Kidney adaptation in nitric oxide-deficient Wistar and spontaneously hypertensive rats

We investigated the renal structural and functional consequences of nitric oxide (NO) deficiency co-treated with angiotensin-converting enzyme inhibitor (ACEi) in 20 adult male Wistar rats and 20 spontaneously hypertensive rats (SHR). The animals were separated into eight groups (n = 5) and treated for 30 days: Control, L-NAME (NO deficient group), Enalapril, L-NAME + Enalapril. The elevated blood pressure in NO deficient rats was partially reduced by enalapril. Serum creatinine was elevated in L-NAME-SHRs and effectively treated with enalapril. The proteinuria was significantly higher only in L-NAME-SHRs, and this was reduced by treatment with ACEi. The glomerular volume density (Vv(gl)) in L-NAME rats, both Wistar and SHR, was greater than in matched control rats, and enalapril treatment effectively prevented this Vv(gl) increase. No significant differences were observed in tubular volume density, Vv(tub), or tubular surface density, Sv(tub), in all Wistar groups. The Vv(tub) was smaller in L-NAME-SHRs than in control SHRs, and this tubular alteration was not prevented by enalapril. The Sv(tub) was not different among the SHR groups. In Wistar rats no changes were seen in vascular surface density, but a greatly increased cortical vascular volume density was seen in the enalapril treated rats. The vascular length density was greatly diminished in NO deficient rats that was effectively prevented with enalapril treatment. The vascular cortical renal stereological indices are normally reduced in SHRs. Administration of enalapril, but not L-NAME, changed this tendency. However, enalapril was not totally effective in preventing vascular damage in SHR NO deficient animals.     

Angiotensin-converting enzyme (kinase II) in pituitary gland of spontaneously hypertensive rats

Angiotensin-converting enzyme (ACE) (kinase II; dipeptidyl carboxypeptidase, EC 3.4.15.1) activity was measured in pituitary gland of young (4-week-old) and adult (18-week-old) male, spontaneously hypertensive rats (SHR) and in age-matched normotensive male Wistar-Kyoto (WKY) control rats. In the three lobes of the pituitary gland ACE activity was significantly higher in young than in adult animals, in both SH and WKY rats. In the anterior lobe, ACE activity was lower in SHR when compared to age-matched Wistar-Kyoto controls. In contrast, ACE activity in the intermediate lobe of the pituitary gland was higher in SHR, and in particular in young animals. The observed differences between young WKY and SH rats in both the intermediate and anterior lobes did not appear to be due to a modified affinity of ACE for the substrate hippuryl-His-Leu, but to alterations in ACE maximal velocity or number of available molecules. No differences in ACE activity were detected between SHR and WKY rats in the posterior lobe. Total protein content was higher in the intermediate lobe and lower in the posterior lobe of young SHR when compared to normotensive controls. The present results suggest the possibility for a role of pituitary ACE in spontaneous (genetic) hypertension in rats.     

Angiotensin II receptor binding in the locus ceruleus of spontaneously hypertensive rats and Wistar-Kyoto rats: A quantitative autoradiographic study with references to hypertension and cardiac/testicular hypertrophy

The locus ceruleus (LC) contains a high density of angiotensin II (All) receptors. The role of All receptors at the LC in genetic hypertension and organ function is unclear. Spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats were studied, and blood pressure of animals was measured using the tail-cuff method. Animals were decapitated and the heart weight (HW) and testicular weight (TW) of animals measured. All receptor binding was carried out by incubating the LC tissue sections with 200 pM [¹²⁵I]-All receptor ligand, and measured using quantitative autoradiography. Results showed that the HW/BW ratio was significantly higher in SHR rats than WKY rats. However, the TW/BW ratio was higher in SHR rats than WKY rats only at two hypertensive stages, whereas All receptor binding capacity in the LC was also statistically higher in SHR rats than WKY rats. Results indicated that cardiac and testicular hypertrophies were related to higher All receptor binding in the LC of SHR rats, when compared with WKY rats. Interestingly, the literature shows that there is an LC-testes axis. In conclusion, this study indicated that All receptors in the LC are associated with genetic hypertension, and testicular weight could be a reasonable index for essential hypertension.     

Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats.

Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP)

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal laminalike material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

Effect of hypertension on angiotensin-(1-7) levels in rats with different angiotensin-I converting enzyme polymorphism

To determine circulating angiotensin-(1-7) [Ang-(1,7)] levels in rats with different angiotensin converting enzyme (ACE) genotypes and to evaluate the effect of hypertension on levels of this heptapeptide, plasma levels of angiotensin II (Ang II) and Ang-(1-7) were determined by HPLC and radioimmunoassay in (a) normotensive F0 and F2 homozygous Brown Norway (BN; with high ACE) or Lewis (with low ACE) rats and (b) in hypertensive F2 homozygous male rats (Goldblatt model). Genotypes were characterized by PCR and plasma ACE activity measured by fluorimetry. Plasma ACE activity was 2-fold higher (p < 0.05) in homozygous BN compared to homozygous Lewis groups. In the Goldblatt groups, a similar degree of hypertension and left ventricular hypertrophy was observed in rats with both genotypes. Plasma Ang II levels were between 300-400% higher (p < 0.05) in the BN than in the Lewis rats, without increment in the hypertensive animals. Plasma Ang-(1-7) levels were 75-87% lower in the BN rats (p < 0.05) and they were significantly higher (p < 0.05) in the hypertensive rats from both genotypes. Plasma levels of Ang II and Ang-(1-7) levels were inversely correlated in the normotensive rats (r = -0.64; p < 0.001), but not in the hypertensive animals. We conclude that there is an inverse relationship between circulating levels of Ang II and Ang-(1-7) in rats determined by the ACE gene polymorphism. This inverse relation is due to genetically determined higher ACE activity. Besides, plasma levels of Ang-(1-7) increase in renovascular hypertension.     

Organization of cells and extracelluar matrix in mesenteric arteries of spontaneously hypertensive rats

Summary Biochemical studies have been used to assess the quantitative changes in elastin and collagen in hypertensive vs. normotensive arteries. However, the relative distribution and organization of these fibrous proteins is likely to be equal in importance to their absolute amounts. In this study we have used scanning electron microscopy in association with selective digestion techniques to assess the organization of cellular and extracellular components of the tunica media of mesenteric arteries of spontaneously hypertensive rats. Superior and small mesenteric arteries were digested with acid, alkali, or bleach to exposure cells, collagen, or collagen and elastin, respectively. We observed that hypertension does not cause a qualitative change in the 3-dimensional arrangement of cells, collagen, or elastin in spontaneously hypertensive arteries when compared to normotensive arteries. However, cells in the superior artery are significantly different in overall shape and surface features when compared to cells of small arteries. These differences in surface morphology of cells are present in hypertensive and normotensive vessels and suggest that superior and small mesenteric artery cells transmit load to the isotropic matrix in different ways. In the elasto-muscular superior artery, force is transmitted across digitations throughout the cell surface. In the muscular small artery, force is transmitted across the tapered, smooth cell surface.     

Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.     

Endogenous angiotensin II enhances atherogenesis in apoprotein E-deficient mice with renovascular hypertension through activation of vascular smooth muscle cells

Renovascular hypertension is one of the most important risk factors in the development of atherosclerosis. However, very little is known about the role of angiotensin II (AII), a key regulator of blood pressure homeostasis, on renovascular hypertension-associated atherogenesis. To study a possible role of AII on atherogenesis, we generated apoE-deficient hypertensive mice with either normal or increased AII production by applying 1-kidney, 1-clip (1K1C) or 2-kidney, 1-clip (2K1C) operation, respectively. Hypertension was successfully achieved in both mice groups, and was persistent for 8 weeks. Atherosclerosis quantification showed a marked increase in lesion area in aortic sinus of 2K1C mice as compared with 1K1C mice, suggesting a potential role of endogenous AII on atherogenesis. In the immunohistochemical analysis, induction of renovascular hypertension with 2K1C for 8 weeks led to an enhanced accumulation of macrophages in the aortic sinus, which was accompanied by a parallel increase in scavenger receptor A (SRA) expression on the macrophages. In in vitro experiments, although treatment of cells with increasing concentrations of AII (0.1 to 10 microM) affects neither SRA expression nor oxLDL uptake by macrophages, conditioned media (CM) derived from AII-stimulated vascular smooth muscle cells (VSMC) increased macrophage uptake of oxLDL in association with an enhanced expression of SRA on the macrophages. These findings suggest that the increased generation of AII in renovascular hypertension may initiate and promote atherosclerosis by activation of VSMC.     

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus

A transgenic mouse model, deficient in kinin B₁ receptor (B₁^−/−) was used to evaluate the role of B₂ receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B₁^−/− fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B₂ receptor, we suggest that this complex was not formed in the stomach deficient in B₁ receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B₁ and B₂ receptors, an interaction between captopril and ACE, B₁ and B₂ receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.     

Unique "delta lock" structure of telmisartan is involved in its strongest binding affinity to angiotensin II type 1 receptor

Angiotensin II type 1 receptor (AT1 receptor) blockers (ARBs) are one of the most popular anti-hypertensive agents. Control of blood pressure (BP) by ARBs is now a therapeutic target for the organ protection in patients with hypertension. Recent meta-analysis demonstrated the possibility that telmisartan was the strongest ARB for the reduction of BP in patients with essential hypertension. However, which molecular interactions of telmisartan with the AT1 receptor could explain its strongest BP lowering activity remains unclear. To address the issue, we constructed models for the interaction between commonly used ARBs and AT1 receptor and compared the docking model of telmisartan with that of other ARBs. Telmisartan has a unique binding mode to the AT1 receptor due to its distal benzimidazole portion. This unique portion could explain the highest molecular lipophilicity, the greatest volume distribution and the strongest binding affinity of telmisartan to AT1 receptor. Furthermore, telmisartan was found to firmly bind to the AT1 receptor through the unique “delta lock” structure. Our present study suggests that due to its “delta lock” structure, telmisartan may be superior to other ARBs in halting cardiovascular disease in patients with hypertension.     

Effects of angiotensin II on the spontaneous activity of rostral ventrolateral medullary cardiovascular neurons and blood pressure in spontaneously hypertensive rats

The interactive role of rostral ventrolateral medulla (RVL) cardiovascular neurons and brain angiotensin II (Ang II) in regulating the arterial blood pressure was examined by recording simultaneously the spontaneous activity of these spinal projecting neurons and the arterial blood pressure in the pentobarbital-anesthetized spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY). It was found that Ang II elicited dose-dependent excitatory responses in a subpopulation of RVL cardiovascular neurons, followed by a subsequent increase in blood pressure. These effects of Ang II were significantly greater in SHR than in WKY. The effects were attenuated or abolished by co-administration of Ang II antagonist, [Sar¹, Ile⁸]-Ang II. Pre-administration of [Sar¹, Ile⁸]-Ang II to RVL using bilateral microinjection attenuated the blood pressure effects of intracerebroventricularly administered Ang II by as much as 70%. These results indicated that spinal projecting RVL cardiovascular neurons are important in mediating the pressor action of Ang II. The enhanced sensitivity and responsiveness of RVL cardiovascular neurons to Ang II may be pertinent to the genesis of hypertension in adult SHR.     

Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.     

Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells

Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.     

Oxidation of Prx2 and phosphorylation of GRP58 by angiotensin II in human coronary smooth muscle cells identified by 2D-DIGE analysis

To study early changes in angiotensin II (Ang II)-induced signaling with post-translational modifications, we analyzed proteins from cultured human coronary smooth muscle cells stimulated with Ang II, using two-dimensional difference gel electrophoresis (2D-DIGE) combined with ProQ Diamond and SYPRO Ruby staining, followed by mass spectrometry or Western blotting. Among 40 proteins identified, peroxiredoxin 2 (Prx2) was oxidized and 58 kDa glucose-regulated protein (GRP58) was phosphorylated after 5 min of Ang II (1 μM) stimulation. Valsartan, a selective Ang II type 1 (AT1) receptor blocker, and N-acetylcysteine, an antioxidant, inhibited both of these modifications, indicating the contribution of AT1 receptor and reactive oxygen species to oxidation of Prx2 and phosphorylation of GRP58 by Ang II.