Glucose tolerance and insulin secretion in conscious and unrestrained normotensive and spontaneously hypertensive rats

We compared the glucose tolerance and insulin responses to intravenous (IV) glucose administration of a dose of 1 g/kg body weight in a conscious and unrestrained state of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) with catheters chronically indwelled into artery and vein. Both plasma glucose levels at two minutes and ten minutes following IV glucose load as well as the incremental and total areas of plasma glucose were slightly but significantly lower in SHR than in WKY. Glucose disappearance rate (K value) was 7.7 +/- 0.3%/min in SHR, being slightly but significantly higher than that of 6.8 +/- 0.3%/min in WKY. On the other hand, insulin responses to the glucose load at ten minutes and 30 minutes as well as incremental and total insulin areas were significantly lower in SHR than in WKY. There was no significant difference in insulinogenic index between SHR and WKY. Our observations suggest that in a conscious and unrestrained state, SHR have the greater glucose tolerance associated with reduced insulin secretion than do WKY.     

Antidiabetic agent englitazone enhances insulin action in nondiabetic rats without producing hypoglycemia

The new antihyperglycemic agent englitazone (CP-68,722) was examined in nondiabetic rats. Administration of englitazone at 50 mg/kg/d for 8 days did not produce overt hypoglycemia but it lowered basal plasma insulin by 59% and 41% in rats fed ad libitum and fasted overnight on the last day, respectively. Drug treatment also lowered (P less than .05) plasma nonesterified fatty acids (1.09 +/- 0.05 to 0.36 +/- 0.05 mmol/L) and cholesterol (2.41 +/- 0.08 to 2.06 +/- 0.07 mmol/L) in fasted rats, and glycerol (0.25 +/- 0.02 to 0.14 +/- 0.02 mmol/L) in fed rats but had no effect on 3-hydroxybutyrate or lactate levels despite the hypoinsulinemia. Disposition of an oral glucose load (1 g/kg) in drug-treated fed rats was identical to that in control rats despite a 40% reduction in the area under the plasma insulin curve. Insulin-stimulated 2-deoxy-D-3H-glucose uptake was significantly (P less than .05) enhanced in adipocytes prepared from both fasted and fed drug-treated rats (0.56 +/- 0.07 to 0.84 +/- 0.03 and 0.79 +/- 0.02 to 1.00 +/- 0.02 nmol/5 min, respectively, at insulin concentration of 2,500 microU/mL). There was also a significant increase in the basal rate of 2-deoxyglucose uptake (0.07 +/- 0.01 to 0.24 +/- 0.07 nmol/5 min) in adipocytes from fasted rats only. Insulin-stimulated lipogenesis from 3H-2-glucose was enhanced in adipocytes from drug-treated fed rats (7.72 +/- 0.09 to 10.19 +/- 0.10 nmol glucose/45 min at insulin concentration of 2,500 microU/mL) but no effect was observed in adipocytes from fasted rats (2.57 +/- 0.30 to 2.33 +/- 0.16 nmol glucose/45 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-stimulated cardiac glucose uptake is impaired in spontaneously hypertensive rats: role of early steps of insulin signalling

OBJECTIVE: Although the heart is one of the target organs of insulin, it is still unknown whether the effect of insulin on cardiac muscle is preserved in essential hypertension, where insulin resistance has been observed in skeletal muscle. METHODS: We evaluated cardiac glucose uptake and the early steps of insulin signalling in spontaneously hypertensive (SHR, 10-12 weeks old) and in age-matched normotensive Wistar-Kyoto (WKY) rats. Cardiac glucose uptake (micromol/100 g per min) was assessed by 2-[14C]deoxyglucose method. After an overnight fast, 16 WKY rats and 17 SHR underwent a hyperinsulinemic euglycemic clamp. In particular, 2-h intravenous (i.v.) infusion of insulin (10 mU/kg per min) or saline (NaCl 0.9%) was administered, followed by an i.v. bolus injection of 2-[14C]deoxyglucose (100 microCi/kg) to measure cardiac glucose uptake. RESULTS: During saline infusion, cardiac glucose uptake was significantly higher in SHR compared to WKY rats (85 +/- 18 versus 8 +/- 3 mg/kg per min, P < 0.01). Furthermore, insulin was able to markedly increase cardiac glucose uptake in WKY rats whereas this insulin action was entirely abolished in SHR; thus, the cardiac glucose uptake became similar in the two rat strains (76 +/- 16 versus 82 +/- 16 mg/kg per min, not significant). More importantly, during saline infusion SHR showed a significantly higher phosphorylation of insulin receptor substance-1 (IRS-1) coupled to enhanced association of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and to an increased PI 3-kinase activity compared to WKY rats. As expected, insulin exposure evoked an activation of its signalling cascade in WKY rats. In contrast, in SHR, the hormone failed to activate post-receptor molecular events. CONCLUSIONS: Our data indicate that the heart of SHR shows an overactivity of the proximal steps of insulin signalling which cannot be further increased by the exposure to the hormone. This abnormality may account for the marked increase of basal cardiac glucose uptake and the loss of insulin-stimulated glucose uptake observed in SHR.     

Tempol selectively attenuates angiotensin II evoked vasoconstrictor responses in spontaneously hypertensive rats

OBJECTIVE : To assess whether superoxide anions mediate vasoconstrictor responses to agonists in blood vessels of spontaneously hypertensive rats (SHRs). METHODS : The effect of the superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol), on responses to angiotensin II (Ang II), endothelin-1, phenylephrine and potassium chloride was determined in aortic rings and perfused mesenteric vascular beds (MVB) of adult male rats of the Sprague-Dawley, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) strains. The effect of tempol on Ang II-evoked superoxide production was assessed in aortic rings. RESULTS : There were no differences in the maximum tension (Emax) attained in response to agonists, but the negative logarithm of the concentration required to produce 50% of the maximal response (EC50) for Ang II was lower (P < 0.05) in aortic rings of SHRs. In the MVBs of SHRs, the Emax but not the EC50 values attained in response to Ang II, endothelin-1 and phenylephrine were greater. Tempol significantly and selectively reduced the Emax of Ang II in both aorta and MVB preparations with intact endothelium. The reduction in Emax attained in response to Ang II was more pronounced in SHRs (P < 0.01) than in WKY rats (P < 0.05) or Sprague-Dawley rats (P < 0.05). The inhibitory effect of tempol was absent when a nitric oxide synthase inhibitor was included or endothelium was denuded. A significant increase in lucigenin chemiluminescence evoked by Ang II in both intact and endothelium-denuded aortic rings of SHRs was abolished when tempol was included in the buffer. CONCLUSIONS : These data suggest that increased superoxide anions mediate vasoconstrictor responses to Ang II, but not to other agonists, in an endothelium-dependent manner, by quenching vasodilatory mediator, nitric oxide. This may account for the exaggerated vasoconstrictor responses to Ang II in SHRs.     

Patterns of insulin secretion and responsiveness in Wistar-Kyoto and spontaneously hypertensive rats.

An intravenous glucose tolerance test (IVGTT) was performed on 16 spontaneously hypertensive rats (SHR) and 15 Wistar-Kyoto rats (WKY) by challenging them with an intravenous glucose load of 0.125 g/100 g body weight. Serum glucose and insulin were measured at 0, 15, 30, 60, 120, and 240 min. In a second experiment, oral glucose tolerance testing (OGTT) was performed with a glucose load of 170 mg/100 g body weight. Serum glucose and insulin were measured at 0, 30, 60, 120, and 240 min. A third experiment examined in vivo insulin-stimulated peripheral glucose uptake via the insulin suppression test. Glucose (8 mg/kg/min), insulin (2.5 to 4.0 mU/kg/min), and somatostatin (1 ng/kg/min) were infused for 3 h. Steady state serum glucose (SSSG) and serum insulin (SSSI) were determined at 0, 140, 160, and 180 min. IVGTT data indicated that serum glucose values were significantly increased in the WKY compared to the SHR at 15, 30, 60, and 120 min. Glucose clearance rates between 15 and 60 min were significantly (P less than .0001) lower in WKY (0.65 +/- 0.07%/min) compared to SHR (1.91 +/- 0.17%/min). The SHR exhibited an exaggerated 15-min peak in insulin secretion that was lacking in the WKY in response to an intravenous glucose load. OGTT data also indicated that the area under the serum insulin curves was higher in the WKY than in the SHR (P less than .017). Peripheral insulin sensitivity showed that despite comparable SSSI levels between the two strains, SSSG values were 31% higher in the SHR compared to the WKY (P less than .001). Thus, it appears that the WKY has a diminished insulin secretory capacity in response to a glucose load, while the SHR has lower tissue responsiveness to insulin's action.     

Aberrant D1 and D3 dopamine receptor transregulation in hypertension

Dopamine plays a role in the regulation of blood pressure by inhibition of sodium transport in renal proximal tubules (RPTs) and relaxation of vascular smooth muscles. Because dopamine receptors can regulate and interact with each other, we studied the interaction of D(1) and D(3) receptors in immortalized RPT cells and mesenteric arteries from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs), and in human coronary artery smooth muscle cells (CASMCs). In WKY rats, the D(1)-like agonist, fenoldopam, increased D(3) receptor protein in a time-dependent and concentration-dependent manner (EC(50)=4.5x10(-9) M, t(1/2)=15.8 hours). In SHRs, fenoldopam (10(-5) M) actually decreased the expression of D(3) receptors. D(1) and D(3) receptor co-immunoprecipitation was increased by fenoldopam (10(-7) M/24 h) in WKY rats but not in SHRs. The effects of fenoldopam in CASMCs were similar as those in WKY RPT cells (ie, fenoldopam increased D(1) and D(3) receptor proteins). Both D(3) (PD128907, Emax=80%+/-6%, pED(50)=5+/-0.1) and D(1)-like receptor (fenoldopam, Emax=81%+/-8%, pED(50)=5+/-0.2, n=12) agonists relaxed mesenteric arterial rings. Co-stimulation of D(1) and D(3) receptors led to additive vasorelaxation in WKY rats, but not in SHRs. D(1) and D(3) receptors interact differently in WKY and SHRs. Altered interactions between D(1) and D(3) receptors may play a role in the pathogenesis of genetic hypertension, including human hypertension, because these receptors also interact in human vascular smooth muscle cells.     

Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells

This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (10(-12) to 10(-6) mol/L) in the absence and presence of PD98059 (10(-5) mol/L). [Ca2+]i and pHi were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]i and pHi and contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9+/-5.1% reduction in cell length relative to basal length) than in WKY (28.1+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]i and pHi effects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 576+/-24 versus 413+/-43 nmol/L; Emax of pHi, 7.33+/-0.01 versus 7.27+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax,19. 3+/-3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i (Emax, 294+/-55 nmol/L) and pHi (Emax, 7. 27+/-0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.     

Hepatic parasympathetic role in insulin resistance on an animal model of hypertension

The hepatic insulin sensitizing substance (HISS) pathway, which includes the hepatic parasympathetic nerves and hepatic nitric oxide (HNO), has been shown to be crucial to the action of insulin on glucose metabolism. Insulin resistance in essential hypertension has been related to parasympathetic dysfunction; thus, we tested the hypothesis that the HISS pathway is impaired in spontaneously hypertensive rats (SHR) when compared with their normotensive controls, Wistar (WIS) and Wistar Kyoto (WKY) rats. A modified euglycemic clamp quantified insulin sensitivity. Differentiation of the HISS-dependent and HISS-independent components of insulin action was achieved by administration of a muscarinic receptor antagonist (atropine, 3 mg/kg) or of a nitric oxide synthase inhibitor (N(g)-methyl-arginine, 0.73 mg/kg). Both SHR and WKY had lower postprandial total insulin action when compared with WIS (209.1 +/- 13.6 for WKY and 217.8 +/- 19.8 for SHR vs 296.1 +/- 16.9 mg glucose/kg body weight for WIS, P < .05). Furthermore, we observed that this is due to a decrease of the HISS-dependent component of insulin action (154.8 +/- 16.4 for WIS vs 87.1 +/- 14.5 for WKY and 55.9 +/- 15.6 mg glucose/kg body weight for SHR; P < .05 and P < .001, respectively; data concerning the atropine protocol). Blockade of HISS action by inhibition of hepatic nitric oxide synthase with N(g)-methyl-arginine showed similar results to those obtained with atropine, suggesting that they indeed act through the same pathway. In conclusion, our results support our hypothesis that impairment of the HISS pathway is responsible for the development of insulin resistance between WIS and SHR.     

Metabolic, hemodynamic, and cardiac effects of captopril in young, spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) demonstrate elevated blood pressure, cardiac hypertrophy, glucose intolerance, and insulin resistance compared with age-matched Wistar-Kyoto rats (WKY). We investigated concurrent effects of captopril on blood pressure, cardiac mass, myocardial enzyme activities, glucose tolerance, and insulin action in young male SHR. At 10 weeks of age, SHR were randomized into two groups, one receiving distilled water, the other a captopril solution (50 mg/kg body weight/day). We also examined age-matched WKY receiving distilled water. Blood pressure was measured by tail-cuff during the 4-week treatment period and oral glucose tolerance was tested at the end of treatment. Hearts were weighed and ventricular tissue was assayed for activities of 3-hydroxyacyl-CoA dehydrogenase, citrate synthase, and hexokinase. Growth rates were similar between captopril-treated and control SHR, but less than those of WKY. Captopril reduced blood pressure (134 +/- 8 v 177 +/- 8 mm Hg, P < .05) and left ventricular mass (-18%, P < .05) in SHR. Cardiac enzyme activities also changed with captopril treatment, reflecting an increased capacity for beta-oxidation of fatty acids and reduced potential for glucose phosphorylation in the left ventricle of SHR. Serum concentrations of glucose, insulin, and free fatty acids after a brief fast and in response to oral glucose were not different after captopril treatment, suggesting no improvement in insulin action or glucose tolerance. In summary, treatment of young male SHR with captopril reduces blood pressure and cardiac mass, and promotes a small but significant increase in cardiac capacity for oxidation of fatty acids and reduction of glucose phosphorylation. In contrast, metabolic effects of captopril on oral glucose tolerance and insulin action were not evident.     

Inhibition of mitogen-activated protein/extracellular signal-regulated kinase improves endothelial function and attenuates Ang II-induced contractility of mesenteric resistance arteries from spontaneously hypertensive rats

OBJECTIVES: Extracellular signal-regulated kinases (ERK1/2) modulate vascular smooth muscle cell (VSMC) growth and contractility, important factors in blood pressure regulation. In the present in vivo study, we investigated whether short-term inhibition of ERK1/2-dependent signaling pathways influences vascular function and blood pressure (BP) in spontaneously hypertensive rats (SHR). METHODS: SHR and Wistar-Kyoto (WKY) rats were injected subcutaneously with either PD98059, selective MEK1/2 inhibitor (20 mg/kg), or vehicle. BP was measured by telemetry. Rats were killed 24 h after injection and small mesenteric arteries mounted as pressurized systems for morphometric analysis and assessment of endothelial function and angiotensin II (Ang II)-induced contractility. ERK1/2 phosphorylation was measured by Western blots, using protein extracts from mesenteric arteries, aorta, heart and kidneys. RESULTS: BP was higher (P < 0.01) in SHR than in WKY rats. PD98059 did not influence BP in either group. Endothelial-dependent relaxation (acetylcholine-induced), which was impaired in SHR, was improved by PD98059 (P < 0.05). Ang II increased contraction, with greater responses in SHR (Emax = 25 +/- 4%) than WKY (Emax = 9 +/- 3%) (P < 0.01). PD98059 reduced Ang II-induced contraction in SHR (Emax = 5.8 +/- 0.4%) and WKY (Emax = 4 +/- 0.4%). Vascular structure was unaltered by PD98059. Vascular and renal ERK1/2 phosphorylation, which was higher in SHR than WKY, was decreased by PD98059 in SHR. CONCLUSION: Short-term treatment with PD98059 improves endothelial function and vascular contractility without influencing BP in SHR. These findings provide evidence that vascular ERK1/2 activity is upregulated and that MEK1/2-sensitive signaling pathways play an important role in the regulation of vascular function in SHR. Acute inhibition of MEK1/2 does not alter blood pressure despite improved endothelial function and reduced arterial reactivity to Ang II.     

High-dose, not low-dose insulin increases the vasoconstrictor effect of norepinephrine in spontaneously hypertensive rats: effects of antihypertensive treatment

The effect of insulin on the vasoconstriction induced by norepinephrine is presently controversial. Therefore, the aims of our study were: (1) to evaluate the effect of low- and high-dose insulin on the concentration-response curve to norepinephrine in small resistance arteries of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) before and after the development of hypertension, and (2) to evaluate the effects of antihypertensive treatment on vascular response to insulin and norepinephrine. Fifty-six rats were included in the study. Six SHR were treated with enalapril and 6 with candesartan cilexetil from the 4th to the 12th week of age, while 10 WKY and 14 SHR were kept untreated. Two additional groups of 10 untreated SHR and 10 WKY were killed at 4 weeks of age, in a prehypertensive phase. Mesenteric small arteries were dissected and mounted on a micromyograph. A dose-response curve to norepinephrine was performed at cumulative concentrations in the presence or absence of low- and high-dose insulin. We found that only high-dose insulin increased the vascular response to norepinephrine in 12-week-old SHR, but not in 4-week-old SHR or in age-matched WKY. The increased responsiveness to norepinephrine disappeared after preincubation of the vessels with a selective inhibitor of endothelin-1 type A receptors. After antihypertensive treatment with enalapril or candesartan cilexetil, the potentiation of the vasoconstrictor response to norepinephrine was abolished. In conclusion, insulin at high, nonphysiological doses seems to induce an increase in the reactivity to norepinephrine in mesenteric small arteries of SHR, possibly mediated by a local production of endothelin-1. Antihypertensive treatment with an ACE inhibitor or an angiotensin II receptor blocker may normalize this altered response. This mechanism may be relevant in the development of hypertension in SHR.     

Streptozotocin-induced diabetes in the spontaneously hypertensive rat.

Spontaneously hypertensive rats (SHR) were more sensitive to the diabetogenic effects of streptozotocin than normotensive Wistar-Kyoto (WKY) rats. Thus, 10 days after intravenous administration of 25 mg/kg streptozotocin in SHR, mean pancreatic insulin content was decreased by 42% (p less than 0.05), and mean plasma glucose concentration was increased from 85 to 215 mg/dl (p less than 0.001), whereas between 37.5 and 50 mg/kg of streptozotocin was required to produce similar effects in normotensive WKY rats. Also, there was a progressive decrease in blood pressure in SHR injected with 25, 35.7, or 50 mg/kg of streptozotocin, whereas blood pressure was progressively increased after streptozotocin in normotensive WKY rats. The opposite effects of streptozotocin-induced diabetes on blood pressure in SHR and WKY rats could be observed at similar degrees of hyperglycemia and are presently unexplained.     

Low-dose streptozotocin-induced diabetes in the spontaneously hypertensive rat

Streptozotocin (STZ, 35 mg/kg body weight) was injected into spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, and plasma glucose and triglyceride concentrations measured 10 days later. Neither mean (+/- SEM) plasma glucose (130 +/- 3 v 136 +/- 3 mg/dL) nor triglyceride (93 +/- 6 v 108 +/- 7 mg/dL) concentrations increased in WKY rats. In contrast, both plasma glucose (141 +/- 3 v 262 +/- 36) and triglyceride (121 +/- 8 v 196 +/- 7 mg/dL) concentrations increased significantly (P less than .01) following administration of STZ in SHR. Furthermore, when SHR previously injected with STZ were fed a diet enriched in fructose, they had a further increase (P less than .01) in both plasma glucose (343 +/- 38 mg/dL) and triglyceride (774 +/- 57 mg/dL) concentrations. Plasma triglyceride concentration also increased significantly (P less than .05) when STZ-injected WKY rats ingested the fructose-enriched diet, but plasma glucose levels still remained within the normal range (152 +/- 5 mg/dL). These results indicate that SHR were more sensitive to the effects of a decrease in pancreatic beta-cell function (STZ) and an increase in insulin resistance (fructose feeding) than WKY rats.     

Dietary alpha-lipoic acid supplementation lowers blood pressure in spontaneously hypertensive rats

BACKGROUND AND OBJECTIVES: In spontaneously hypertensive rats (SHRs), excess endogenous aldehydes bind sulfhydryl groups of membrane proteins, altering membrane Ca2+ channels and increasing cytosolic free calcium and blood pressure. The thiol compound, N-acetyl cysteine, normalizes elevated blood pressure in SHRs by binding excess endogenous aldehydes and normalizing membrane Ca2+ channels and cytosolic free calcium. The aim of the present study was to investigate whether a dietary supplementation of an endogenous fatty acid, alpha-lipoic acid, another thiol compound that is known to increase tissue cysteine and glutathione, can lower blood pressure and normalize associated biochemical and histopathological changes in SHRs. METHODS AND RESULTS: Starting at 12 weeks of age, animals were divided into three groups of six animals each. Animals in the Wistar- Kyoto (WKY) rat control group and the SHR control group were given a normal diet, and the SHR-lipoic acid group was given a diet supplemented with lipoic acid (500 mg/kg feed) for the next 9 weeks. After 9 weeks, systolic blood pressure, platelet [Ca2+]i, plasma insulin and liver, kidney and aortic aldehyde conjugates were significantly higher in SHR controls as compared with WKY rat controls and the SHR lipoic acid group. SHR controls also showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidneys. CONCLUSIONS: Dietary alpha-lipoic acid supplementation in SHRs lowered the systolic blood pressure, cytosolic [Ca2+]i, blood glucose and insulin levels, and tissue aldehyde conjugates, and attenuated adverse renal vascular changes.     

Interaction between AT1 and alpha1-adrenergic receptors in cardiomyopathic hamsters

BACKGROUND: Cross talk between angiotensin AT1 and alpha1-adrenergic receptors has been reported previously and points to the existence of physiologic regulation between the renin-angiotensin system and the sympathetic nervous system at the receptor level. This regulation may play an important role in the control of blood pressure and may be modified in different cardiovascular pathologies. Nevertheless, neither the physiologic actions nor the clinical relevance of the interaction between these 2 receptors has yet been established. To reveal these aspects in relation to heart failure, the interaction between vascular AT1 and alpha1-adrenergic receptors was evaluated in the Syrian cardiomyopathic hamster model. METHODS AND RESULTS: The vascular response of each individual receptor to vasoactive agonist was assessed in the presence and absence of antagonists of the other receptor using aortic rings from 11-month-old Syrian cardiomyopathic hamsters. Age-matched golden hamsters were used as controls. In control hamsters, concentration-response curves for the norepinephrine (NE)-induced contraction were significantly displaced to the left after 100 mmol/L losartan incubation. The maximal tension achieved (Emax) values increased by 26+/-4.3% after incubation (P < .05). Similar results were obtained when 20 micromol/L enalapril was used to block angiotensin II (Ang II) synthesis. NE concentration-response curves were also displaced to the left and Emax increased by 27%+/-8.0% (P < .05). The concentrations that induce 50% of the maximal contraction (EC50) were 22.2+/-0.2 nmol/L for untreated and 27.1+/-2.0 nmol/L for losartan-treated aortic rings (n = 8, P > .05). However, EC50 values were significantly reduced in aortic rings treated with enalapril (7.51+/-0.16 nmol/L, n = 8, P < .05). Blockade of alpha1 receptor with 10 micromol/L prazosin increased the response to Ang II by 32% (n = 6, P < .05). In contrast, when these experiments were repeated in aortic rings from cardiomyopathic animals, no interaction between the 2 receptors was observed. NE concentration-response curves, Emax (9.6%+/-2.8% increase after enalapril. and 5.8%+/-6.5% increase after losartan, P > .05) and EC50 values (14.7+/-0.7 nmol/L without treatment, 17.5+/-1.5 nmol/L with enalapril and 11.1+/-0.8 with losartan, n = 8, P > .05) were similar. Furthermore, in cardiomyopathic animals, prazosin did not modify the vascular response to Ang II. CONCLUSIONS: An interaction exists between vascular AT1 and alpha1-adrenergic receptors in control hamsters but not in cardiomyopathic animals. This interaction seems to be bidirectional and counterregulatory. The lack of this regulation may promote a state of enhanced vascular wall activity, which could contribute to the increased vasoconstriction and total peripheral resistance characteristic of heart failure.     

Effects of dihydropyridines on tension and calcium-45 influx in isolated mesenteric resistance vessels from spontaneously hypertensive and normotensive rats

Contractile tension responses to norepinephrine and depolarizing potassium (80 mM K+), as well as calcium-45 influx stimulated by these agents, were studied in isolated mesenteric resistance vessels (each 100 microM internal diameter) from spontaneously hypertensive rats (SHRs) and from normotensive Wistar Kyoto rats (WKYs). Inhibitory effects of 2 dihydropyridine Ca++ antagonists, PN 200-110 (isradipine) and nisoldipine, on these parameters were also determined. Contractile responses to 80 mM K+ were inhibited by both Ca++ antagonists with the same potency and efficacy in SHR compared with WKY vessels (PN 200-110 IC50 = 2.8 +/- 1.3 X 10(-8) M in SHRs and 2.5 +/- 1.5 X 10(-8) M in WKYs; nisoldipine IC50 = 1.1 +/- 0.4 X 10(-8) M in SHRs and 1.2 +/- 0.9 X 10(-8) M in WKYs). However, contractile responses to norepinephrine (10(-4) M) were inhibited less potently by nisoldipine in SHR vessels (IC50 = 2.2 +/- 0.3 X 10(-9) M) compared with WKY vessels (IC50 = 1.6 +/- 0.6 X 10(-10) M). Similarly, PN 200-110 tended to be less (but not significantly less) potent in SHR vessels (IC50 = 3.3 +/- 1.8 X 10(-8) M) than in WKY vessels (IC50 = 3.4 +/- 0.9 X 10(-9) M); its efficacy was significantly depressed in the SHR vessels (by approximately 20%). When norepinephrine-stimulated calcium-45 influx was determined in the presence of these Ca++ antagonists, a similar profile emerged with respect to a comparison of SHR and WKY vessels. These results support a previously hypothesized alteration in receptor-activated Ca++ influx pathways in SHR mesenteric resistance vessels.     

Reversal of interstitial fibroblast hyperplasia via apoptosis in hypertensive rat heart with valsartan or enalapril

OBJECTIVE: Renin-angiotensin system inhibitors transiently induce apoptosis at the onset of cardiac hypertrophy regression in spontaneously hypertensive rats (SHRs). The focus of this study is to evaluate the cell selectivity of this response. METHODS: SHRs were treated with valsartan or enalapril (30 mg kg(-1) day(-1)) or placebo for 1 to 4 weeks. Stereological and morphological data were obtained from immunohistological analyses. Apoptosis was quantified by DEVDase (caspase-3-like) activity assay and immunoblot analysis of apoptosis-regulatory proteins (Bax and Bcl-2). Identification of the apoptotic cell type was conducted using in situ TUNEL labeling, in conjunction with alpha-sarcomeric actin or lectin immunoreactivity as markers for cardiomyocytes and endothelial cells, respectively. RESULTS: Stereological analysis of the left ventricle revealed significant non-cardiomyocyte hyperplasia in placebo-treated SHRs (239+/-29x10(6) nuclei) as compared to untreated age-matched normotensive Wistar-Kyoto (WKY) rats (107+/-12x10(6)). In contrast, the number of cardiomyocyte nuclei was comparable between untreated SHRs (48+/-4x10(6)) and WKY rats. After 4 weeks of valsartan or enalapril treatment, SHRs showed significant reductions in systolic blood pressure (>28%), left ventricular hypertrophy (>9%) and cardiomyocyte cross-sectional area (>17%). Moreover, these treatments abolished non-cardiomyocyte hyperplasia in SHR left ventricle without affecting cardiomyocyte number, capillary density or number of capillary per cardiomyocyte nucleus. As a mechanism of cell deletion consistent with apoptosis induction, ventricles showed increased caspase-3 activation (>4.5-fold) as well as Bax to Bcl-2 protein ratio (>3.2-fold) within 2 weeks of valsartan or enalapril treatment. Immunohistological analysis revealed a significant increase in TUNEL-positive, lectin-negative non-cardiomyocytes, suggesting a rise in apoptotic interstitial fibroblasts in the left ventricle within 2 weeks of treatment with valsartan or enalapril (>63%), with a return to baseline (0.033+/-0.003%) at 4 weeks. Treatments did not affect right ventricular mass, apoptosis or cellularity. CONCLUSION: Cardiac apoptosis induction during regression of left ventricular hypertrophy reverses interstitial fibroblast hyperplasia in SHRs treated with inhibitors of the renin-angiotensin system.     

Dissociation between calcium influx blockage and smooth muscle relaxation by nifedipine in spontaneously hypertensive rats

Aortic smooth muscle isolated from spontaneously hypertensive rats (SHR) and normotensive, age-matched Wistar Kyoto rats (WKY) was precontracted by potassium chloride. The relaxant effect of nifedipine (NIF) was much more pronounced in SHR than in WKY, while the relaxation produced by nitroglycerin (NTG) was similar in both tissues. EC50s were (in - log [M]) NIF:SHR 13.1 +/- 0.4 and WKY 9.4 +/- 0.2 (p less than 0.05); NTG:SHR 7.35 +/- 0.3 and WKY 7.26 +/- 0.18 (NS). Aortas from SHR were less sensitive to the contractile effect of Ca2+ than their WKY controls (EC50 was 3.18 +/- 0.03 in WKY and 2.76 +/- 0.13 in SHR, p less than 0.05). The relaxant effect of NIF was dissociated from its effect on Ca2+ influx in SHR aortas. NIF 10(-10) M relaxed the muscle by 100% without producing Ca2+ influx blockage, and NIF 10(-9) and 10(-8) M induced Ca2+ influx blockage while the muscle continued in the relaxed state. Chemically skinned aortic fibers from SHR were less sensitive to the contractile effect of Ca2+ than their normotensive (NR) controls (pCa for EC50 was 5.91 +/- 0.05 in SHR and 6.20 +/- 0.03 in NR, p less than 0.05). NIF 10(-10) M depressed the contractile response to Ca2+ significantly more in SHR than in NR skinned fibers (pCa for EC50 for 5.62 +/- 0.09 in SHR and 6.07 +/- 0.07 in NR, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-dependent inhibition by ghrelin of insulin secretion in the mouse

Ghrelin is produced by stomach oxyntic cells and thought to be involved in the regulation of body weight and food intake. We demonstrate here that the peptide inhibits insulin secretion from overnight-incubated mouse islets in the presence of 8.3, 11.1, and 22.2 mmol/liter glucose. Ghrelin was most efficient at 1 nmol/liter and its effect disappeared by raising the dose more than 25 nmol/liter. Also, insulin secretion in the presence of high K(+) concentrations (20 mmol/liter) was inhibited by ghrelin. Furthermore, when administered iv to mice together with glucose (1 g/kg), ghrelin (50 nmol/kg) inhibited both the rapid 1-min insulin response (364 +/- 90 vs. 985 +/- 114 pmol/liter in controls, P < 0.001) and the area under the 50 min curve of insulin concentration (12.6 +/- 1.2 vs. 15.6 +/- 1.2 nmol/liter x 50 min; P = 0.046) without affecting the glucose disposal rate, insulin sensitivity or glucose effectiveness, i.e. glucose disposal independent from any dynamic change in insulin. The insulinostatic effect of ghrelin was inversely related to insulin sensitivity. In contrast, ghrelin had no influence at the lower dose of 5 nmol/kg and only slightly inhibited insulin secretion at the higher dose of 150 nmol/kg. These findings therefore show that ghrelin inhibits glucose-stimulated insulin secretion in the mouse. The effect is dependent on the dose and elicited on distal signaling steps in islet cells. The results suggest that the islet beta-cells are targets for ghrelin.     

Decreased levels of cytochrome P450 2E1-derived eicosanoids sensitize renal arteries to constrictor agonists in spontaneously hypertensive rats

We compared renal interlobar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in terms of cytochrome P450 (CYP) 4A and CYP2E1 protein expression; levels of 20-HETE, 19-HETE, and 18-HETE; and responsiveness to phenylephrine in the absence and presence of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 mumol/L), a CYP4A inhibitor. Relative to data in WKY, arteries of SHR exhibited diminished (P<0.05) CYP2E1 and levels of 19-HETE (66.7+/-6.0 versus 44.9+/-2.8 pmol/mg) and 18-HETE (13.8+/-1.6 versus 7.9+/-0.5 pmol/mg), whereas CYP4A and 20-HETE levels (99.3+/-9.1 versus 98.9+/-12.8 pmol/mg) were unchanged. Phenylephrine contracted vascular rings of SHR and WKY; the R(max) was similar in both strains, but SHR vessels were more sensitive as denoted by the lower (P<0.05) EC50 (0.28+/-0.07 versus 0.71+/-0.12 mumol/L). DDMS decreased 20-HETE and, to a lesser extent, 19-HETE, while increasing (P<0.05) the EC50 for phenylephrine by 475% and 54% in vessels of SHR and WKY, respectively. The desensitizing effect of DDMS was reversed by 20-HETE. Notably, the minimal concentration of 20-HETE that decreased the EC50 for phenylephrine in DDMS-treated vessels was smaller in SHR (0.1 micromol/L) than WKY (10 micromol/L), and the sensitizing effect of 20-HETE was blunted (P<0.05) by the (R) stereoisomers of 19-HETE and 18-HETE. We conclude that the increased sensitivity to phenylephrine in arteries of SHR is attributable to a vasoregulatory imbalance produced by a deficit in vascular CYP2E1-derived products, most likely 19(R)-HETE and 18(R)-HETE, which condition amplification of the sensitizing action of 20-HETE.