Spontaneously Hypertensive Rats Adenylyl cyclase activity, pmol camp/mg protein for 10 minutes Additions None 10 umol/l GTP -CT CT Stimulation, % -CT +CT Stimulation, % indicates Wistar-Kyoto rats;, spontaneously hypertensive rats; -CT, absence of cholera toxin (CT); and +CT, presence of CT. Values are mean+sem of six separate experiments. Platelet membranes from and age-matched (12 weeks old) were incubated with CT at 30 C for 30 minutes as described in "Materials and Methods," and adenylyl cyclase activity was determined, as described in "Materials and Methods."

6 Anand-Srivastava Adenylyl Cyclase/cAMP Signal Transduction in Hypertension 1037 PLATELETS PT T - + Th - + PLATELET 94 cn 0 67x jf-- 0 LUi 47cc :D LU ac FIG 5. Left, Autoradiograph illustrates the pertussis toxin (PT)-catalyzed ADP-ribosylation of platelet membranes from 12-week-old Wistar-Kyoto () rats and age-matched spontaneously hypertensive rats (). Platelet membrane protein (40 Mg) from rats (lanes 1 and 2) and (lanes 3 and 4) were incubated with [cv-32p]nad in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 5 gg/ml PT at 30 C for 30 minutes. The 32P-labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography as described in 'Materials and Methods." The autoradiograms shown are representative of four separate experiments. Right, Immunoblots are from platelet membranes of 12-week-old rats and age-matched. The membrane proteins (40 gg) from rats and were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using AS/7 antibody as described in "Materials and Methods." The detection of the inhibitory G protein a-subunit (Ct40) was performed by using diaminobenzidine and hydrogen peroxide technique as described in "Materials and Methods." The autoradiograms shown are representative of four separate experiments. associated with a downregulation of the defective receptor-effector coupling,3 and an enhanced expression of Ga'2' and/or impaired catalytic subunit of adenylate cyclase. However, in the present studies, we demonstrate a differential regulation of adenylyl cyclase by hormones and guanine nucleotides in platelets from. An augmented stimulation of adenylyl cyclase and camp levels by GTP, PGEJ, and NECA in as compared with rats is in contrast with the observations made in heart and aorta, where the stimulatory responses of hormones and guanine nucleotides on adenylyl cyclase were significantly attenuated.21 However, these results are in agreement with the studies of Hamet et al,27 who reported a potentiation of the stimulatory effect of PGE, on adenylyl cyclase in platelets from. In addition, the increased responsiveness of adenylyl cyclase to stimulatory hormones has also been demonstrated in splenocytes from.41" The enhanced stimulation of the adenylyl cyclase activity and camp formation by PGE, in platelets may explain the augmented attenuation of platelet aggregation by PGE, in.27 The increased responsiveness of PGE, and NECA to adenylyl cyclase stimulation may be explained by the upregulation of hormone receptors; however, various crease was receptor.39' a reports have shown a downregulation or no change in the number of stimulatory hormone receptors in several tissues from various cardiovascular diseases such as hypertension and congestive heart failure.39 Furthermore, our results on the CT-catalyzed ADP-ribosylation and immunoblotting with antibodies against Gsa did not show any alterations in Gsa in as compared with platelets and suggest that Gsa may not be responsible for the augmented ability of GTP, PGEI, and NECA to stimulate adenylyl cyclase in. In addition, the stimulation of the GTP-sensitive adenylyl cyclase activity to a similar extent by CT treatment in and platelets further substantiates the notion that the functions of the Gsa may not be altered in. These results are in agreement with our previous studies demonstrating no alterations in the levels and functions of GSa in the heart and aorta of as compared with rats.21 Similar results have also been reported in the failing human heart.'s However, the decreased functions of GUa in femoral arteries4' and the decreased levels and functions of have been 6, reported in compensated left ventricular hypertrophy,42 pressure-overload left ventricular failure,43 and myocar- dial ischemia,44 whereas such studies have not been reported in platelets.

7 1038 Circulation Research Vol 73, No 6 December 1993 A decreased labeling of 40/41 kd protein in as compared with platelets by PT-catalyzed ADPribosylation suggests that platelets from have decreased levels of G,a 2 and/or Gia3 proteins and not of Giai or Go, because these proteins have been reported to be absent in platelets.45 That the levels of Gia2 are decreased in platelets is further substantiated by immunoblotting studies using AS/7 antibodies, which recognized only one protein, G,,2, and the levels of this protein were significantly attenuated in platelets but were not completely abolished. The decreased levels of Gia were also correlated with the decreased function, such as an attenuation of the inhibitory responses of Ang II and ANF on adenylyl cyclase and camp levels. However, it appears that a partial decrease in the levels of Gia2 observed in as compared with platelets may be enough to uncouple the receptors from adenylyl cyclase and thereby completely abolish the inhibitory responses of hormones on adenylyl cyclase, or alternatively, some other mechanisms at the receptor level, such as receptor downregulation, may also be responsible for a complete attenuation of inhibitory responses on adenylyl cyclase. In this context, the ANF receptor downregulation in various models of hypertension and congestive heart failure has been demonstrated in various tissues including platelets.4647 Taken together, it is suggested that the decreased levels of Gia2 in platelets may partly be responsible for the attenuated inhibition of adenylyl cyclase by Ang II and ANF in platelets. However, our results indicating decreased levels of Gia2 in platelets are in contrast to the studies reported earlier in tissues such as heart, aorta,21 and brain striatum,48 where the levels of Gia2 were significantly increased in and may suggest that the G proteins are differentially regulated in platelets as compared with the other tissues. This apparent discrepancy may be attributed to the tissue difference. A differential regulation of G proteins in lymphocytes and heart from congestive heart failure has also been reported; the levels of Gs were decreased in lymphocytes and were unaltered in heart.41,49 It is also possible that the augmentation of the stimulatory responses of PGE, and NECA on adenylyl cyclase and camp levels in platelets in the present studies may also be attributed to the decreased levels of Gi,2. In this regard, it has been demonstrated that PT and amiloride, which inactivate the G, protein, resulted in the augmentation of stimulatory responses of hormones on adenylyl cyclase Similarly, the enhanced formation of camp by FSK in as compared with platelets in the present studies may be due to the hypersensitivity of the catalytic subunit of the adenylyl cyclase system per se or to the decreased expression of Gia2 or both. The Gi-mediated regulation of FSKstimulated enzyme activity can be further supported by the results of various studies that have been performed involving the effect of PT treatment on adenylyl cyclase activity and that have resulted in an augmentation of the FSK-stimulated enzyme activity.33 Similarly, the overexpression of Gia has also been shown to result in the inhibition of the FSK-stimulated adenylyl cyclase activity.2' On the other hand, the requirement of G, and guanine nucleotides for the FSK activation of adenylyl cyclase has also been reported.5' Since the present studies do not demonstrate any alteration in GS, the Chem. 1988;263: increased sensitivity of adenylyl cyclase to FSK stimulation in hypertension cannot be attributed to the G, activity. Taken together, these data indicate that the decreased levels of G,a2 in hypertensive platelets may partly be responsible for the observed augmentation of the FSK-sensitive adenylyl cyclase activity. In conclusion, we have demonstrated that, unlike the response in heart and aorta, the G protein in platelets is differentially regulated; the expression of Gia2 is decreased in platelets from, whereas the levels of G,, were not altered. The decreased expression of G,,- appears to explain, in part, the attenuated responsiveness of adenylyl cyclase to inhibitory hormones and augmented responsiveness to stimulatory hormones and agents that activate adenylyl cyclase by receptor-independent mechanisms. It is suggested that the decreased expression of Gia-2 in platelets from may be one of the mechanisms responsible for the altered platelet aggregation in. Acknowledgments This study was supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada. Dr Anand-Srivastava is a recipient of the Medical Research Council Scientist Award from the Medical Research Council of Canada. The author would like to thank Sylvie Picard, Line Pilon, and Yuen Le Minh Quynh for their excellent technical assistance and Christiane Laurier for her valuable secretarial help. References 1. Amer MS. Cyclic nucleotides in disease, on biochemical etiology of hypertension. Life Sci. 1975;17: Ramanathan S, Shubata S, Tashaki TK, Ichord RN. Cyclic AMP metabolism in the cardiac tissue of the spontaneously hypertensive rat. Biochem Pharmacol. 1976;25: Anand-Srivastava MB. Altered responsiveness of adenylate cyclase to adenosine and other agents in the myocardial sarcolemma and aorta of spontaneously hypertensive rats. Biochem Pharmacol. 1988;37: Gilman AG. G-protein and dual control of adenylate cyclase. Cell. 1984;36: Gilman AG. G-proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56: Stryer L, Bourne HR. G-proteins: a family of signal transducers. Annu Rev Cell Biol. 1986;2: Sternweis PC, Northup JK, Smigel MD, Gilman AG. 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