Transgenic rats (TGRs) TGR(mREN2)27 are characterized by fulminant hypertension, an inverse circadian blood pressure rhythm, and severe hypertensive target organ damage. In the present study, we evaluated cardiovascular risk factors, renal function, and urinary protein loss in transgenic rats before and after treatment with the calcium channel blocker amlodipine. Amlodipine was injected intraperitoneally in a dose of 5 mg/kg/day, either once daily at 8.00 h or twice daily in divided doses at 8.00 and 20.00 h. Untreated TGRs and Sprague-Dawley rats served as hypertensive and normotensive controls, respectively. Before and after 5 weeks of treatment, rats were placed in metabolic cages for sampling of urine. Prior to treatment, urinary excretion rates of protein, albumin, and Ca2+ were significantly higher in TGRs than in Sprague-Dawley controls. Urinary excretion of protein and albumin was reduced by 5 weeks of amlodipine treatment, whereas the excretion of Ca2+ was not affected. The reductions in renal proteinuria and albuminuria by amlodipine treatment were significantly correlated with the treatment-induced decrease in blood pressure. These findings indicate that blood pressure itself is an important contributor to albumin loss by the kidney in renin-dependent hypertension of TGRs.

Transgenic rats (TGRs) TGR(mREN2)27 are characterized by fulminant hypertension, an inverse circadian blood pressure rhythm, and severe hypertensive target organ damage. In the present study, we evaluated cardiovascular risk factors, renal function, and urinary protein loss in transgenic rats before and after treatment with the calcium channel blocker amlodipine. Amlodipine was injected intraperitoneally in a dose of 5 mg/kg/day, either once daily at 8.00 h or twice daily in divided doses at 8.00 and 20.00 h. Untreated TGRs and Sprague-Dawley rats served as hypertensive and normotensive controls, respectively. Before and after 5 weeks of treatment, rats were placed in metabolic cages for sampling of urine. Prior to treatment, urinary excretion rates of protein, albumin, and Ca2+ were significantly higher in TGRs than in Sprague-Dawley controls. Urinary excretion of protein and albumin was reduced by 5 weeks of amlodipine treatment, whereas the excretion of Ca2+ was not affected. The reductions in renal proteinuria and albuminuria by amlodipine treatment were significantly correlated with the treatment-induced decrease in blood pressure. These findings indicate that blood pressure itself is an important contributor to albumin loss by the kidney in renin-dependent hypertension of TGRs.

It was the aim of the present study to evaluate whether, in physiological cardiac tissue, long and short splice variants of G(s)alpha (the alpha subunit of the stimulatory G-protein) differ in their susceptibility to guanine nucleotide-mediated activation. As a measure of G(s)alpha activation, we determined the proportion of G(s)alpha subunits which were released from the plasma membrane upon stimulation. Membrane preparations from heart ventricles of Wistar rats were incubated with increasing concentrations of the non-hydrolyzable GTP analogue guanylyl-imidodiphosphate (GppNHp, 0-100 micromol/L) in the absence or presence of the beta-adrenoceptor agonist isoprenaline (1 micromol/L). The 45 and 52 kDa forms of G(s)alpha (G(s)alpha-S and -L, respectively) were measured in the supernatant of the incubation mixture by immunoblotting and densitometry. The increase in cyclic AMP induced by GppNHp was measured in the same supernatant. In the absence of isoprenaline, GppNHp increased cyclic AMP formation and the concentration-dependent release of G(s)alpha-L (Friedman test, P < 0.05), whereas the amount of soluble G(s)alpha-S was not affected. After addition of isoprenaline, the redistribution of G(s)alpha-S into the soluble fraction could be stimulated by GppNHp in a concentration-dependent manner (P < 0.05). Kinetic experiments revealed that activation of G(s)alpha-S by GppNHp was rather slow, but could be markedly enhanced by isoprenaline. Thus, it is likely that the different susceptibilities of G(s)alpha-S and -L towards GppNHp reflects differences in the rate of spontaneous GDP release.

The effects of amlodipine on blood pressure profiles, cardiac hypertrophy, and beta-adrenergic signal transduction were studied in transgenic hypertensive TGR(mREN2)27 rats (TGRs), which are characterized by an inverse circadian blood pressure rhythm. Cardiovascular parameters were monitored by radiotelemetry; beta-adrenoceptor density and function were measured by radioligand binding and by determination of beta-adrenergic stimulation of adenylyl cyclase. Ventricular weight and the activity of cardiac sarcolemmal 5-nucleotidase were used as measures of hypertrophy. Acute i.p. injection of amlodipine (1, 3, 10 mg/kg body weight) either at 8:00 or at 20:00 h dose-dependently reduced blood pressure irrespective of the dosing time. For long-term treatment, TGRs were divided into three groups: untreated; amlodipine, once-daily, 5 mg/kg; and amlodipine, twice daily, 2.5 mg/kg. Both treatment schedules resulted in decreased 24 h means in systolic and diastolic blood pressure and a reduction in ventricular hypertrophy but had no effects on cardiac beta-adrenergic signaling. Once-daily dose of amlodipine at 8:00 h decreased blood pressure predominantly during the daily resting period of the rats, whereas twice-daily dosing induced a bimodal blood pressure pattern. However, even after 5 weeks of treatment, typical circadian profiles could not be observed with either treatment, indicating a short duration of action of amlodipine in rats. Thus it remains an open question whether pharmacologic normalization of the circadian blood pressure pattern in TGRs will more effectively reduce myocardial hypertrophy and restore beta-adrenergic signaling than a reduction in 24-h blood pressure per se.

1. In human congestive heart failure beta-adrenoceptor antagonists improve exercise tolerance and cardiac contractility. These beneficial effects are thought to reflect an up-regulation of cardiac beta-adrenoceptors, involving mainly the beta1-subtype. In the present study we evaluated the functional contribution of beta-adrenoceptor subtypes to stimulation of adenylyl cyclase in an animal model of dilated cardiomyopathy, and compared the effects of treatment with propranolol on cardiac beta-adrenergic signal transduction in myopathic and control hamsters. 2. Cardiomyopathic BIO TO2 hamsters and BIO F1B controls aged 270 days were used. In the treatment study, hamsters received drinking water with or without propranolol 40 mg kg(-1) d(-1) for 4 weeks prior to sacrifice. Density and subtype distribution of beta-adrenoceptors were determined in radioligand binding studies. Functional contributions of beta-adrenoceptors were evaluated by subtype-selective stimulation of adenylyl cyclase. Cardiac G-protein content was determined by immunoblotting. 3. Compared to BIO F1B controls, myopathic hamsters showed increases in cardiac total beta- and beta2-adrenoceptor density, G(s alpha) and G(i alpha) content. In BIO TO2 ventricles, beta1-adrenoceptors were almost completely uncoupled from adenylyl cyclase stimulation despite an unchanged density. Treatment of hamsters with propranolol resulted in increased density of beta1-adrenoceptors in both strains, but had no effect on their functional efficacy. Moreover, beta2-adrenergic stimulation of adenylyl cyclase was even reduced in propranolol-treated animals, which could not be explained by changes in cardiac G-protein content. 4. Cardiomyopathic BIO TO2 hamsters showed functional uncoupling of cardiac beta1-adrenoceptors, which could not be normalized by propranolol and, therefore, is unlikely to be solely due to agonist-dependent desensitization. The paradoxical reduction in beta2-adrenergic efficiency in propranolol-treated myopathic and control hamsters deserves further investigation.

The circadian time-dependent, acute effects of routine animal house manipulations on cardiovascular parameters were studied in Sprague-Dawley rats. Blood pressure, heart rate and motor activity a ere monitored telemetrically in 5 min intervals in male Sprague-Dawley rats (n = 6, aged 28 weeks). Animals were kept in LD 12:12 (lights on at 07:00 h) under constant temperature and had free access to food and water. Three different routine manipulations were tested during the rats' rest period (08:45 h) as well as during the activity period (20:35 h): 1) refill of food and water, 2) i.p. injections of NaCl 0.9% (vehicle injection), and 3) cage cleaning. Supplying food and Mater had almost no effect on the parameters studied. In contrast, new housing and vehicle injections resulted in increased values in blood pressure, heart rate and motor activity. The effects of either manipulation on cardiovascular parameters were more pronounced during the daily rest period. We conclude that (a) telemetry is a suitable method to evaluate and quantify cardiovascular effects of animal house routines in freely moving rats and (b) the short term cardiovascular response to routine animal house manipulations depends on the nature of the stressor and the time of day when the stress occurrs.

Transgenic hypertensive TGR(mREN2)27 (TGR) rats, carrying an additional mouse renin gene, have been found to show inverse circadian blood pressure profiles compared to normotensive Sprague-Dawley rats. In order to evaluate the contributions of the suprachiasmatic nucleus (SCN) and the neurohormone melatonin to cardiovascular circadian regulation in TGR(mREN2)27 rats and Sprague-Dawley (SPRD) controls, we investigated the effects of melatonin agonist and antagonist treatment in SCN-lesioned and nonlesioned rats, which were kept under conditions of alternating light and darkness (LD). After destruction of the SCN, circadian rhythmicity in blood pressure, heart rate (HR), and motor activity (MA) was almost abolished in rats of both strains. One week of treatment with a synthetic melatonin agonist S-21634 was not able to restore circadian variation in the parameters monitored. In nonlesioned TGR(mREN2)27 rats and Sprague-Dawley control rats, the melatonin antagonist S-22365 had no suppressive effect on LD-synchronized circadian rhythmicity, indicating that LD itself may have a stronger influence on the SCN than endogenous melatonin.

Adenylyl cyclase and soluble guanylyl cyclase activities were measured in cardiac and aortic tissue from transgenic hypertensive TGR(mREN2)27 and normotensive Sprague-Dawley rats. Cardiac basal and stimulated adenylyl cyclase activity was significantly lower in TGR(mREN2)27 than in Sprague-Dawley rats except after uncoupling of G-proteins by Mn2+-ions. Aortic cAMP formation did not differ between both strains, indicating that the disturbance of cardiac adenylyl cyclase activity was due to local rather than systemic factors. Vascular cGMP formation was significantly reduced in TGR(mREN2)27 aortae under basal conditions and after stimulation with sodium nitroprusside, indicating that there is a subsensitive vasodilating second messenger pathway in the transgenic strain.

In myopathic BIO 8262-hamsters beta1-adrenergic stimulation of cardiac adenylyl cyclase has been found to be markedly reduced compared to that of healthy controls. In order to test the hypothesis that the functional uncoupling of beta1-adrenoceptors in diseased hamster hearts is due to agonist-dependent desensitization, we investigated the effects of prolonged treatment with beta-adrenoceptor antagonists on cardiac beta-adrenergic signaling. Groups of hamsters aged 240 days received either drinking water, or drinking water containing metoprolol (10 or 100 mg/kg/day) or propranolol (4 or 40 mg/kg/day). After 4 weeks' treatment animals were killed and heart ventricles were prepared for determination of beta1- and beta2-adrenoceptor densities and their functional contribution to stimulation of adenylyl cyclase. Markers of myocardial hypertrophy, i.e. absolute and relative ventricular weight and 5-nucleotidase activity, were not affected by the different treatment regimens. Neither absolute densities nor relative proportions of beta-adrenoceptor subtypes differed between untreated and treated hamster groups. Metoprolol had no effects on the functional efficacy of beta1- and beta2-adrenoceptors. Hamsters treated with high dose propranolol showed unchanged beta1-adrenoceptor function but reduced beta2-adrenergic stimulation of adenylyl cyclase. The findings of the present study demonstrate that the disturbed coupling of cardiac beta1-adrenoceptors to adenylyl cyclase cannot be reversed by in vivo treatment with beta-adrenoceptor antagonists and, therefore, is unlikely to be due to agonist-dependent desensitization.

In an attempt to further investigate circadian changes in kidney function, the planar surface area (PSA) (mu m(2)) of renal isolated glomeruli from normal rats was monitored using a computerized image analyzer method. Eight male Sprague-Dawley (SPRD) rats, aged 12-14 weeks, were entrained to a 12-h light/12-h dark cycle in controlled environmental conditions of temperature (22+/-2 degrees C) two weeks before the experiments started. Isolated glomerular preparations were obtained, using a mechanical sieving technique, at four different circadian times: 07:00, 13:00, 19:00 and 01:00. It was the finding of the present study that the PSA of the glomeruli varied significantly over the 24-h period, but showed a weak amplitude. The size of the glomeruli reached the highest values at night (21358.59+/-456.72 mu m(2) at 21:17) with an acrophase at 21:39, as do all the other renal parameters, but also blood pressure and many vasoactive compounds involved in the regulation of the mesangial cell physiology, contractile element of the glomerulus. Such chronobiological data not only provided a clear example of complementarity between in vivo and ex vivo experiments, but evidenced that temporal changes do exist in kidney structure and could be correlated with rhythms in renal physiology.

Patients with secondary hypertension frequently display abnormal circadian blood pressure profiles, characterized by a failure to decrease blood pressure at night. The transgenic TGR(mRen-2)27 rat strain, developing fulminant hypertension after the mouse salivary Ren-2 renin gene has been integrated into its genome, provides a fundamental model of genetic hypertension, Because of an inverse circadian blood pressure profile and an unchanged rhythmic pattern of heart rate compared with the normotensive Sprague-Dawley (SPR) strain, it was proposed to serve as an animal model of genetic hypertension. It was the aim of the present study to investigate the circadian rhythmicity in renal function of the transgenic rat to determine whether hypertension and disturbed circadian blood pressure profile would affect kidney function. Urinary water, electrolyte, and protein excretion, as well as glomerular filtration rate and renal plasma flow, were determined in unrestrained freely moving transgenic hypertensive (TGR) and SPR normotensive control rats by collecting urine and arterial blood every 4 h. Significant and similar circadian rhythms were found in renal excretion and hemodynamics in both normotensive and hypertensive strains. Peaks occurred in the active dark period, whereas troughs were found in daytime for all parameters. However, it has to be pointed out that, although the circadian profiles were not grossly perturbed in hypertensive animals, some small differences between SPR and TGR strains did exist in renal function. These discrepancies were precisely related to acrophase, showing a slight phase delay, and also to relative amplitude in TGR. This study demonstrates that the inverted circadian blood pressure profile affected only slightly the circadian rhythms in kidney function in TGR compared with SPR. These findings support the notion that time-dependent changes in systemic blood flow may be of greater importance for circadian regulation of kidney function than systemic blood pressure.

OBJECTIVES:The study was performed in order to elucidate whether or not the nitric oxide-guanylyl cyclase system is involved in the circadian regulation of blood pressure. METHODS:Basal and sodium nitroprusside-stimulated guanylyl cyclase activity was studied in vitro in aortic tissue from normotensive Wistar-Kyoto rats sacrificed at 7 circadian times. Effects of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) on blood pressure and heart rate of rats were measured telemetrically after acute injection of L-NAME either in the morning or in the evening as well as after chronic application in drinking water. RESULTS:Guanylyl cyclase activity exhibited significant circadian rhythmicity under basal conditions and after stimulation by sodium nitroprusside 10 microM. Peaks in cGMP formation were observed in the daily resting period, i.e. the period of low blood pressure in rats. Acute application of L-NAME dose-dependently increased blood pressure; effects were more pronounced after injection in the morning than in the evening, leading to an inverse blood pressure profile after the highest dose (30 mg/kg i.p.). Chronic treatment with L-NAME resulted in a dose-dependent upward shift of the control blood pressure profile. Only at the highest dose applied in drinking water (100 mg/kg per day) was the circadian pattern in blood pressure altered, showing a reduced fall in the morning. CONCLUSIONS:Circadian rhythmicity in basal and stimulated soluble guanylyl cyclase activity and the reversed blood pressure profile after acute injections of L-NAME indicate an involvement of the nitric oxide-guanylyl cyclase system in circadian blood pressure regulation. After chronic inhibition of nitric oxide synthase additional and counterregulatory mechanisms have to be considered.