Spontaneously Hypertensive Rats Vascular Smooth Muscle Cells Research Articles

Functional alterations in cerebrovascular K+ and Ca2+ channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.

Gene expression and functional activity of sodium/calcium exchanger enhanced in vascular smooth muscle cells of spontaneously hypertensive rats.

Effects of hypertension on the function of the Na+/Ca2+ exchanger (NCX) were investigated by analyzing vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Angiotensin II-induced 45Ca2+ efflux from VSMCs mediated by NCX was enhanced by up to 3-fold in SHR compared with WKY, whereas ionomycin-induced Ca efflux mediated by NCX was not different between SHR and WKY. The decline rate from the peak value of intracellular 45Ca2+ concentration ([Ca2+]i) mobilized by angiotensin II was decelerated by removal of extracellular sodium (Na+o) in SHR but not in WKY. Gene expressions of NCX subtype 1 and angiotensin II receptor type1A assessed by quantitative RT-PCR were increased by 1.3- and 1.5-fold, respectively in SHR compared with WKY. NCX protein was also increased 1.6-fold in SHR compared with WKY. MEK inhibitor, PD98059, partly blocked the Nao-dependent acceleration of the [Ca2+]i recovery rate and tyrosine kinase inhibitor, genistein, diminished it in SHR. Genistein decreased angiotensin II-induced Nao- dependent 45Ca2+ efflux. However, angiotensin II did not enhance the tyrosine phosphorylation of NCX. These results suggest that acceleration of Ca2+ efflux from VSMCs of SHR was at least partly due to the enhancement of functional activity of NCX via increased gene expression and tyrosine phosphorylation in connection with hypertension.

Altered insulin-like growth factor-1 and nitric oxide sensitivities in hypertension contribute to vascular hyperplasia

Vascular medial thickening, a hallmark of hypertension, is associated with vascular smooth muscle cell (VSMC) hypertrophy and hyperplasia. Although the precise mechanisms responsible are elusive, we have shown that strain induced regulation of autocrine insulin-like growth factor-1 (IGF-1) and nitric oxide (NO) reciprocally modulate VSMC proliferation. Therefore, we investigated potential IGF-1 and NO abnormalities in young (10-week-old) spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and their respective VSMC ex vivo. The SHR had increased mean arterial pressure (173 ± 2 v 128 ± 3 mm Hg, n = 24, P < .05) but similar pulse pressures (31 ± 2 v 30 ± 3 mm Hg; P > .05) v WKY. The SHR exhibited increased aortic wall thickness in comparison with WKY (523 ± 16 v 355 ± 17μm; P < .05). No differences were seen in plasma combined NO 2 and NO 3 (NO x) (0.48 ± 0.11 mmol/L for WKY v 0.58 ± 0.18 mmol/L for SHR) or plasma IGF-1 (1007 ± 28 ng/mL for WKY v 953 ± 26 ng/mL for SHR). Aortic VSMC from SHR displayed enhanced proliferation in comparison with WKY ( P < .05). Underlying this enhanced proliferation was altered SHR VSMC sensitivity to the antiproliferative NO donor 2,2"[Hydroxynitrosohydrazono] bis-ethanimine (DETA-NO) (ID 50: 270 ± 20 mmol/L for SHR; 150 ± 11 mmol/L for WKY; P < .05). Basal cyclic guanosine monophosphate (cGMP) secretion from SHR VSMC was 65-fold greater than that seen from WKY ( P < .001). In response to DETA-NO, cGMP secretion from SHR VSMC increased modestly (1.5-fold; P < .01), whereas treatment of WKY VSMC resulted in a 26-fold ( P < .001) increase in cGMP. The SHR VSMC did not respond to exogenous IGF-1, whereas WKY VSMC exhibited a dose dependent increase in proliferation with IGF-1 (10 −10 to 10 −7 mol/L). These data suggest that VSMC hyperplasia in early hypertension is not reflected by imbalances in plasma IGF-1 or NO. Rather, altered SHR VSMC sensitivity to NO is likely responsible in part for the observed hyperproliferation seen in early stages of hypertension.

Coculture of Vascular Endothelial Cells and Smooth Muscle Cells from Spontaneously Hypertensive Rats

The effect of endothelium‐released vasoactive factors on vascular smooth muscle cell (VSMC) proliferation was studied in a coculture system. Isolated aortic endothelial cells and smooth muscle cells from 4‐week‐old spontaneously hypertensive rats (SHR) and age‐matched Wistar–Kyoto (WKY) rats were cocultured. After coculture, the VSMC proliferation rate was examined by 3H‐thymidine incorporation assay and the levels of the vasoactive factors in medium were determined by enzyme immunoassay (EIA). The results indicate that the proliferation rate of VSMCs in SHR was significantly higher than in WKY rats when VSMCs were cultured alone. When SHR vascular endothelial cells (VECs) were cocultured with VSMCs, the proliferation rate of SHR VSMCs was enhanced; however, there was no growth promoting effect in WKY VSMCs. When WKY VECs were cocultured with VSMCs, no VSMC proliferation effect was observed. When VSMCs were cultured alone, the endothelin‐1 (ET‐1) secretion in SHR was significantly higher than in WKY rats. When VECs and VSMCs were cocultured, the ET‐1 concentration increased in both SHR VEC and WKY VEC coculture groups in a similar manner; but the SHR VECs tended to release more thromboxaneA2 (TXA2) and less PGI2 than WKY VECs. These results suggest that some kind of interaction between SHR VSMCs and SHR VECs is responsible for the high proliferation of SHR VSMCs but not the effects of SHR VECs per se.

Proteoglycans production by aortic vascular smooth muscle cells from hypertensive rats

Remodeling of large and small arteries contributes to the development and complications of hypertension. Artery structural changes in chronic sustained hypertension include vascular smooth muscle cells (VSMC) proliferation and extracellular matrix (ECM) modifications. Extracellular constituents such as proteoglycans (PGs), may modulate vascular stiffness and VSMC growth and differentiation. We examined the effect of growth factors on secreted and membrane-bound PGs synthesis by cultured aortic smooth muscle cells (SMC) from 12- to 14- week-old spontaneously hypertensive rats (SHR) and age-matched Wistar rats. After stimulation with platelet-derived growth factor (PDGF-BB), 10% fetal calf serum (FCS) or 0.1% FCS as control, PGs synthesis (dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method using both [3H]-glucosamine and [35S]-sodium sulfate which are incorporated into all complex carbohydrates or only into sulfated dysaccharides, respectively. Data are presented as percent of the control (0.1% FCS). SHR VSMC displayed a significantly greater synthesis of M-ECM [3H]-PGs than Wistar rat cells, with both treatments, but no differences in M-ECM [35S] uptake were found in any case. In the P-ECM, both PDGF-BB and 10% FCS produced a greater effect on [3H]-PGs and sulfated PGs synthesis in VSMC from SHR. An important change seen in SHR cells was a significant decreased sulfation, assessed by [35S]/[3H] ratio, in basal and stimulation conditions. Present results indicate the existence of changes in PGS synthesis and modulation in VSMC from a conduit-artery of SHR and support the pathophysiological role proposed for matrix proteoglycans in the vascular wall changes associated to hypertension and related vascular diseases as atherosclerosis.

Altered PYK2 phosphorylation by ANG II in hypertensive vascular smooth muscle

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased cell growth compared with normotensive Wistar-Kyoto rats (WKY). ANG II stimulates growth via G(q)-protein-coupled signaling that involves changes in cytosolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. This study examines the role of the proline-rich tyrosine kinase 2 (PYK2) in hypertensive VSMC. Basal PYK2 phosphorylation in SHR VSMC was increased compared with WKY (0.44 +/- 0.02 vs. 0.20 +/- 0.02-fold). ANG II-induced activation of PYK2 in SHR VSMC was of greater magnitude (2.2 +/- 0.2-fold in SHR; 1.4 +/- 0.1-fold in WKY) and occurred more rapidly (peak activation at 2 min in SHR vs. 5 min in WKY). This effect was blocked by pretreatment with the [Ca(2+)](i) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the PKC inhibitor chelerythrine. Basal and ANG II-stimulated c-Fos expression was increased in SHR versus WKY VSMC. PYK2 downregulation with antisense oligonucleotides blocked ANG II-induced c-Fos expression. Increased PYK2 activation may be altered signaling cascades that regulate cell growth in hypertensive VSMC.

Effects of superoxide on signaling pathways in smooth muscle cells from rats.

The effects of hypoxanthine and xanthine oxidase-induced superoxide anion were evaluated on various signal transduction pathways in aortic smooth muscle cells (SMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Superoxide increased inositol 1,4,5-tris-phosphate (IP(3)) formation in a concentration- and time-dependent manner in both strains but more markedly in SMCs from SHR. Various antioxidants significantly decreased the superoxide-induced IP(3) formation in both strains. In addition, tyrosine kinase inhibitors, genistein and tyrphostin A25, inhibited the superoxide-induced IP(3) formation more markedly in SHR than in WKY. Moreover, superoxide decreased the basal level of cGMP to a greater extent in SHR and also suppressed the rise in cGMP induced by S-nitroso-N-acetylpenicillamine. In addition, the superoxide-induced increase in IP(3) formation was significantly inhibited by guanylyl cyclase stimulator S-nitroso-N-acetylpenicillamine but was potentiated by ODQ (a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one) and KT5823 (a cGMP-dependent protein kinase inhibitor), with a greater effect in SHR. Finally, the superoxide-enhanced IP(3) formation was not accompanied by simultaneous changes in cAMP levels, and inhibition of the adenylyl cyclase pathway did not modify the superoxide-induced IP(3) formation. Our results thus demonstrate a stimulatory effect of superoxide on IP(3) formation, mediated by the tyrosine kinase-coupled phospholipase C(gamma) activity, and an inhibitory effect of superoxide on cGMP formation in vascular SMCs. The increased reactivity of the phospholipase C pathway and the decreased cross inhibition of the IP(3) pathway by cGMP in the presence of superoxide may underlie the altered functions of vascular SMCs in SHR.

Comparison of calcium import as a function of contraction in the aortic smooth muscle of Sprague-Dawley, Wistar Kyoto and spontaneously hypertensive rats.

Genetic variations of far-reaching consequences have been established between spontaneously hypertensive rats (SHR) and their controls, Wistar Kyoto rats (WKY). The SHR strain is the most widely used model for the study of genetic hypertension. Calcium homeostasis in the vascular smooth muscle (VSM) is controlled by calcium channels and calcium pumps located in both VSM and the overlying endothelial cells that line the large blood vessels and the heart. Hypertension adversely affects calcium homeostasis. Investigations on the import of calcium from extracellular spaces with alpha1-adrenergic stimulation as a function of contractility of VSM cells in SHR and WKY were made and compared with the contractility observed in VSM cells of Sprague-Dawley (CD) rats. Experiments were performed on rings from thoracic aortas of three strains with endothelial lining intact or removed to discern the paracrine control of endothelium on contractility in response to calcium import. The internal stores of Ca2+ were depleted by repeated alpha 1-adrenergic stimulation with phenylephrine (PE) and refilling of these stores was prevented by cyclopiazonic acid (CPA) and/or thapsigargin (TG), two known inhibitors of Ca2+ATPase, the enzyme that drives sarcoplasmic calcium pumps. The two components of tonic muscular contraction, T I and T II, which are known to be due to the flow of Ca2+ from the extracellular gradient controlled via the poly-phosphoinositide cascade and nifedipine sensitive Ca2+ channels were found to be variable among these strains. Implications of these variations are discussed in this report

Mechanisms of Increased NGF Production in Vascular Smooth Muscle of the Spontaneously Hypertensive Rat

The spontaneously hypertensive rat (SHR) was developed as a genetic model of essential hypertension.In vivoandin vitroevidence demonstrates that vascular smooth muscle cells (VSMCs) from the SHR produce more nerve growth factor (NGF) than the normotensive Wistar-Kyoto (WKY) control strain. This increased NGF production is accompanied by excessive innervation of target tissues in the SHR. In the present study, a sensitive, competitive, quantitative, reverse-transcriptase polymerase chain reaction (C Q RT-PCR) assay is characterized and used to analyze levels of NGF mRNA in cultured VSMCs derived from the SHR and WKY strains as well as bladder tissue. Differences in NGF secretion rates between SHR and WKY VSMCs were partially due to an increased stability of NGF mRNA in SHR VSMCs. Following treatment with platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β1) to elevate NGF production, the half-life of the NGF mRNA was 104.5 ± 18.0 min in SHR VSMCs, compared to only 36.5 ± 11.6 min in WKY VSMCs. Sequence analysis of the 3′ untranslated region (UTR) revealed no strain differences incis-acting sequences potentially involved in determining mRNA stability. Thus, it seems unlikely to be a 3′UTR mutation that prolongs mRNA lifetime. Rather, differential regulation of an RNA-binding protein may play a role in the abnormal NGF mRNA stability in SHR VSMCs. SHR VSMCs also demonstrate an increased translational efficiency of NGF protein; more NGF protein is synthesized per unit of NGF mRNA. The use of a C Q RT-PCR assay has allowed the determination that abnormal NGF mRNA stabilization as well as altered translational efficiency may contribute to excess NGF synthesis and progressive hypertension in the SHR.

Transforming Growth Factor-β and Receptor Tyrosine Kinase–Activating Growth Factors Negatively Regulate Collagen Genes in Smooth Muscle of Hypertensive Rats

Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-beta (TGF-beta) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-beta on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-beta isoforms -beta1, -beta2, or -beta3 induced rapid, transient elevations in mRNAs encoding collagens alpha1(I), alpha2(I), and alpha1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen alpha1(III)] to ninefold [collagen alpha1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-beta isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-beta1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-beta isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-beta did not affect the time course or magnitude of the changes in collagens alpha1(I), alpha2(I), or alpha1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-beta1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-beta, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-beta responsiveness.

A 90-kD Na + -H + Exchanger Kinase Has Increased Activity in Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.

Endothelium stimulates the vascular smooth muscle cell Na/K pump of normotensive and hypertensive rats by a protein kinase C pathway.

To investigate the mechanisms involved in the endothelial stimulation of the vascular smooth muscle cell Na/K pump and their possible alteration by hypertension. The Na/K pump activity of vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) was studied using the radioactive analogue of K+ 86Rb+. Conditioned medium of bovine endothelial aortic cells was used to investigate the endothelial modulation of VSMC Na/K pump activity. Conditioned medium enhanced VSMC Na/K pump activity (ouabain-sensitive 86Rb+ uptake), this effect being higher in SHR cells. This stimulatory effect was neither modified in Na(+)-loaded cells from both rat strains nor inhibited by the Na/H exchange blocker amiloride. Permeable analogues of cyclic adenosine and guanosine monophosphates did not modify the baseline VSMC Na/K pump activity of WKY rats and SHR, and subsequently the guanylate cyclase inhibitor methylene blue did not alter the conditioned medium-induced stimulation of the pump. However, the Ca(2+)-channel inhibitor nifedipine reduced the Na/K pump stimulation by conditioned medium, this decrease being higher in WKY rat than in SHR VSMC. Moreover, treatment with phorbol 12,13-dibutyrate for 24 h or with the protein kinase C inhibitor staurosporine for 15 min reduced the conditioned medium-induced Na/K pump activation in both VSMC cultures. Na/K pump stimulation by conditioned medium of endothelial cells is mediated mainly via activation of protein kinase C in VSMC from either WKY or SHR VSMC. However, SHR VSMC show some alterations in their intracellular signalling pathways.

Inducible nitric oxide synthase in vascular smooth muscle cells from prehypertensive spontaneously hypertensive rats

The inducible nitric oxide synthase (iNOS) present in vascular smooth muscle cells (VSMC) may play a role in the generation of nitric oxide (NO) in the vascular wall, regulating blood vessel tone in normotension and hypertension. In this study the effect of interleukin (IL)-1β, a cytokine that induces iNOS, on NO generation (measured as nitrite), cyclic guanosine monophosphate (cGMP) generation, and steady-state abundance of iNOS mRNA were examined in VSMC from 3 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, during the period preceding the elevation of blood pressure. With cell density dependent variations in nitrite production eliminated, VSMC from SHR and WKY did not differ in NO generation except after prolonged incubation (30 h), when SHR cells produced less NO. However, cGMP concentrations associated with IL-1β stimulation were significantly smaller in SHR VSMC than in cells from WKY. IL-1β stimulation resulted in increased abundance of iNOS mRNA to the same extent in both WKY and SHR VSMC. Inhibitors of NOS, N G-monomethyl- l-arginine (L-NMMA) and N ω-nitro- l-arginine methyl ester (L-NAME), did not block the induction of iNOS mRNA, although nitrite production and cGMP generation were inhibited. The protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin-D almost completely blocked the production of nitrite in cells from both strains of rats. Actinomycin-D completely blocked the induction of iNOS mRNA by IL-1β in cells from both strains of rats, whereas cycloheximide partially blocked its synthesis in WKY, but had no significant effect on IL-1β induced expression of iNOS mRNA in SHR VSMC. Thus, IL-1β controls iNOS gene expression at the transcriptional level, and an intermediate labile protein, whose synthesis is inhibited by cycloheximide, is required for IL-1β stimulated induction of iNOS mRNA transcription in WKY cells but not in SHR. We conclude that although iNOS is expressed to similar extent in VSMC of prehypertensive SHR and WKY and similar amounts of NO are initially generated, there are differences between the VSMC of SHR and WKY in the regulation of the transcription of iNOS mRNA, there is a lower sustained production of NO, and there is a reduced generation of cGMP in response to IL-1β stimulated NO production. These differences between VSMC from prehypertensive SHR and WKY may indicate a pathophysiological role of iNOS in early blood pressure elevation in SHR.

Vascular effects of metformin. Possible mechanisms for its antihypertensive action in the spontaneously hypertensive rat.

Metformin, an antidiabetic agent, potentiates insulin action and reduces insulin resistance. We examined the antihypertensive effects and vascular effects of metformin in spontaneously hypertensive rats (SHR). Wistar-Kyoto normotensive (WKY) and SHR were injected with metformin (100 mg/kg) or saline subcutaneously twice daily for 4 weeks. Blood pressure was recorded by a tail-cuff plethesmographic method. Metformin treatment significantly attenuated (P < .05) the increase in blood pressure in metformin treated SHR versus untreated control SHR. At the end of the experimental period of 4 weeks, metformin-treated SHR had a mean blood pressure that was 34 mm lower than that of untreated SHR. Metformin treatment had no significant effect on blood pressure in WKY rats. Treatment of SHR aortic smooth muscle (SM) cells with metformin (2 micrograms/mL) for 24 h significantly decreased (P < .05) arginine vasopressin- and thrombin- stimulated increase in [Ca2+]i. However, metformin treatment did not have a significant effect on the basal [Ca+]i. Incubation of SHR aortic SM cells with OH-L-arginine (25 to 100 mumol/L) for 24 h increased nitrite production in a dose dependent manner. Metformin (5 micrograms/mL) treatment of SM cells increased nitrite production at all concentrations of OH-L-arginine; however, differences were significant (P < .05) only at 25 and 50 mumol/L OH-L-arginine. These results suggest that metformin may be decreasing arterial pressure in the SHR, at least in part, by attenuating the agonist-stimulated [Ca2+]i response in SHR vascular smooth muscle cells.

Activation of Nitric Oxide Synthesis in Vascular Smooth Muscle Cells and Macrophages During Development in Spontaneously Hypertensive Rats

We previously showed that the lymphocyte proliferation response was significantly suppressed in spontaneously hypertensive rats (SHR) and that this depressed response was due to excessive production of nitric oxide (NO) in macrophages and vascular smooth muscle cells (VSMC). Whether lymphocyte depression and activation of NO synthesis are related to age and development of hypertension remains unclear. The present study addresses such a correlation by examining the time course of development of hypertension, NO synthesis alteration and lymphocyte depression in SHR. Our results show that 1) SHR spleen cell proliferation responses are depressed at 4, 8, and 12 weeks and 1 year of age, with the lowest response occurring at 4 weeks of age; 2) this depressed response is corrected by either NO synthase inhibitor or removal of macrophages from spleen cells; 3) NO production by SHR spleen macrophages is significantly higher in all age groups; 4) upon stimulation with lipopolysaccharide or cytokines, SHR VSMC produce a significantly greater amount of NO in all age groups; 5) the increase in NO synthesis in VSMC correlates significantly with the rise in blood pressure in SHR. However, statistical correlation analysis suggests that lymphocyte depression and the alteration of NO synthesis in macrophages were not associated with either age or increased blood pressure in SHR. On the contrary, the activation of NO synthesis in VSMC can be statistically correlated with elevated blood pressure throughout the development of hypertension in SHR. Nevertheless, the results also suggest that a general alteration in the NO synthesis system may exist in SHR.

Sodium ionophore converts growth manner of vascular smooth muscle cells from spontaneously hypertensive rats

Vasoconstrictor peptides such as endothelin (ET) cause hypertrophy of vascular smooth muscle cells (VSMC) in Wistar Kyoto rats (WKY) and hyperplasia in spontaneously hypertensive rats (SHR). They also induce an increase in Na+ concentration ([Na+]i) and activate protein kinase C (PKC) independently. Therefore, we tested the hypothesis that the increase in [Na+]i may be involved in the conversion of growth manner under activated PKC in SHR VSMC. 10(-7) M phorbol ester (TPA) increased the diameter and protein content of VSMC from both strains under 18% serum conditions. Further addition of 10(-6) M gramicidin (Na+ ionophore) converted TPA-induced hypertrophy to hyperplasia, which was due to the quick transition from S to G2/M phase, only in SHR VSMC. Western blot analysis showed that serum- and TPA-induced tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was potentiated by 10(-6) M gramicidin in SHR. [Na+]i, which was measured by sodium-binding benzofuran isophthalate (SBFI), was increased about 35 mM by 10(-6) M gramicidin in both strains, but TPA did not affect basal [Na+]i and the gramicidin-induced increase in [Na+]i. We conclude that sodium ionophore may convert hypertrophy to hyperplasia synergistically with activated PKC in SHR VSMC, possibly by MAP kinase phosphorylation.

Altered signalling in vascular smooth muscle from spontaneously hypertensive rats may link medial hypertrophy, vessel hyperinnervation and elevated nerve growth factor.

1. Secretion of nerve growth factor (NGF) by cultured vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto (WKY) strain was measured via two site immunoassay (ELISA). 2. Basal NGF secretion rates of quiescent SHR VSMC in serum-free culture medium were elevated compared to similar WKY VSMC. 3. SHR VSMC displayed increased NGF secretion in response to activation of sympathetic neurotransmitter receptors while VSMC of WKY were largely unresponsive to the agents (phenylephrine, isoproterenol, alpha-beta-methyl ATP, neuropeptide Y). 4. Mitogenic stimulation with platelet-derived growth factor (PDGF) raised SHR NGF secretion rates almost three times more than PDGF increased WKY secretion. 5. SHR VSMC also failed to demonstrate normal inhibitory control over NGF secretion seen in WKY and previously in Sprague-Dawley and Wistar strain VSMC with adenylate cyclase activation and down-regulation of protein kinase C. High concentrations of forskolin stimulated, instead of inhibiting, secretion in SHR. Stimulation was also seen after pretreatment with phorbol ester for 24 h while this inhibited secretion in the WKY. 6. These results confirm that the SHR VSMC are hyperresponsive to growth stimuli such as contractile agonists and mitogens. This hyperresponsiveness includes an abnormal control over NGF secretion such that normally inhibitory treatments stimulate NGF output in the SHR. 7. Because the SHR demonstrates important defects in the major intracellular growth-signalling systems that also regulate NGF output and vessel innervation, the predicted result of the defects is a destructive feed-forward cycle of growth and innervation. This is the SHR phenotype in vivo.

Angiotensins differentially activate phospholipase d in vascular smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats

We previously showed that angiotensin (Ang) II activates phospholipase D (PLD) through AT1 receptors in vascular smooth muscle cells (VSMC) isolated from Sprague-Dawley rats [Freeman and Tallant, Biochem J. 304:543-548, (1994)]. In the present study, we compared activation of PLD by angiotensin peptides in VSMC from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats. Ang II caused a dose-dependent increase in PLD activity in VSMC from both rat strains. However, the response to Ang II in VSMC from hypertensive rats was approximately three times higher than that observed in VSMC from normotensive controls. Furthermore, Ang II-induced activation of PLD in VSMC from hypertensive rats was significant within 1 min, whereas significant increases in PLD activity in cells from normotensive rats were not seen until 10 min after exposure to Ang II. Ang-(2-8) caused a similar increase in PLD activity which was three times higher in SHR VSMC than in WKY controls. In contrast, Ang-(1-7) did not affect PLD activity in either smooth muscle cell population. The Ang II-mediated increases in PLD activity in VMSC from both rat strains were completely blocked by AT1 receptor antagonists (EXP 3174 or L-158,809). Conversely, the AT2 receptor antagonist PD 123177 (1 mumol/L) was ineffective. Thus Ang II stimulation of PLD in VSMC derived from both the hypertensive and normotensive rat aorta and the accumulation of its metabolites (e.g., phosphatidic acid and diacylglycerol) is coupled to activation of AT1 receptors predominantly and occurs in response to Ang II or Ang-(2-8) but not Ang-(1-7). Moreover, activation of PLD by angiotensins in VMSC from the SHR is significantly more robust than that observed in VSMC from the normotensive WKY rat. We conclude that increased activation of PLD by Ang II in genetically-induced hypertension may reflect an additional mechanism linking enhanced contractile responses to enhanced growth.

Altered regulation of nerve growth factor secretion by cultured VSMCs from hypertensive rats.

Vascular tissues from spontaneously hypertensive rats (SHR) exhibit increased nerve growth factor (NGF) levels and increased density of sympathetic innervation compared with those from normotensive Wistar-Kyoto (WKY) rats. The present study asked whether basal NGF secretion or secretion elicited by agents analogous to sympathetic neurotransmitters differ in cultured vascular smooth muscle cells (VSMCs) from SHR and WKY rats. VSMCs were maintained in serum-free medium (SFM) for 72 h and then treated and sampled at 4, 6, 8, and 24 h. Conditioned medium was assayed for NGF using a two-site enzyme-linked immunoassay. NGF secretion by SHR (19.2 +/- 4.6 pg.well-1.48 h-1) and WKY VSMCs (16.7 +/- 5.4 pg.we..-1.48 h-1) was similar in cultures grown in serum-containing medium, whereas SHR VSMCs maintained in SFM secrete more NGF than WKY VSMCs (9.1 +/- 1.9 vs. 2.9 +/- 0.4 pg.well-1.24 h-1, respectively). Treatment of cultures with phenylephrine (0.1-10 microM), neuropeptide Y (1-1,000 nM), or alpha beta-methyleneadenosine 5'-triphosphate (10 and 100 microM) had no effect on NGF secretion by WKY VSMCs, while increasing NGF secretion by SHR VSMCs. Treatment with isoproterenol (0.1-10 microM) decreased NGF secretion by WKY VSMCs but not SHR VSMCs. These data indicate that the regulation of NGF secretion by sympathetic neurotransmitter receptors is different for cultured VSMCs from SHR and WKY rats. If similar differences exist in vivo, they could account for the alterations in NGF levels and sympathetic innervation that are observed.

Altered β-adrenergic regulation of Na-K-Cl cotransport in cultured smooth muscle cells from the aorta of spontaneously hypertensive rats role of the cytoskeleton network

To verify the hypothesis of Na-K-Cl cotransport (COTR) involvement in ion transport abnormalities as revealed in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR), we compared the rate of ouabain-insensitive, bumetanide-inhibited 86Rb influx in quiescent and growing cultures of VSMC from the aorta of SHR and normotensive (BN.lx) rats and its regulation by the cAMP signaling system. Basal COTR was not altered in quiescent cells from SHR but was decreased by 30% to 40% (P < .02) in growing SHR VSMC as compared to BN.lx rats. In quiescent BN.lx VSMC, isoproterenol inhibited COTR by 50% and induced cell shape transition of > 90% of cells, resulting in the appearance of rounded VSMC with arborized cytoplasms. In contrast, isoproterenol elicited cell shape transition in only 50% of quiescent SHR VSMC and did not modify COTR. In growing cells, it decreased COTR by 85% to 95% and altered cell morphology in > 95% of VSMC without differences between SHR and BN.lx rats. Neither inhibitors of protein kinase A (H-89 and KT-5720) nor an inhibitor of phosphoprotein phosphatase (okadaic acid) affected cell shape transition and COTR suppression in isoproterenol-treated VSMC. The COTR suppression and cytoplasm arborization was also demonstrated by the addition of cytochalasin B, a disintegrator of microfilament bundles, and staurosporine, an inhibitor of protein kinase C. The effects of these compounds on COTR and SHR and BN.lx VSMC morphology were not different. The calmodulin antagonist R24571 decreased COTR by 60% to 70% in quiescent BN.lx VSMC and did not modify this carrier in SHR VSMC.(ABSTRACT TRUNCATED AT 250 WORDS)