Effects of Renin-Angiotensin Blockade on Sympathetic Reactivity and β-Adrenergic Pathway in the Spontaneously Hypertensive Rat
Abstract As interactions between the renin-angiotensin and sympathetic nervous systems have been suggested in the pathogenesis of hypertension, we wanted to investigate the effect of chronic renin-angiotensin blockade with losartan and enalaprilat on the sympathetic reactivity to hypotension and on the cardiac β-adrenergic–coupled adenylyl cyclase pathway in 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Both treatments, exerting equipotent shifts of angiotensin-pressure responses, lowered blood pressure and attenuated cardiac hypertrophy similarly in SHR. The nitroprusside-induced hypotension was similar in both strains, but the associated increases in plasma catecholamines and heart rate were higher in SHR. In SHR treated with losartan and enalaprilat, the nitroprusside-induced hypotension was greater and associated with markedly attenuated increases in norepinephrine and heart rate. The binding affinity of cardiac β-adrenoceptors was significantly lower, and β2-adrenoceptor subtype was dominant in untreated SHR in contrast to WKY, in which β1-adrenoceptor subtype was dominant. Enalaprilat treatment increased total β-adrenoceptor density, whereas both treatments restored the binding affinity and β1- and β2-adrenoceptor proportions to normal in SHR. Isoproterenol-, guanylylimidodiphosphate [Gpp(NH)p]–, and forskolin-stimulated adenylyl cyclase reactivity was increased in SHR. Enalaprilat restored adenylyl cyclase reactivity to normal in SHR and reduced the sensitivity (EC50) of Gpp(NH)p-induced cAMP formation in both strains. The present study supports the possibility that functional alterations of the renin-angiotensin and sympathetic systems are involved in hypertension in SHR. The antihypertensive action of losartan and enalaprilat in SHR may be partly mediated through the normalization of sympathetic hyperreactivity and the restoration of β-adrenergic signaling pathway sensitivity.
- hypertension, genetic
- autonomic nervous system
- renin-angiotensin system
- receptors, adrenergic, beta
- adenylyl cyclase
The RAS plays an important role in maintaining cardiovascular homeostasis. The RAS was originally thought to be a circulating endocrine system, but it is now known that components of the RAS are also present in local tissues, such as blood vessel walls, the heart, the kidney, the adrenal, the brain, and other tissues, and exert autocrine and paracrine influences on local tissue functions.1 2 The local RASs may be involved in the pathophysiology of hypertension. The inhibition of local angiotensin production may explain the observation that the long-term BP-lowering effect of ACE inhibitors is often unrelated to pretreatment plasma renin levels.3 There are numerous interactions between the RAS and other BP control systems, for instance, the sympathetic nervous system. Ang II has been shown to facilitate neurotransmission at the nerve endings, ganglia, adrenal medulla, and central nervous system. More specifically, Ang II has been suggested to decrease prejunctional reuptake, increase catecholamine biosynthesis, facilitate catecholamine release through AT1 presynaptic mechanisms, and sensitize postjunctional structures.4 5
Orally active AT1 receptor antagonists are a new class of drugs currently being used in the treatment of hypertension. Losartan, by interfering with the RAS via AT1 receptor blockade, could provide some advantages over ACE inhibitors in blocking specifically AT1-mediated effects of circulating and tissue Ang II. The differences between these two therapeutic approaches could be accounted for by the differences in neurohormonal activation, in bradykinin potentiation, and in the inhibition of the action of Ang II derived from the classic pathway and also from alternative synthesis pathways.6
SHR have been widely used as a model for the study and development of antihypertensive drugs. The etiology of hypertension in SHR is not fully elucidated, but treatment with ACE inhibitors has been shown to lower BP effectively. Since both the RAS and sympathetic nervous system have been implicated in the development and maintenance of hypertension, the objectives of our study were to evaluate the effect of chronic RAS blockade on BP and sympathetic reflex reactivity. We also investigated the effect of chronic RAS blockade on cardiac postsynaptic functions such as the characteristics of β-adrenergic receptors (total number and affinity, subtype distribution, and guanine nucleotide–sensitive functional coupling) as well as the basal and induced formation of cAMP by the β-adrenergic receptors, guanine nucleotide regulatory (G) proteins, and the catalytic subunit adenylyl cyclase. To determine whether losartan and enalaprilat (an ACE inhibitor) possess different mechanisms of action, we compared their effects on sympathetic functions using equipotent doses of both drugs in displacing angiotensin-pressure response curves.
Experiments were carried out in accordance with institutional animal ethics committee guidelines. Male WKY and SHR were used at 10 weeks of age (Charles River Laboratories, St-Constant, Québec, Canada). Rats were anesthetized with sodium pentobarbital (50 mg/kg IP, Somnotol, MTC Pharmaceuticals), and osmotic pumps (model 2002, Alza Corp) were implanted subcutaneously in the flank region for 12 days. WKY and SHR control groups underwent the same surgical procedure without implantation of an osmotic pump. Twenty-four hours before implantation, osmotic pumps were filled with losartan (dissolved in isotonic saline) to provide an infusion rate of 5 mg/kg per day or with enalaprilat (dissolved in saline [0.3% Na2CO3]) to provide an infusion rate of 1 mg/kg per day. At the end of the treatments, when the animals were 12 weeks old, systolic arterial BP was measured in conscious rats by tail-cuff plethysmography (Harvard Apparatus Ltd) and was recorded on a MacLab/8 system (AD Instruments Pty Ltd). Six to seven BP readings were obtained for each rat and averaged.
In Vivo MAP Response Curves to Ang I and Ang II
Animals assigned to in vivo experiments in conscious, unrestrained, resting conditions were catheterized. After 11 days of their respective treatments, the rats were anesthetized, and polyethylene tubings (PE-10) welded to PE-50 catheters were inserted into the left femoral artery and vein and positioned in the abdominal aorta and vena cava. Both catheters were tunneled subcutaneously and extruded at the back of the neck through L-shaped PE-280 tubing and were protected from the rat by insertion into a stainless steel tether. The catheters were filled with heparinized saline, and the rats were placed in individual cages, where they were allowed to recover for at least 24 hours. The arterial catheter was coupled to a pressure transducer (Statham P23ID, Gould Statham Inc), and the signal was amplified and recorded by a Biopac data-acquisition system (MP100WS, Harvard Apparatus Canada). MAP and heart rate were monitored with a computerized analysis program (Acknowledge 3.0, Harvard Apparatus). When MAP and heart rate had reached stable basal levels, after at least 20 minutes of continuous recording, basal values were measured and 0.4 mL arterial blood was withdrawn for measurement of basal aortic plasma catecholamine levels. The volume withdrawn was replaced with heparinized saline. Ang I and Ang II dose-MAP response curves were constructed. Bolus injections of increasing and noncumulative doses of Ang I and Ang II (0.003 to 1.0 μg/kg) were administered at 5-minute intervals through the venous catheter. A blood sample (0.4 mL) was withdrawn from the arterial catheter for measurement of plasma levels of losartan and its metabolite EXP 3174 (see below). From each logarithmic dose-response curve, the ED50 was calculated as the dose resulting in 50% of the maximal response.
Reflex Activation of the Sympathetic Nervous System
In other groups of rats different from those in the above-described protocol, basal MAP and heart rate were recorded, and a blood sample of 0.4 mL was withdrawn after a stabilization period. A 5-minute intravenous infusion of sodium nitroprusside (50 μg/kg per minute) was administered at 0.05 mL/min. Hemodynamic parameters and plasma catecholamine levels were measured at the end of the 5-minute infusion period.
Measurement of Plasma and Tissue Catecholamine Levels
Aortic blood samples (0.4 mL) were placed in ice-chilled tubes containing a preservative solution (0.25 mol/L EGTA, 0.2 mol/L reduced glutathione, pH 7). The samples were rapidly centrifuged at 14 000g for 5 minutes at 4°C. The plasma was frozen at −80°C until the assay. Parts of the left and right ventricles were rapidly dissected and weighed and kept at −80°C until the assay. Plasma and tissue catecholamine concentrations were measured by radioenzymatic assay.7
For in vitro experiments, the rats were weighed and killed by decapitation. The hearts were rapidly removed and tissues isolated from the left and right ventricular free walls. Cleaned and minced myocardium was homogenized in ice-cold buffer (mmol/L: sucrose 250, Tris 5, MgCl2 1, EDTA 1, dithiothreitol 1, phenylmethylsulfonyl fluoride [PMSF] 0.01, pH 7.4) with a Polytron homogenizer (Brinkmann) at a setting of 9, two times for 10 seconds and once for 5 seconds. The homogenate was filtered through three layers of cheesecloth and centrifuged at 1000g for 10 minutes at 4°C. The supernatant filtered through two layers of cheesecloth was centrifuged at 45 000g for 30 minutes at 4°C to yield membranes. The pellet was washed twice in a second ice-cold buffer (incubation buffer, mmol/L: Tris 50, MgCl2 10, EDTA 1, dithiothreitol 1, PMSF 0.01, pH 7.4). The final plasma membrane pellet was resuspended to give a final concentration of 1 mg/mL protein. The protein concentration was determined by the method of Lowry et al8 using bovine albumin serum as the standard.
Characteristics of β-Adrenergic Receptors
ICYP binding to β-adrenergic receptors was studied in purified cardiac membrane preparations. For saturation binding experiments, membrane preparations (approximately 40 μg protein) were incubated with ICYP (10 to 250 pmol/L) in incubation buffer either alone or with alprenolol (10 μmol/L), which was used for determination of nonspecific binding. The incubation was carried out at room temperature (25°C) for 2 hours in a total volume of 250 μL. The reaction was terminated by the addition of ice-cold incubation buffer and rapid vacuum filtration through type A/C glass fiber filters (Gelman Sciences). The density (Bmax) and affinity (Kd) of binding sites for ICYP were determined by linear regression analysis of saturation isotherm data, linearly transformed according to the method of Scatchard.9 The proportions of β1- and β2-adrenoceptors subtypes were assessed from displacement curves of ICYP binding (50 pmol/L) by ICI 118,551 (a β2-selective antagonist, 10 pmol/L to 200 μmol/L). Competition experiments of ICYP binding with increasing concentrations of isoproterenol (500 pmol/L to 1 mmol/L) in the absence and presence of Gpp(NH)p (50 μmol/L) were performed to determine the proportions of the high- and low-affinity states of the receptor for the agonist as an index of receptor–G protein coupling, since the guanine nucleotide–sensitive high-affinity state of the receptors is believed to represent the complex agonist–β-adrenoceptor–Gs. All experiments were performed in duplicate. The binding data of the displacement curves for each animal, expressed in picomoles per liter, were analyzed with an iterative nonlinear regression program (Allfit).10
Adenylyl Cyclase Activity
Adenylyl cyclase activity and sensitivity in cardiac membrane preparations were assessed by the method of Salomon.11 Basal adenylyl cyclase activity and concentration-response curves to isoproterenol, Gpp(NH)p, and forskolin (1 nmol/L to 1 mmol/L) as well as cAMP formation in the presence of NaF (10 mmol/L) were determined. Membrane samples containing 4 μg protein were incubated 15 minutes at 37°C in (mmol/L) GTP 0.06, ATP 0.1, cAMP 1, 3-isobutyl-1-methylxanthine 0.1, and phospho(enol)pyruvate 2.8 as well as 2.5 U/mL pyruvate kinase and 19 U/mL myokinase with 10 μCi/mL of [α-32P]ATP in a total volume of 50 μL. All assays were performed in duplicate. The reactions were stopped by the addition of 1 mL ice-cold solution consisting of 50 μCi/L [3H]cAMP, 0.35 ATP, and 0.25 mmol/L cAMP. Reaction products were separated by sequential column chromatography on Dowex and alumina columns (Bio-Rad Laboratories). The [32P]cAMP counts were corrected for column recovery of [3H]cAMP (approximately 85%).
Measurement of Plasma Concentrations of Losartan and EXP 3174
Aortic blood samples placed in ice-chilled tubes were rapidly centrifuged at 14 000g and 4°C for 5 minutes. The plasma was frozen at −80°C until the assay. Plasma concentrations of losartan and its metabolite EXP 3174 were determined in the laboratory of Dr Chantal Lambert (Department of Pharmacology, Université de Montréal) by high-performance liquid chromatography as described by Furtek and Lo12 using the internal standard L-158,854.
Solutions and Chemicals
All chemicals were supplied by Sigma Chemical Co, except for pyruvate kinase (Calbiochem Co) and sodium nitroprusside (Hoffmann–La Roche Ltd). Radioisotopes ICYP and 5′,8-[3H]cAMP were supplied by DuPont-NEN. [α-32P]ATP was supplied by ICN Biomedicals. Losartan and enalaprilat were kindly provided by DuPont Merck Pharmaceutical Co and Merck Frosst Canada Inc. The internal standard L-158,854 was kindly provided by Dr Man-Wai Lo of Merck Sharpe & Dohme Research Laboratories.
Data are given as mean±SEM; n is the number of rats used. Maximal responses, ED50, EC50, Vmax, Bmax, Kd, and Ki values were determined for each concentration-response curve. These parameters were used for statistical comparison by ANOVA followed by Scheffé’s F test or Student’s unpaired t test when appropriate. A value of P<.05 was considered statistically significant. All statistical tests were performed with StatView and SuperAnova statistical packages (Abacus Concepts Inc).
BP, Myocardial Hypertrophy, and Catecholamine Levels
As shown in Table 1⇓, systolic arterial BP measured by tail-cuff plethysmography was significantly increased in 12-week-old SHR compared with age-matched WKY. Chronic 12-day treatment with losartan or enalaprilat reduced significantly and similarly BP in SHR, whereas these treatments had no significant effect on BP in WKY. The hearts of SHR demonstrated left ventricular hypertrophy, and the antihypertensive effect of losartan and enalaprilat was accompanied by the normalization of left ventricular relative weight in SHR to values similar to those in control WKY (Table 1⇓). Neither losartan nor enalaprilat treatment had a significant effect on body weight in the different groups (Table 1⇓). Plasma epinephrine levels appeared to be higher in untreated SHR, and both antihypertensive treatments tended to increase plasma epinephrine levels in WKY and SHR, but those differences were not significant. Plasma norepinephrine concentrations were similar in control and treated WKY and SHR (Table 1⇓). Cardiac tissue contents of norepinephrine and epinephrine were similar in untreated WKY and SHR. Treatment with losartan in SHR increased left ventricular epinephrine (P<.05) and norepinephrine concentrations (P=.05), whereas enalaprilat had no effects on tissue catecholamine levels (Table 1⇓).
Ang I– and Ang II–MAP Response Curves
The dose-MAP curves constructed with Ang I were similar in untreated WKY and SHR and were similarly shifted to the right by chronic treatment with losartan or enalaprilat (Fig 1⇓). In fact, both treatments caused similar ED50 increases in WKY and SHR (P<.05 versus respective controls, Table 2⇓). The maximal increases in MAP after Ang I challenge were not significantly altered by either treatment in both strains. The Ang II dose–MAP response curves were similar in untreated WKY and SHR, and chronic treatments with losartan caused a similar rightward shift in both strains (Fig 1⇓). The ED50 values in untreated WKY and SHR were similarly increased after losartan treatment (P<.05 versus respective controls, Table 2⇓). Losartan did not affect the maxima of the dose-pressure curves to Ang II in both strains (Table 2⇓). As expected, chronic treatment with enalaprilat had no effect on the MAP response curves to Ang II in WKY and SHR (Fig 1⇓). The plasma levels of losartan and its major metabolite EXP 3174 measured after chronic treatment in both strains revealed no significant differences in treated WKY and SHR (Table 2⇓).
Sympathetic Reflex Activation
Baseline MAP and heart rate are shown in Table 3⇓ and basal aortic plasma norepinephrine and epinephrine levels in Table 1⇑. Direct MAP measurements in conscious rats confirmed the higher BP in SHR and the potent and similar hypotensive effects of losartan and enalaprilat, as demonstrated by indirect systolic BP measurements (Table 1⇑). Basal heart rate did not differ significantly in WKY and SHR, and this parameter was not affected by losartan and enalaprilat treatments (Table 3⇓). The hypotension induced by sodium nitroprusside infusion was similar in WKY and SHR (ΔMAP, −42.6±4.8 and −31.4±3.8 mm Hg). In SHR treated with losartan and enalaprilat, the hypoten- sion was significantly greater (ΔMAP, −53.4±6.4 and −52.2±6.5 mm Hg), although these treatments in WKY did not alter MAP responses (Fig 2A⇓). The concomitant increases in norepinephrine, epinephrine, and heart rate were expressed as a ratio to the hypotension induced in each animal (Fig 2B⇓, 2C⇓, and 2D⇓). The calculated ratio of the changes from baseline values over the changes in MAP is a better representation of the reflex activation of the sympathetic system per millimeter of mercury and thereby allows a better comparison between the groups as they showed different hypotensive responses. The associated increases in plasma norepinephrine (9.6±0.7 versus 4.9±1.3 [Δpg/mL]/Δmm Hg, P<.05), plasma epinephrine (2.8±0.7 versus 1.4±0.3 [Δpg/mL]/Δmm Hg, P<.05), and heart rate (4.9±1.0 versus 3.0±0.5 Δbeats per minute/Δmm Hg) were higher in control SHR versus WKY although the hypotensive responses induced by nitroprusside infusion were similar. In SHR treated with losartan or enalaprilat, the greater nitroprusside-induced hypotension was associated with smaller increases in plasma norepinephrine levels (1.7±0.4 and 2.8±0.7 [Δpg/mL]/Δmm Hg, P<.05) and heart rate (0.8±0.4 and 0.4±0.3 Δbeats per minute/Δmm Hg, P<.05), whereas these responses were unchanged in treated WKY. These results suggest an attenuation of sympathetic reactivity in SHR (Fig 2⇓).
Myocardial β-Adrenergic Receptors
Total specific β-adrenergic receptor density (Bmax) and their dissociation constant (Kd) are summarized in Table 4⇓. β-Adrenoceptor density was similar in untreated WKY and SHR, but β-adrenergic receptor affinity was significantly reduced in control SHR as expressed by the increased Kd. In SHR, both antihypertensive treatments normalized receptor affinity to values similar to those calculated in control WKY. Moreover, in SHR treated with enalaprilat, total β-adrenergic receptor density was significantly increased and affinity was increased to a greater extent than in SHR treated with losartan. In WKY, losartan and enalaprilat treatments had no significant effect on total β-adrenergic receptor density and affinity.
β1- and β2-Adrenergic Receptor Subtypes
ICI 118,551 inhibited ICYP binding, with shallow biphasic curves that could be best resolved by a model with two affinity binding sites in all animal tissues—the high-affinity site representing the binding of the ligand to β2-adrenoceptors and the low-affinity site to β1-adrenoceptors (Fig 3A⇓). Thus, calculation of the relative subtypes was possible in all individual preparations (Table 4⇑). None of the treatment modified Ki values for the low- and high-affinity sites of ICI 118,551. Computer analysis of these data revealed a significant decrease in the proportion of β1-adrenoceptors and a significant increase in the proportion of β2-adrenoceptors in control SHR. Losartan and enalaprilat treatments restored the relative proportions to values similar to those calculated in control WKY (Table 4⇑ and Fig 3B⇓). Indeed, the absolute densities of left ventricular β1- and β2-adrenoceptors calculated for each animal revealed a greater number of β1-adrenoceptors than β2-adrenoceptors in WKY, whereas in SHR, the number of β2-adrenoceptors was increased compared with β1-adrenoceptors. There was a significant increase in β2-adrenoceptor density in control SHR versus WKY (20.4±4.2 versus 7.8±2.8 fmol/mg, P<.05) and a significant reduction in β2-adrenoceptor density after treatment with either losartan or enalaprilat in SHR (4.4±1.8 and 7.0±1.6 fmol/mg, respectively). In WKY, neither treatment affected the proportion of β1- and β2-adrenoceptor subtypes (Table 4⇑ and Fig 3B⇓).
β-Adrenergic Receptor Functional Coupling
Agonist competition curves in the absence of Gpp(NH)p were analyzed according to a two-site model. The competition curves in the presence of guanine nucleotides were analyzed according to a one-site model, and the Ki in that case corresponded to the Ki of low-affinity sites determined in isoproterenol competition curves in the absence of guanine nucleotides. Computer analysis revealed a similar proportion of the high-affinity component in untreated WKY and SHR (Table 4⇑). This suggests that β-adrenoceptors are functionally coupled to Gs to the same extent in left ventricles of WKY and SHR. Treatment with losartan or enalaprilat had no effect on β-adrenoceptor functional coupling to Gs in either group (Table 4⇑).
Adenylyl Cyclase Activity
The activity of the β-adrenergic–coupled adenylyl cyclase signaling pathway was studied in the same membrane preparations as those that served for β-adrenergic receptor binding studies. cAMP production in the presence of different agonists is represented as dose-response curves in Fig 4⇓, and the values of the respective parameters are presented in Table 5⇓. Basal cAMP production was similar in control and treated WKY and SHR. The sensitivity of the adenylyl cyclase activity response curves in the presence of the different agonists is expressed by the EC50, and the maximal rate of conversion of ATP to cAMP expressed by the Vmax was calculated from a double reciprocal of each curve. In isoproterenol-stimulated cAMP formation curves, the EC50 was similar in both strains, treated and untreated. The Vmax was slightly but significantly increased in untreated SHR compared with WKY, and treatment with enalaprilat reduced significantly the Vmax to values near those of untreated WKY. In the presence of Gpp(NH)p, adenylyl cyclase sensitivity was similar in control WKY and SHR, but Vmax was greater in SHR. Losartan treatment had no effect in either group, whereas in both strains treated with enalaprilat, the curves were significantly shifted to the right (EC50 decreased by half a log, Fig 4⇓). Vmax was reduced only in SHR treated with enalaprilat to values similar to values in control WKY (Table 5⇓). The sensitivity of the adenylyl cyclase to forskolin was similar in control and treated WKY and SHR. However, Vmax was markedly greater in SHR, suggesting an increased activity of the catalytic enzyme itself. Enalaprilat treatment significantly reduced the forskolin-induced production of cAMP in SHR to values similar to those in untreated WKY. Thus, chronic treatment with enalaprilat in SHR reduced the increased maximal rate of conversion of ATP to cAMP stimulated by isoproterenol, Gpp(NH)p, and forskolin and decreased the sensitivity of the adenylyl cyclase pathway to Gpp(NH)p.
When the maximal formation of cAMP induced by isoproterenol (0.1 mmol/L) and Gpp(NH)p (1.0 mmol/L) was normalized as a ratio of the maximal formation of cAMP stimulated by forskolin (0.1 mmol/L) for each animal, there were no differences between untreated WKY and SHR and between untreated SHR and SHR treated with enalaprilat (Table 5⇑). This implies an altered adenylyl cyclase activity in isoproterenol and Gpp(NH)p responses. The ratio of cAMP formation induced by sodium fluoride, which activates Gs directly, to forskolin-induced cAMP formation was similar in WKY and SHR, and the treatments had no effects on the Gs–adenylyl cyclase pathway (Table 5⇑).
Chronic treatments with losartan and enalaprilat displaced MAP response curves to exogenous Ang II and Ang I to a similar degree in WKY and SHR. Therefore, achieving a similar blockade of the RAS with equipotent doses allowed us to better compare the effects of these two drugs on hemodynamic and presynaptic and postsynaptic sympathetic functions. Since the plasma levels of losartan and its major metabolite EXP 3174 were similar and losartan increased the ED50 similarly in both strains, the efficacy of Ang II receptor blockade was reflected by the plasma levels of the drug and its metabolite. In normotensive rats treated with various doses of losartan and enalaprilat, we have previously demonstrated that the doses chosen in the present study produced equally efficient maximal angiotensin blockade and that higher doses of losartan were not more effective in blocking vascular Ang II receptors.13 In addition, both treatments attenuated angiotensin-induced inositol phosphate formation in both WKY and SHR (data not shown).14
Both chronic treatments with losartan and enalaprilat similarly reduced BP and cardiac mass, suggesting that the RAS is implicated in the development and/or maintenance of hypertension and cardiac hypertrophy in SHR. Other studies have also reported that RAS blockade lowered BP in SHR, although this model is generally considered to be a normal- or low-renin type of hypertension. This suggests that angiotensin blockade interferes with BP control systems other than that expressed by circulating renin or angiotensin levels. In SHR, losartan and enalaprilat were found to reduce BP and increase plasma renin activity and Ang II levels, but left ventricular Ang II content decreased concomitantly with the regression of left ventricular hypertrophy.15 16 17 It has been demonstrated that SHR have increased levels of angiotensinogen mRNA in the hypertrophied ventricle, suggesting enhanced expression of the cardiac RAS.18 Several studies have reported that even at doses that had no effect on BP, ACE inhibitors produced a regression of cardiac hypertrophy, whereas others have suggested that Ang II induced an AT1 receptor–mediated cardiac hypertrophy independent of mechanical stimuli.19 20 These results provide evidence that the blockade of tissue Ang II plays an important role in the pharmacological effects of RAS inhibitors, such as BP reduction and regression of cardiac hypertrophy.
Kinin accumulation and production of vasodilator prostaglandins also have been proposed to be responsible for the beneficial effect of ACE inhibitors.21 Moreover, since aldosterone has been suspected to be a mediator of cardiac hypertrophy, a theoretical advantage of AT1 receptor antagonists could be through achieving a greater aldosterone suppression compared with ACE inhibitors.22 However, in the present study, treatment with an AT1 antagonist or an ACE inhibitor given at equipotent doses resulted in similar hypotensive and cardiac effects. Thus, no advantages of one drug over the other were observed, indicating that the putative kinin hypotensive mechanism of the ACE inhibitor was not sufficiently important to result in additional hemodynamic effects. This observation also confirms the results of other researchers, who reported that bradykinin is not involved in the effects of ACE inhibitors in SHR.23
Basal plasma and cardiac norepinephrine and epinephrine levels did not differ significantly in untreated normotensive and hypertensive animals and yet constitute an abnormality indicating a reduced sensitivity of baroreceptor mechanisms in SHR. Moreover, the similar fall in BP after sodium nitroprusside infusion in untreated WKY and SHR was associated with markedly greater increases in plasma norepinephrine and epinephrine and a slightly greater increase in heart rate in SHR, suggesting an important sympathetic hyperreactivity. This could be due to an impaired sensitivity of the baroreflex control and/or an increased sensitivity of the presynaptic facilitative mechanisms, as has been postulated in young SHR.24 In support of that latter mechanism, we have reported a greater sensitivity of the presynaptic modulation by Ang II, via AT1 receptors, of adrenergic neurotransmission in isolated rat atria of untreated SHR, whereas chronic treatment with losartan inhibited the effect of Ang II on norepinephrine release.25
Liard26 reported that the arterial baroreflex is reset toward higher BP levels in SHR. One putative central effect of Ang II on the cardiovascular system could be through the attenuation of the baroreceptor reflex–mediated decrease in sympathetic nerve activity. It has been reported that the baroreflex control of heart rate is impaired after central administration of Ang II in SHR, resulting in a blunted response to the pressor effect of phenylephrine and an exaggerated response to hypotension.4 27 This experimental evidence suggests the contribution of a hyperactivity of the brain RAS to hypertension and to the impairment of the baroreceptor reflex control of heart rate in SHR. If endogenous Ang II thus acts on cardiovascular centers to attenuate the baroreceptor reflex, then the reduction in Ang II biosynthesis or the blockade of its action in the brain stem could lead to the sensitization of the baroreceptor reflex. The degree to which the brain RAS is blocked by enalaprilat or losartan depends on the capacity of the drugs to reach the brain from the blood. This is likely to happen since the angiotensin-sensitive sites in the rat brain are densely vascularized and devoid of a blood-brain barrier, which makes those structures reactive to both blood-borne and brain angiotensin.28 Chronic treatment of SHR with EXP 3174 decreased MAP and restored baroreflex function to levels similar to those in control WKY.29 However, it is still not known whether functional blockade of central AT1 receptors is associated with the restoration of baroreflex function in SHR.
Despite an important BP reduction, neither losartan nor enalaprilat treatment elicited a reflex tachycardia or rise in plasma catecholamine levels in SHR. This lack of reflex sympathetic activation could indicate that the baroreflex set point was changed or that the peripheral sympathetic reactivity was attenuated. Moreover, we observed a blunted response to nitroprusside-induced hypotension in SHR treated with losartan and enalaprilat, thus reversing the sympathetic hyperreactivity observed in SHR. Such sympatholytic actions could play a significant role in the antihypertensive effect of losartan and enalaprilat. These results suggest that the RAS is directly implicated in the sympathetic hyperresponsiveness in SHR.
Although the total population of cardiac β-adrenoceptors was similar in SHR and WKY, a reduction of their affinity constant in untreated SHR was associated with changes in the relative density of β-adrenoceptor subtypes. β1-Adrenoceptors were predominant in WKY, whereas cardiac β2-adrenoceptors were predominant in SHR. The physiological role of cardiac β2-adrenoceptors is still not completely understood in rats. It has been proposed that β2-adrenoceptors are involved in the positive chronotropic effects of β-adrenergic agonists.30 Since norepinephrine is the major peripheral sympathetic neurotransmitter acting almost exclusively at cardiac β1-adrenoceptors under normal physiological conditions, only β1-adrenoceptors would regulate heart rate and contractility. Whereas epinephrine stimulates both β1- and β2-adrenoceptors, in the situation of increased epinephrine levels, cardiac β2-adrenoceptor stimulation would also contribute to increases in heart rate or contractility. Similar differential regulation of β-adrenoceptor subtype seems to occur in SHR, deoxycorticosterone acetate–salt hypertension in rats, and chronic heart failure in humans.30 31 32 33 In human heart failure, cardiac β2-adrenoceptors have been suggested to compensate for the loss of β1-adrenoceptors, maintaining contractility. Since the majority of rat cardiac β2-adrenoceptors are found on endothelial cells and prejunctional nerve endings, β2-adrenoceptor–mediated facilitation of sympathetic neurotransmission was suggested to be involved in mediating the pro-hypertensive effects of circulating epinephrine in the SHR.34 Transgenic mice overexpressing cardiac β2-adrenoceptors demonstrated increases in adenylyl cyclase activity, atrial contractility, and in vivo left ventricular function.35 Therefore, the overexpression of β2-adrenoceptors in SHR might result in the maintenance of a physiological response. The treatments with losartan and enalaprilat were associated with the normalization of β-receptor affinity and with the restoration of the proportion of β1 and β2 subtypes. Moreover, the total population of β-adrenoceptors was slightly increased in SHR treated with enalaprilat. Since Ang II potentiates sympathetic neurotransmission, an angiotensin blockade–induced decrease in neurohumoral reactivity could contribute to the restoration of β-adrenoceptors affinity. In accordance with these results, we have previously reported an increased number of human lymphocytic β-adrenergic receptors after chronic trandolapril treatment in association with a decreased sympathetic reactivity to isometric exercise and postural changes.36 It has also been reported that ACE inhibitors can resensitize the β-adrenergic receptor system in heart failure37 38 and after chronic catecholamine exposure.39 Although the resensitization of β-adrenoceptors could be explained by the sympatholytic effect of RAS blockade, it is unknown why ACE inhibitors and the AT1 receptor antagonist exerted a different effect. The role of kinins needs to be explored further in this respect.
The hyperresponsiveness of adenylyl cyclase catalytic activity observed in SHR seemed to be responsible for the enhanced production of β-adrenoceptor–coupled second messengers since functional coupling was similar in WKY and SHR hearts. Our results confirm those of other researchers who reported an enhanced myocardial adenylyl cyclase activity in SHR.40 The increased responsiveness of the β-adrenergic signaling system could be associated with the development of hypertension through an increased cardiac output, as was demonstrated in young human subjects with labile hypertension and in neonatal SHR.40 The increased adenylyl cyclase activity in our studies suggests that modifications of the catalytic unit activity itself occurred. In contrast, other studies have reported that the adenylyl cyclase activity was greater in WKY than in SHR probably because of alterations in Gs function or of enhanced Gi activity in SHR.41 42 However, we and others have observed no qualitative differences in Gs or Gi proteins as measured by immunoblotting studies in both strains before and after losartan and enalaprilat treatments (data not shown).43 44 Another hypothesis could imply a reduced inhibitory influence of Gi on adenylyl cyclase activity, as was reported in platelets from SHR, independent of the similar levels of G protein subunits in WKY and SHR.45 A further question that will need to be investigated is whether the expression of the type of cardiac adenylyl cyclase is altered in hypertension and/or by the treatments, as each type of adenylyl cyclase is differently regulated by G protein βγ- and α-subunits.
Only the chronic treatment with enalaprilat significantly restored adenylyl cyclase activity to values similar to those in control WKY. On the other hand, the sensitivity of the nucleotide regulatory protein–catalytic subunit complex was decreased in both WKY and SHR treated with enalaprilat, indicating a possible interaction of the drug on this coupling via alterations at the level of Gs, the catalytic subunit, or both. However, the apparent unchanged sensitivity of the receptor-stimulated signaling pathway might be due to the potentiating effect of enalaprilat on the number and affinity of total cardiac β-adrenergic receptors, thus masking the defective coupling between the nucleotide regulatory protein and adenylyl cyclase. In right ventricular membrane preparations from WKY and SHR treated with enalaprilat, we also observed a similar significant reduction in adenylyl cyclase activity and an uncoupling between G proteins and adenylyl cyclase (data not shown). Thus, enalaprilat seems to interact with the components of the β-adrenergic signaling pathway by reducing the hyperactivity of the catalytic subunit, by the uncoupling of the Gs–adenylyl cyclase complex, and by increasing β-adrenergic receptor number and affinity. In contrast, losartan seems to exert an effect only on β-adrenergic receptor affinity.
The results of the present study support the possibility that functional alterations of the sympathetic system and RAS are involved in the pathogenesis of hypertension in SHR. Indeed, the reflex reactivity of the sympathetic system was exaggerated in conscious hypertensive animals compared with WKY, and an increased adenylyl cyclase activity concomitant with a higher proportion of β2-adrenoceptors was observed in left ventricular membrane preparations of SHR. The contribution of the RAS in this form of experimental hypertension was supported by the marked BP-lowering effect accompanied by the inhibition of the sympathetic hyperreactivity in SHR after chronic treatments with losartan and enalaprilat. The treatments with RAS inhibitors attenuated the sympathetic pathway hyperresponsiveness and restored the proportions of β1- and β2-adrenoceptors to values similar to those observed in WKY, suggesting an important interaction with the sympathetic system in the maintenance of an elevated BP in this hypertension model. Our study thus supports the hypothesis that the local RAS contributes to the alterations of presynaptic or postsynaptic adrenergic function associated with the development or maintenance of hypertension in SHR.
Selected Abbreviations and Acronyms
|Ang I||=||angiotensin I|
|Ang II||=||angiotensin II|
|AT1||=||angiotensin II type 1 (receptor)|
|MAP||=||mean arterial pressure|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by grants from the Medical Research Council of Canada and from the Quebec Heart and Stroke Foundation. A.K.-Laflamme held a studentship from the Canadian Heart and Stroke Foundation (CHSF). L. Oster held a fellowship from CHSF/Merck Frosst. R. Cardinal held a scholarship from the Fonds de la Recherche en Santé du Québec. J. de Champlain holds a career investigatorship from the J.C. Edwards Foundation. The authors would like to express their gratitude to Jo-Anne Le Guerrier, Diane Papin, and Carole Champagne for their technical assistance.
- Received September 19, 1996.
- Revision received October 15, 1996.
- Accepted December 31, 1996.
Dzau VJ. Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation. 1988;77(suppl I):I-4-I-13.
Keuneke C, Yacullo R, Metzger R, Hellmann T, Peters J, Ganten D. The role of tissue renin-angiotensin systems in hypertension and effects of chronic converting-enzyme inhibition. Eur Heart J. 1990;11(suppl D):11-16.
Saxena PR. Interaction between the renin-angiotensin-aldosterone and sympathetic nervous systems. J Cardiovasc Pharmacol. 1992;19(suppl 6):S80-S86.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
DeLéan A, Munson PJ, Rodbard D. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response curves. Am J Physiol. 1978;235:E97-E102.
Salomon Y. Adenylate cyclase assay. In: Brooker G, Greengard P, Robison GA, eds. Advances in Cyclic Nucleotide Research. New York, NY: Raven Press Publishers; 1979;10:35-55.
Moreau P, Oster L, Laflamme AK, Lambert C, de Champlain J. Relationship between the plasma and the efficacy of losartan during a chronic therapy in the rat. Can J Physiol Pharmacol. 1994;72(suppl 1):178. Abstract.
Oster L, Laflamme AK, Moreau P, de Champlain J. Effect of losartan and enalaprilat in inositol phosphate accumulation in aorta of SHR. J Hypertens. 1994;12(suppl 3):S88. Abstract.
Mizuno K, Tani M, Hashimoto S, Niimura S, Sanada H, Watanabe H, Ohtsuki M, Fukuchi S. Effects of losartan, a nonpeptide angiotensin II receptor antagonist, on cardiac hypertrophy and the tissue angiotensin II content in spontaneously hypertensive rats. Life Sci. 1992;51:367-374.
Li C, Prakash O, Re RN. Altered expression of angiotensinogen gene expression in the left ventricles of the hypertensive rats. Circulation. 1989;80(suppl II):II-450. Abstract.
Van Gilst WH, Scholtens E, De Graeff PA, De Langen CDJ, Wesseling H. Differential influences of angiotensin converting-enzyme inhibitors on the coronary circulation. Circulation. 1988;77(suppl I):I-24-I-29.
Foucart S, Patrick S, Oster L, de Champlain J. Effects of chronic treatment with losartan and enalaprilat on [3H]-norepinephrine release from isolated atria of Wistar-Kyoto and spontaneously rats. Am J Hypertens. 1996;9:61-69.
Liard JF. The baroreceptor reflexes in experimental hypertension. Clin Exp Hypertens. 1980;2:479-498.
Guo GB, Abboud FM. Angiotensin II attenuates baroreceptor control of heart rate and sympathetic activity. Am J Physiol. 1984;246:H80-H89.
Unger T, Badoer E, Ganten D, Lang R, Rettig R. Brain angiotensin: pathways and pharmacology. Circulation. 1988;77(suppl I): I-40-I-54.
Yamada S, Ishima T, Tomita T, Hayashi M, Okada T, Hayashi E. Alterations in cardiac alpha and beta adrenoceptors during the development of spontaneous hypertension. J Pharmacol Exp Ther. 1984;228:454-460.
Nguyen CT, de Champlain J, Bouvier M. Tissue specific changes in β-adrenergic receptors subtypes in DOCA-salt hypertensive rats. Biomed J. 1995;2:34-42.
Milano CA, Allen LF, Rockam HA, Dolber PC, McMinn TR, Chien KR, Johnson TD, Bond RA, Lefkowitz RJ. Enhanced myocardial function in transgenic mice overexpressing the β2-adrenergic receptor. Science. 1994;264:582-586.
Horn E, Corwin SJ, Steinberg SF, Chow YK, Neuberg GW, Cannon PJ, Powers ER, Bilezikian JP. Reduced lymphocyte stimulatory guanine nucleotide regulatory protein and β-adrenergic receptors in congestive heart failure and reversal with angiotensin converting enzyme inhibitor therapy. Circulation. 1988;78:1373-1379.
Gilbert EM, Sandoval A, Larrabee P, Renlund DG, O’Connell JB, Bristow MR. Effect of lisinopril on cardiac adrenergic drive and myocardial β-receptor density in heart failure. Circulation. 1988;78(suppl II):II-576. Abstract.
Maisel AS, Phillips C, Michel MC, Ziegler MG, Carter SM. Regulation of cardiac β-adrenergic receptors by captopril: implications for congestive heart failure. Circulation. 1989;80:669-675.
Hamet P, Orlov SN, Tremblay J. Intracellular signaling mechanisms in hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. 2nd ed. New York, NY: Raven Press Publishers; 1995:575-607.
Anand-Srivastava MB. Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats. Biochem J. 1992;288:79-85.
Clark CJ, Milligan G, McLellan AR, Connell JMC. Guanine nucleotide regulatory proteins in the spontaneously hypertensive rat. Hypertension. 1993;21:204-209.