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(Hypertension. 1996;28:83-90.)
© 1996 American Heart Association, Inc.

Articles

Aberrant Adenylyl Cyclase/cAMP Signal Transduction and G Protein Levels in Platelets From Hypertensive Patients Improve With Antihypertensive Drug Therapy

Josee Marcil; Ernesto L. Schiffrin; Madhu B. Anand-Srivastava

the Department of Physiology, Faculty of Medicine, Groupe de recherche sur le systeme nerveux autonome (J.M., M.B.A.-S.), and Clinical Research Institute of Montreal (E.L.S.), University of Montreal (Canada).

Correspondence to Dr Madhu B. Anand-Srivastava, Department of Physiology, Faculty of Medicine, University of Montreal CP 6128, Succursale A, Montreal, Quebec, Canada H3C 3J7.

	Abstract

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We have previously demonstrated a decreased expression of G_i2 protein in platelets from spontaneously hypertensive rats that was associated with an altered responsiveness of adenylyl cyclase to hormone stimulation and inhibition. In the present studies, we have used platelets from hypertensive patients and examined the hormonal regulation of adenylyl cyclase as well as the levels of G proteins and their modulation by antihypertensive drug therapy. We performed these studies in platelets from four groups of subjects: normotensive subjects (group 1), untreated mildly essential hypertensive patients (group 2), and treated moderately to severely hypertensive patients whose blood pressure was uncontrolled (group 3) or controlled with drug treatment (group 4). GTPS, 5'-(N-ethylcarboxamido)adenosine (NECA), and prostaglandin E₁ stimulated adenylyl cyclase activity to a greater extent in hypertensive patients (group 2). This effect was partially corrected (by approximately 50% to 80%) in the patients under antihypertensive drug therapy (groups 3 and 4). In addition, inhibition of adenylyl cyclase mediated by a ring-deleted analogue of atrial natriuretic factor (C-ANF_4-23) observed in control normotensive subjects was blunted in hypertensive patients (group 2) and was not corrected in treated patients. G_s levels determined by immunoblotting were in the same range for the four groups, whereas G_i2 and G_i3 levels were decreased by 70% and 60%, respectively, in hypertensive patients (group 2) compared with normotensive subjects. Antihypertensive drug therapy (groups 3 and 4) partially restored G_i2 levels toward normal (group 1) by about 60% and 70%, respectively; however, the reduced G_i3 levels in group 2 hypertensive patients were not improved in group 3 but were raised toward normal levels in group 4 by about 55%. These results suggest that the altered responsiveness of platelet adenylyl cyclase to hormones in hypertension and the normalization of the response with antihypertensive drug therapy could partly be due to the ability of the latter to modulate G_i protein expression. These effects on platelet function may underlie the beneficial effects of antihypertensive agents on some of the complications of hypertension.

Key Words: adenyl cyclase • G proteins • platelets • hypertension, essential • antihypertensive therapy

	Introduction

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The adenylyl cyclase-cAMP signal transduction system plays an important role in the regulation of a variety of physiological functions, such as cardiovascular function, including arterial tone and reactivity, and platelet function, including platelet aggregation, secretion, and clot formation. Several abnormalities in adenylyl cyclase activity and cAMP levels, which may be involved in blood pressure elevation, have been reported in cardiovascular tissues from genetic SHR and different models of experimentally induced hypertension in rats.^{1 2 3 4 5} Impaired platelet function—including abnormal, augmented, or decreased platelet aggregation in response to various stimuli, such as ADP, epinephrine, thrombin, prostaglandin, and the Ca²⁺ ionophore A23187—have been shown in hypertensive patients as well as in SHR.^{6 7 8 9} These defects may be attributed to altered adenylyl cyclase activity and cAMP levels,¹⁰ a signal transduction system that has been implicated in the regulation of platelet aggregation. Abnormal platelet function in hypertensive patients may participate in the progression of atherosclerosis and triggering of myocardial or cerebral ischemia complications, which are frequently present in hypertension.

The adenylyl cyclase system is composed of three components: an extracellular receptor, a catalytic subunit, and G proteins. The hormonal stimulation and inhibition of adenylyl cyclase are mediated by two G proteins: stimulatory (G_s) and inhibitory (G_i), respectively.¹¹ G proteins are heterotrimeric and composed of three distinct subunits: , ß, and .¹² The -subunit (G_s) binds and hydrolyzes GTP and confers specificity in receptor and effector interactions.¹³ Two different forms of the G_s protein with molecular sizes of 45 and 52 kD have been characterized¹⁴ ; however, a third form of the G_s protein with a molecular size of 47 kD has also been detected in the heart.¹⁵ These different forms of G_s arise from several species of mRNA that appear to be the products of alternative splicing of a common precursor.^{16 17} On the other hand, three distinct forms of G_i (G_i1, G_i2, and G_i3) have been identified and characterized and have been shown to be the products of different genes.^{18 19 20} All three forms of G_i have been shown to be implicated in adenylyl cyclase inhibition.²¹ Alterations in G_s or G_i protein expression have been demonstrated in various pathophysiological conditions, such as heart failure, diabetes, and hypothyroidism.^{22 23 24} We have recently shown an enhanced expression of G_i2 at protein and mRNA levels and its relation with adenylyl cyclase inhibition in heart and aorta from SHR and deoxycorticosterone acetate-salt hypertensive rats, in which the levels of G_s protein and G_s mRNA were not altered.^{4 5 25}

In addition, we have shown a differential regulation of G proteins in platelets from SHR¹⁰ compared with that in heart and aorta.⁴ Unlike heart and aorta, G_i2 levels were decreased in platelets without any change in G_s levels.¹⁰ This decrease in G_i2 was associated with decreases in G_i function, in which the inhibitory effects of ANF and angiotensin II on adenylyl cyclase and cAMP levels were completely attenuated and the stimulatory effects elicited by PGE₁ and NECA were augmented.¹⁰ We undertook the present studies to investigate (1) whether G protein levels and adenylyl cyclase activity from platelets of hypertensive patients are regulated in a way similar to the regulation in platelets from SHR, and (2) whether antihypertensive drug therapy is associated with a reversal of the alterations in G protein levels and adenylyl cyclase activity.

To our knowledge, this is the first study demonstrating that G_i2 and G_i3 levels, which are decreased in platelets from hypertensive patients, return toward normal during antihypertensive drug therapy. It suggests that one of the possible mechanisms by which antihypertensive drugs may affect blood pressure and platelet function, and through the latter may favorably affect some of the complications of hypertension that result in part from abnormal platelet function, could be due to an ability to modulate G_i protein expression.

	Methods

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ATP, cAMP, isoproterenol, oxotremorine, glucagon, and CT were purchased from Sigma Chemical Co. Creatine kinase (EC 2.7.3.2), creatine phosphate, myokinase (EC 2.7.4.3), GTPS, GTP, and adenosine deaminase (EC 3.5.4.4) were purchased from Boehringer-Mannheim. NECA was from Research Biochemicals. The peptide C-ANF_4-23 was from Peninsula Laboratories. [-³²P]ATP and the Western blotting detection kit were from Amersham. AS/7, EC/2, and RM/1 antibodies were from DuPont.

Patients and Protocol
The protocol was approved by the Ethics Committee of the Clinical Research Institute of Montreal. Written informed consent was obtained from each subject. Ambulatory patients with essential hypertension were recruited from the Hypertension Clinic of the Clinical Research Institute of Montreal and the Internal Medicine Clinic of Hotel-Dieu Hospital of Montreal. The criterion for acceptance into the study was that on more than two occasions patients had a systolic pressure greater than 150 mm Hg or a diastolic pressure greater than 90 mm Hg. The diagnosis of essential hypertension was established before the study by the absence of any clinical evidence of secondary hypertension; normal serum electrolytes, creatinine, and urinalysis; and normal abdominal echogram, intravenous pyelogram, and where indicated, renal arteriogram and computed abdominal tomography. Subjects were divided into four groups. Group 1 consisted of healthy normotensive volunteers who were not taking any drugs and who were asked to serve as controls. Subjects were included in the study if after a normal physical examination, they were shown to have a normal complete blood count and normal blood chemistries (usual automated laboratory techniques) and urinalysis. They were excluded if their systolic pressure was greater than 145 mm Hg or diastolic pressure was greater than 85 mm Hg. Group 2 were patients with mild hypertension who were studied before medication was begun if they had not been previously treated with antihypertensive agents. If they were receiving antihypertensive treatment, medication was discontinued at least 2 weeks before the study. If blood pressure increased to greater than 170 mm Hg systolic or 100 mm Hg diastolic after medication was withdrawn, treatment was begun again and the patient considered within the treated groups described below. Moderately and severely hypertensive patients excluded from group 2 because of increases in blood pressure were studied under antihypertensive treatment. Group 3 included patients who had blood pressure within the hypertensive range under treatment, who were in the process of increasing antihypertensive medication, or who were resistant to two or more drugs. Group 4 included those patients whose blood pressure was less than 150 mm Hg systolic and less than 90 mm Hg diastolic under treatment and whose hypertension was considered to be under control. Antihypertensive drugs that patients were taking included ß-blockers, diuretics, calcium channel antagonists, angiotensin-converting enzyme inhibitors, and -adrenergic antagonists. Patients were excluded if their fasting blood glucose level was not normal, if they had been previously diagnosed as having diabetes mellitus, or if they had a serum creatinine concentration greater than 150 µmol/L or a severe systemic disease.

On the day of the study, the subjects arrived between 7:30 and 9 AM, having fasted since the previous evening. Blood pressure (standard mercury sphygmomanometer) was measured after subjects had rested 15 minutes in the supine position. Diastolic pressure was read as phase V of the Korotkoff sounds. After 30 minutes, blood was withdrawn and platelets were isolated. Height without shoes and weight with light clothes were measured.

Fifty-four subjects were studied: 14 normotensive control subjects, 17 untreated mildly to moderately essential hypertensive patients, 11 treated hypertensive patients with controlled blood pressure, and 12 treated hypertensive patients with uncontrolled blood pressure. The patients were relatively young during the stable phase of hypertension. Patients in whom treatment could not be stopped were characterized clinically as being moderately to severely hypertensive, and mildly hypertensive patients were those who were newly diagnosed or were off drug treatment; this was ethically acceptable because they had mild hypertension. The duration of hypertension was 2 to 10 years.

Preparation of Platelet Membranes
Platelet membranes were prepared as described previously.^{10 26} Blood was drawn from each subject into polypropylene tubes containing 75 mmol/L trisodium citrate, 42 mmol/L citric acid, and 139 mmol/L dextrose, pH 6.8, as an anticoagulant (1 part to 6 parts blood). The whole blood was centrifuged at 350g for 10 minutes, and 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 nitrogen and stored at -80°C. Before the assay, the frozen platelet suspension was thawed and homogenized, and this freeze-thawing was repeated twice. This preparation was used for adenylyl cyclase determination and G protein levels. Ten milliliters of heparinized blood was used for analysis of serum biochemistry.

Adenylyl Cyclase Activity Determination
Adenylyl cyclase activity was determined by measurement of [³²P]cAMP formation from [-³²P]ATP, as described previously.^{4 10} Typical assay medium contained 50 mmol/L glycylglycine (pH 7.5), 0.5 mmol/L MgATP, 1x10⁶ to 1.5x10⁶ cpm [-³²P]ATP, 5 mmol/L MgCl₂, 0.5 mmol/L cAMP, 5 U/mL adenosine deaminase (or otherwise as indicated), 10 µmol/L GTP (or otherwise as indicated), 100 mmol/L NaCl, 1 mmol/L 3-isobutyl-1-methylxanthine (or otherwise as indicated), 0.1 mmol/L EGTA, and an ATP-regenerating system consisting of 2 mmol/L creatine phosphate, 0.1 mg/mL creatine kinase, and 0.1 mg/mL myokinase in a final volume of 200 µL. Incubations were initiated by addition to the membranes of 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 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 addition of 0.5 mL of 144 mmol/L Na₂CO₃ and subsequent chromatography by the double-column system as described by Salomon et al.²⁷ Recovery of [³²P]cAMP was monitored by unlabeled cAMP and measurement of absorbance at 259 nm. Under the assay conditions used, adenylyl cyclase activity was linear with respect to protein concentration and incubation time. Protein was determined essentially as described by Lowry et al,²⁸ with crystalline bovine serum albumin as standard.

CT Treatment of Platelet Membrane
Membranes were treated with CT as described earlier.¹⁰ CT (500 µg/mL) was preactivated for 20 minutes at 37°C in a mixture containing 20 mmol/L dithiothreitol, 1 µg/mL bovine serum albumin, and 25 mmol/L KH₂PO₄ (pH 8.0). For study of the effect of CT on adenylyl cyclase activity, platelet membranes were pretreated with and without CT for 30 minutes at 30°C in a reaction mixture containing 250 mmol/L KH₂PO₄ (pH 6.8), 1 mmol/L MgCl₂, 0.5 mmol/L EDTA (pH 8.0), 5 mmol/L ATP, 15 mmol/L thymidine, 0.15 mmol/L GTP, 20 mmol/L dithiothreitol, and 1 mmol/L NAD. 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were electrophoretically transferred to nitrocellulose paper (Schleicher & Schuell) with a semidry transblot apparatus (Bio-Rad) at 15 V for 45 minutes as described previously.^{4 10} After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and incubated in PBS containing 3% dehydrated milk at room temperature for 2 hours. The blots were then incubated with antisera against G proteins in PBS containing 8% dehydrated milk and 0.1% Tween 20 at room temperature for 2 hours. The antigen-antibody complexes were detected by incubation of 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 enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham).

Autoradiograms and immunoblots were quantified by densitometric scanning with an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was one dimensional and scanned the entire area of protein bands in autoradiograms and immunoblots.

Data Analysis
Results are expressed as mean±SE. Comparisons between groups were made with either Student's t test or ANOVA where appropriate. Results were considered significant at a value of P<.05.

	Results

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Patient Characteristics
The demographics of the control subjects and hypertensive patients studied are presented in Table 1. The untreated (group 2) and treated but uncontrolled (group 3) hypertensive patients had significantly higher blood pressures compared with the normotensive subjects (group 1) and patients whose blood pressure was under control with drug treatment (group 4). Serum biochemistry, including plasma ANP levels (data not shown), did not differ significantly among the groups. No effort was made to further categorize patients into potential physiopathologically distinct subsets.

View this table: [in this window] [in a new window]   Table 1. Demographics of Study Subjects

GTPS-Mediated Stimulation of Adenylyl Cyclase
To investigate whether an impaired response of guanine nucleotides to adenylyl cyclase exists in platelets from hypertensive patients, we studied the effect of GTPS on adenylyl cyclase activity in platelets from the four groups. GTPS stimulated adenylyl cyclase activity in a concentration-dependent manner in all four groups (Fig 1); however, responses were significantly enhanced in hypertensive patients (group 2) compared with normotensive subjects (group 1). GTPS at 10 µmol/L stimulated adenylyl cyclase activity by about fivefold in platelets from normotensive subjects and about eightfold in platelets from hypertensive patients. In addition, platelets from hypertensive patients treated with antihypertensive drugs (groups 3 and 4) showed a significantly decreased stimulation of adenylyl cyclase by GTPS compared with platelets from hypertensive patients (group 2). At 10 µmol/L GTPS, the augmented stimulation of adenylyl cyclase returned toward normal by about 70% in groups 3 and 4.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of GTPS on adenylyl cyclase activity in human platelets. Group 1 is normotensive subjects; group 2, mildly essential hypertensive patients; group 3, moderately to severely hypertensive patients with uncontrolled blood pressure under drug therapy; and group 4, moderately to severely hypertensive patients with controlled blood pressure under drug therapy. Adenylyl cyclase activity was determined in the absence or presence of various concentrations of GTPS as described in "Methods." Adenosine deaminase was omitted from the assay medium. Values are mean±SE of seven patients. *P<.05 compared with group 1; P<.05 compared with group 2. Basal enzyme activities (pmol cAMP/mg protein per 10 minutes) in the absence of GTPS (10 µmol/L) were 79.5±16.4, 60.1±4.6, 9.9±13.9, and 85.7±13.4 in groups 1 through 4, respectively.

Hormonal Stimulation of Adenylyl Cyclase Activity
To examine whether G_s-mediated hormonal responsiveness was augmented in platelets from hypertensive patients, we studied the effect of PGE₁ and NECA, which inhibit platelet aggregation, on adenylyl cyclase activity. NECA and PGE₁ stimulated adenylyl cyclase activity to different degrees in group 1 (Fig 2). The extent of stimulation was higher in hypertensive patients (group 2) compared with normotensive subjects (group 1). NECA and PGE₁ stimulated adenylyl cyclase activity by about 2.5- and 15-fold, respectively, in normotensive subjects and by about 4.5- and 23-fold in hypertensive patients. In platelets from treated patients (groups 3 and 4), the augmented responsiveness of adenylyl cyclase to NECA was restored partially toward that of normotensive subjects by about 50% and 55%, whereas the hyperresponsiveness of adenylyl cyclase to PGE₁ was not restored in group 3 and was normalized in group 4.

View larger version (41K): [in this window] [in a new window]   Figure 2. Effect of stimulatory hormones on adenylyl cyclase activity in human platelets from subjects from the four groups described in Fig 1 legend. Adenylyl cyclase activity was determined as described in "Methods" in the presence of 5 U/mL adenosine deaminase and 10 µmol/L GTP alone (basal), taken as 100%, and in combination with 10 µmol/L NECA or 1 µmol/L PGE1. Basal enzyme activities (pmol cAMP/mg protein per 10 minutes) in the presence of GTP were 43.2±16.1, 40.2±8.8, 67.0±17.1, and 48.9±15.9 in groups 1 through 4, respectively. In these studies, isobutylmethylxanthine was replaced by Ro 20-1724. Values are mean±SE of six patients in each group. *P<.05 compared with group 1; P<.05, P<.01 compared with group 2; P<.01 compared with group 3.

G Protein Levels
To examine whether the changes found were the consequence of increased G_s levels, we performed immunoblotting experiments with specific antibodies against G_s. Fig 3A shows that RM/1 antibodies recognized a single 45-kD protein in platelets from normotensive subjects and hypertensive patients. The relative amount of immunodetectable G_s did not differ significantly in these two groups (Table 2). Also, G_s protein levels were unaltered in groups 3 and 4, as shown in Fig 3A and Table 2.

View larger version (47K): [in this window] [in a new window]   Figure 3. A, Quantification of Gs protein by immunoblotting in human platelet membranes from subjects from the four groups described in Fig 1 legend. Membrane proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, which was then immunoblotted with RM/1 antibody as desribed in "Methods." The autoradiogram is representative of eight separate experiments. Detection of the Gs protein -subunit (Gs45 in the figure) was performed with chemiluminescence Western blotting detection reagents. B and C, Quantification of Gi2 and Gi3 by immunoblotting in human platelet membranes from subjects from the four study groups. Membrane proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, which was immunoblotted with antibody AS/7 for Gi1 and Gi2 (B) or EC/2 antibody for Gi3 (C) as described in "Methods." The autoradiogram is representative of eight separate experiments. Detection of the Gi protein -subunit was performed with chemiluminescence Western blotting detection reagents.

View this table: [in this window] [in a new window]   Table 2. Quantitative Analysis of G Protein Levels in Platelets

To corroborate these results with G_s function, we analyzed the effect of CT treatment on GTP-sensitive adenylyl cyclase. CT treatment augmented the GTP-sensitive adenylyl cyclase activity in platelets from the four groups. The extent of stimulation was not similar in the four groups (group 1: basal+GTP [10 µmol/L], 133.7±17.3 pmol cAMP per milligram protein per 10 minutes; CT, 937.2±5.5; group 2: basal+GTP, 124.0±0.5; CT, 888.9±7.7; group 3: basal+GTP, 163.5±34.8; CT, 1103.5±41.9; group 4: basal+GTP, 118.3±36.4; CT, 848.2±16.1).

Thus, G_s levels may not be responsible for the hyperresponsiveness of adenylyl cyclase to GTPS and stimulatory hormones as well as for the recovery of response found in patients under antihypertensive drug treatment. Since modulation of G_s functions by G_i has been reported,²² we examined G_i protein levels by an immunoblotting technique using specific antibodies. The results shown in Fig 3B illustrate that AS/7 antibodies, which react with both G_i1 and G_i2 proteins,²⁹ recognized a single protein of approximately 40 kD, referred to as G_i2 (G_i1 is absent from platelets) and EC/2 antibodies specific for G_i3 detected a single protein of 39/40 kD in platelets from the four groups. The relative amounts of immunodetectable G_i2 (Fig 3B) and G_i3 (Fig 3C) were significantly decreased in hypertensive patients by 70% and 60%, respectively (Table 2), compared with normotensive subjects (group 1). On the other hand, G_i2 levels returned toward normal by about 60% and 70% in groups 3 and 4, respectively (group 1 taken as 100%). G_i3 levels were not restored in group 3 but were restored by about 55% in group 4 compared with normotensive subjects (group 1 taken as 100%).

Hormonal Inhibition of Adenylyl Cyclase
Since G_i2 and G_i3 proteins are implicated in the hormonal inhibition of adenylyl cyclase,²¹ we investigated the inhibitory effects of hormones on adenylyl cyclase mediated through the G_i regulatory protein^{26 30} . C-ANF_4-23, which inhibits adenylyl cyclase activity in rat platelets,²⁶ inhibited adenylyl cyclase activity by about 50% in platelets from normotensive subjects (group 1) (Fig 4). This inhibition was almost abolished in hypertensive patients (group 2). Antihypertensive drug treatment (groups 3 and 4) was ineffective in eliciting any alterations in the responsiveness of adenylyl cyclase to C-ANF_4-23 inhibition compared with untreated hypertension. Fig 5 shows the effect of various concentrations of C-ANF_4-23 on adenylyl cyclase activity in platelets from the four study groups. C-ANF_4-23 inhibited adenylyl cyclase activity in a concentration-dependent manner in the normotensive group, with an apparent K_i of about 10^-8 mol/L. The maximal inhibition observed was approximately 50%. In platelets from hypertensive patients, the degree of C-ANF_4-23-mediated inhibition was significantly attenuated. The inhibitory effect of C-ANF_4-23 on adenylyl cyclase remained blunted in groups 3 and 4.

View larger version (73K): [in this window] [in a new window]   Figure 4. Effect of C-ANF4-23 on adenylyl cyclase activity in human platelets from subjects from the four groups described in Fig 1 legend. Adenylyl cyclase activity was determined as described in "Methods" in the presence of 10 µmol/L GTPS alone (basal), taken as 100%, and in the presence of 10-7 mol/L C-ANF4-23. Basal enzyme activities (pmol cAMP/mg protein per 10 minutes) in the presence of 10 µmol/L GTPS alone were 277.4±36.9, 238.1±48.0, 271.3±47.4, and 351.9±43.6 in groups 1 through 4, respectively. Values are mean±SE of five patients in each group. *P<.05 vs group 1.

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of various concentrations of C-ANF4-23 on adenylyl cyclase activity in human platelets from subjects from the four groups described in Fig 1 legend. Adenylyl cyclase activity was determined as described in "Methods" in the presence of 5 U/mL adenosine deaminase and 10 µmol/L GTPS alone (basal), taken as 100%, and in the presence of various concentrations of C-ANF4-23. Basal enzyme activities (pmol cAMP/mg protein per 10 minutes) in the presence of 10 µmol/L GTPS were 104.07±29.5, 124.9±40.2, 147.8±25.5, and 175.4±28.6 in groups 1 through 4, respectively. Values are mean±SE of five patients in each group. *P<.05 vs group 1.

Forskolin-Stimulated Adenylyl Cyclase Activity
Forskolin, which interacts with the catalytic subunit of adenylyl cyclase, stimulated the enzyme activity in normotensive subjects and hypertensive patients by about 150- and 130-fold respectively, and antihypertensive drug therapy was not associated with any significant alteration in this response in groups 3 and 4 (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present studies, we demonstrate for the first time that adenylyl cyclase activity and its responsiveness to various hormones, as well as G_i protein levels but not G_s protein levels, are altered in platelets from hypertensive patients and that these alterations were restored toward normal during antihypertensive drug therapy.

Our results on unaltered expression of G_s and decreased expression of G_i2 and G_i3 levels in platelets from hypertensive patients are in agreement with studies reported in platelets from SHR.¹⁰ Effective antihypertensive drug therapy was associated with a significant increase in both G_i2 and G_i3 levels in hypertensive patients whose blood pressure was under control with drug treatment (group 4), whereas in treated hypertensive patients who had uncontrolled hypertension (group 3), the G_i2 levels but not G_i3 levels were normalized. Antihypertensive drugs have previously been reported to suppress abnormal platelet aggregation.^{31 32} Taken together, these data could suggest that G_i2 and G_i3 proteins may be important in signal transduction mechanisms involved not only in the regulation of blood pressure but also in platelet function. Since platelet dysfunction may contribute to the severity of hypertensive complications, it is important to elucidate the mechanism by which antihypertensive agents may affect platelet function.

A significant increase in the responsiveness of adenylyl cyclase to GTPS stimulation in platelets from hypertensive patients compared with those from normotensive subjects suggests that G_s protein may be hypersensitive or that G_s protein levels may be increased in platelets from hypertensive patients. However, G_s protein levels were unaltered in hypertensive patients, which suggests that G_s may not be responsible for the increased sensitivity of adenylyl cyclase to GTPS stimulation. In addition, the stimulation of GTP-sensitive adenylyl cyclase to a similar extent by CT treatment in platelets from normotensive subjects and hypertensive patients substantiated the notion that the function of G_s may not be altered in hypertensive patients. These results are in agreement with our previous studies performed on platelets from SHR¹⁰ and suggest a potential similarity at this level between this genetic model of hypertension and human hypertension. Furthermore, the improvement of the enhanced sensitivity of adenylyl cyclase to GTPS stimulation found in patients treated with antihypertensive drugs without any changes in G_s protein levels and function supports the hypothesis that G_s is not responsible for the observed augmentation of the stimulatory responses to GTPS of adenylyl cyclase in hypertensive patients. Since G_i2 and G_i3 levels were significantly decreased in platelets from hypertensive patients, this could be the mechanism underlying the enhanced stimulation of adenylyl cyclase by GTPS in hypertensive patients. The results obtained in patients receiving antihypertensive drug therapy, in whom the augmented response to GTPS of adenylyl cyclase was corrected to the same extent in both groups 3 and 4 and in whom the levels of either G_i2 and G_i3 (group 4) or only of G_i2 (group 3) were normalized toward normal values, suggest that G_i2 alone may be responsible for the improvement of GTPS-mediated stimulation of adenylyl cyclase.

Hyperresponsiveness of adenylyl cyclase of platelets from hypertensive patients to PGE₁ and NECA stimulation is in agreement with studies performed with platelets and splenocytes from SHR^{10 33} and may be attributed to upregulation of hormone receptors or increased levels of G_s. However, several studies have shown downregulation or no change in the number of stimulatory hormone receptors in various cardiovascular diseases, including hypertension and congestive heart failure.^{22 34} Since no alteration in G_s levels as well as G_s function in hypertensive patients was observed, the enhanced stimulation of adenylyl cyclase by NECA and PGE₁ cannot be explained by G_s. This is further substantiated by the results in patients treated with antihypertensive drugs, in whom NECA- and PGE₁-stimulated adenylyl cyclase activity was restored almost to control normotensive values in the absence of a change in G_s protein levels. Thus, antihypertensive drug-mediated modulation of hormonal responses may occur at the level of receptors, G_i protein, or both. Since G_i2 and G_i3 levels were significantly decreased in hypertensive patients compared with normotensive subjects, the augmented responsiveness of adenylyl cyclase to NECA and PGE₁ in hypertensive patients can be attributed to decreased levels of G_i2 proteins, G_i3 proteins, or both. This notion is supported by the partial correction of the responsiveness of adenylyl cyclase to NECA stimulation together with restoration of G_i2 levels (group 3) and complete restoration of PGE₁ stimulation together with the recovery of G_i3 levels alone or of a combination of G_i2 and G_i3 (group 4) during antihypertensive drug therapy.

Attenuated inhibition of adenylyl cyclase by C-ANF_4-23 in platelets from hypertensive patients may be due to decreased G_i2 and G_i3 levels as well as to downregulation of ANF receptors. Since normalization of G_i2 and G_i3 levels by antihypertensive drugs was unable to correct the C-ANF_4-23-mediated inhibition of adenylyl cyclase, this suggests that the decreased levels of G_i2, G_i3, or both may not be the only factor. Downregulation of ANF receptors in various models of hypertension and congestive heart failure has been demonstrated in various tissues, including platelets,^{35 36} which may also contribute to the blunted inhibition of adenylyl cyclase by C-ANF_4-23. In addition, attenuation of C-ANF_4-23-mediated inhibition of adenylyl cyclase in platelets from SHR has also been reported.¹⁰

Normal sensitivity of adenylyl cyclase to forskolin stimulation in platelets from hypertensive patients untreated and treated with antihypertensive drugs suggests that the catalytic subunit of adenylyl cyclase is not altered in platelets in essential hypertension. This is in contrast to earlier observations showing an enhanced ability of forskolin to increase cAMP levels in platelets from SHR compared with those from Wistar-Kyoto rats.¹⁰ This apparent discrepancy may be due to species differences. Increased G_i2 levels may decrease forskolin-mediated stimulation of adenylyl cyclase,⁴ whereas decreased G_i2 levels resulted in augmentation of forskolin-mediated stimulation of adenylyl cyclase and cAMP levels.¹⁰ However, the present results do not show any involvement of G_i proteins in forskolin-mediated adenylyl cyclase activation in human platelets, which may be due to the possibility that the isoform of adenylyl cyclase present in platelets may not be modulated by G_i proteins.

Some important points remain to be emphasized. Since platelet activation occurs in hypertensive patients, it is possible that the functional and molecular differences demonstrated in the present study reflect different turnover rates of platelets and thus the different mean ages of platelets in the different groups of subjects. Correction of platelet function during antihypertensive treatment, which has already been demonstrated for other platelet functional parameters,³⁷ may result in a decrease in the potential for platelet-vascular wall interactions that may contribute to vasoconstriction, endothelial and vascular damage, progression of atherosclerosis, and thrombotic events, particularly in the cerebral and coronary circulations, with normalized turnover. As a result, the molecular and functional differences may disappear. Also, it is important to emphasize that the molecular and functional changes reported in the present study in platelets need not occur also in smooth muscle cells in the vascular wall, since platelets may have a completely different behavior than smooth muscle cells. These findings help us understand platelet mechanisms that may contribute to vascular complications of hypertension resulting from platelet activation rather than mechanisms potentially occurring in the vascular wall.

In conclusion, we have shown that the levels of G_i2 and G_i3 but not G_s protein are decreased in platelets from hypertensive patients, which may partly explain the attenuated responsiveness of adenylyl cyclase to C-ANF_4-23 and augmented responsiveness to NECA and PGE₁. Decreased expression of G_i protein may be a possible mechanism for altered platelet aggregation in hypertension. The partial correction of G_i2 and G_i3 levels by antihypertensive drug therapy suggests that a potential mechanism whereby antihypertensive drugs may contribute to blood pressure regulation and platelet function could be attributed to their ability to modulate G_i2 and G_i3 protein expression. Altered platelet activation in hypertension may participate in complications of hypertension, such as atherosclerosis and myocardial ischemia. Normalization of platelet function by antihypertensive therapy may be a mechanism by which antihypertensive drugs indirectly contribute to reduce the progression of atherosclerosis and decrease the incidence or severity of myocardial ischemia. Whether the observed normalization of G_i proteins and adenylyl cyclase activity by antihypertensive therapy in the present study is the result of specific effects of some individual agents—diuretics, ß-blockers, angiotensin-converting enzyme inhibitors, calcium channel blockers, or -adrenergic antagonists—is not known and needs to be further investigated.

	Selected Abbreviations and Acronyms

 

C-ANF4-23 = ring-deleted analogue of atrial natriuretic factor CT = cholera toxin NECA = 5'-(N-ethylcarboxamido)adenosine PGE1 = prostaglandin E1 SHR = spontaneously hypertensive rat(s)

	Acknowledgments

 
This work was supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada. M.B.A.-S. is a recipient of the Medical Research Council Scientist Award from the Medical Research Council of Canada. We would like to thank Christiane Laurier for her valuable secretarial help.

Received June 16, 1995; first decision August 30, 1995; first decision March 11, 1996;

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