Spontaneously Hypertensive Rats Vascular Smooth Muscle Cells Research Articles

Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo. An autocrine mechanism promoting hypertensive vascular hypertrophy.

To clarify the role of PDGF A-chain in hypertensive vascular hypertrophy of spontaneously hypertensive rats (SHRs), we studied levels of PDGF A-chain gene expression and transcription factors related to the gene in vascular smooth muscle cells (VSMCs) of SHRs in vivo. RNase protection assay and in situ hybridization showed that PDGF A-chain mRNA levels in VSMCs of SHRs were twofold higher than in those of normotensive Wistar-Kyoto rats. Gel retardation assays showed that levels of Sp1 and AP-2 in VSMCs of SHRs were twofold more abundant than in those of Wistar-Kyoto rats. Treatment with four pharmacologically different species of antihypertensive drugs for 2 wk decreased the levels of both PDGF A-chain mRNA and Sp1, but not AP-2 level in VSMCs of SHRs with regression of aortic hypertrophy, indicating that increases in levels of both PDGF A-chain mRNA and Sp1 in VSMCs of SHRs were associated with high blood pressure. These results suggest that high blood pressure is a stimulus which upregulates PDGF A-chain gene expression in VSMCs of SHRs, resulting in an autocrine enhancement in hypertensive vascular hypertrophy, and that the activation of the gene may be mediated through increases in Sp1 in these cells.

Carvedilol, a novel cardiovascular agent, inhibits development of vascular and ventricular hypertrophy in spontaneously hypertensive rats.

The effects of carvedilol, a novel cardiovascular agent, were evaluated in developing spontaneously hypertensive rats (SHR) for effects on hemodynamics, and the ability to effect the development of left ventricular, and vascular hypertrophy associated with chronic hypertension. Chronic oral administration of low dose carvedilol (20 mg/kg/day) was initiated when rats were 5 weeks of age, and experiments progressed until 14 weeks of age. Carvedilol-treated SHR had significantly reduced systolic blood pressures and heart rates throughout the duration of the experiment, and had significantly reduced ventricle/body weights by approximately 9.0%. Morphologic analysis of tertiary branches of the mesenteric artery revealed that carvedilol-treated SHR had significant reductions in medial cross-sectional area. Carvedilol produced concentration-dependent inhibition of basal [3H]thymidine incorporation in cultured SHR vascular smooth muscle cells, as well as by stimulation produced by PDGF (1 nM), EDGF (1 nM), thrombin (0.5 U/ml), or endothelin-1 (1 nM), indicating that carvedilol had direct anti-mitogenic activity. The present studies demonstrate that low dose carvedilol produced sustained reductions in blood pressure and heart rate in developing SHR that were accompanied by significant inhibition in the development of vascular and myocardial hypertrophy. The morphological changes induced by carvedilol may be mediated by a combination of hemodynamic effects, as well as by direct anti-mitogenic effects on vascular smooth muscle.

Platelets, Growth Factors, and Vascular Smooth-Muscle Cells in Hypertension and Diabetes

Inappropriate vascular smooth-muscle cell (VSMC) growth is the hallmark of vascular pathology in essential hypertension and diabetic macroangiopathy, whereas platelets constitute an important regulator of vessel wall homeostasis because of their content of various growth factors. Numerous abnormalities exist in platelet functions in diabetes and hypertension, such as enhanced activity and altered adhesion and aggregation. Increased thromboxane (TX2) production is characteristic of diabetes, and an elevation of intracellular free Ca2+ is found in platelets of hypertensive patients. By studying the growth patterns of VSMC from spontaneously hypertensive rats (SHRs) vs. those obtained from their normotensive counterparts, Wistar-Kyoto (WKY) rats, we have demonstrated that VSMC from SHRs exhibited a higher specific growth rate, abnormal contact inhibition, and accelerated entry into the S phase of the cell cycle. Moreover, they were hyperresponsive to many growth factors such as calf serum, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor beta 1 (TGF beta 1), and insulin. Additive effects were observed for EGF and PDGF or EGF and insulin. These intrinsic growth anomalies in cells of hypertensive origin persist in culture indicating their putative primary role in the pathogenesis of hypertension. Endogenous TGF beta 1 revealed an augmented expression of its message levels in SHR VSMC, the difference in mRNA between both strains being more pronounced at high cell density. Further, TGF beta 1 protein synthesis and secretion in VSMC culture were confirmed by immunoprecipitation of de novo labeled TGF beta 1. At high cell density, which most likely represents the physiological state of VSMC, plasmin, an activator of TGF beta 1, significantly stimulated DNA synthesis of VSMC in both strains. The reverse effect was obtained at low cell density. Yet, the fold stimulation was higher in WKY rats, suggesting that TGF beta 1 may be partially activated in SHR VSMC. This is supported by the inhibition of baseline DNA synthesis by TGF beta 1 neutralizing antibody in VSMC of hypertensive origin and not of normotensive controls. TGF beta 1 antisense oligodeoxynucleotide (ODN) nearly normalized the increased proliferation of SHR VSMC in culture. On the other hand, growth-promoting activity (GPA) in platelets of either diabetic or hypertensive patients was higher than in platelets of healthy controls and was found to be normalized by intensive insulin therapy in insulin-dependent diabetic patients. In hypertensive patients, however, hydrochlorothiazide (HCTZ)--even in low doses (25 mg/day)--enhanced the GPA in platelets, whereas other antihypertensive agents such as indapamide, atenolol, and captopril, had neutral effects.(ABSTRACT TRUNCATED AT 400 WORDS)

OPC-13340, a new dihydropyridine calcium channel blocker attenuates rapid vascular smooth muscle cell growth in spontaneously hypertensive rats.

We investigated the mechanism of the antimitotic effects of calcium channel blockers in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). VSMC from SHR exhibited rapid proliferation through a quick transition from the G0/G1 to the DNA synthetic (S) phase and from the S to the G2/mitotic (M) phase, whereas the DNA synthetic rate itself was equal to that of Wistar-Kyoto rats (WKY). OPC-13340, a new dihydropyridine calcium channel blocker, dose-dependently decreased incorporation of [3H]thymidine into the DNA fragments in randomly cycling VSMC in SHR. Cell cycle analysis showed that the rapid transition from the S to the G2/M period was restored by OPC-13340 to the control level in WKY, whereas the quick transition from G0/G1 to S was unaffected. This antimitotic effect of OPC-13340 was reflected by attenuation of enhanced cellular protein synthesis during the G2/M period. Protein synthesis in the G0/G1 period was not influenced by OPC-13340. Thus, these data indicate that the calcium channel blocker OPC-13340 mitigates the enhanced proliferation observed in randomly cycling VSMC from SHR and that this effect is primarily due to normalization of the premature mitosis during the G2/M period.

Vascular smooth muscle growth in genetic hypertension

To gain insight into the mechanisms which contribute to the development of vascular hypertrophy in the spontaneously hypertensive rat (SHR). These experiments were performed under conditions which most closely mimic the growth of smooth muscle in blood vessels, i.e. once cell-cell contact has been achieved. A comparison of the growth characteristics (growth rates and cell density at quiescence) of vascular smooth muscle cells (VSMC) from SHR and normotensive Wistar-Kyoto (WKY) rats. In the presence of foetal calf serum (1, 2.5, 5 and 10%), early passaged VSMC from SHR exhibited higher growth rates and reached higher densities at quiescence than VSMC from WKY rats. Accelerated growth rates could not be attributed to differences in cell-cell interactions. Also, growth rates and cell density at quiescence appear to be regulated by distinct mechanisms. Transforming growth factor-beta 1 (TGF-beta 1) caused an inhibition of serum-stimulated proliferation of confluent VSMC from WKY rats. In contrast, TGF-beta 1 had little, if any, inhibitory action upon the growth of VSMC from SHR. Scatchard analysis of 125I-TGF-beta 1 binding to VSMC from both strains yielded a single class of high affinity binding sites. VSMC from SHR exhibit enhanced proliferation, attain a higher cell density at quiescence and are less susceptible to growth inhibition by TGF-beta 1 than VSMC from WKY rats. All these characteristics of SHR VSMC may contribute to the development of vascular hypertrophy in this strain.

Alterations of calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats.

We examined the possible alterations in calcium handling through the calcium channels of spontaneously hypertensive rats (SHR) using 45Ca2+ uptake measurements in cultured aortic cells. Primary cultures of vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the thoracic aortas from 8-week-old SHR and age-matched Wistar-Kyoto rats (WKY). The functions of voltage sensitive calcium channels (VSCC) and receptor operated calcium channels (ROCC) were estimated from the activated 45Ca2+ uptake in VSMC with high K+ depolarization and arginine vasopressin (AVP), respectively. Compared to basal conditions, depolarization with 55 mM KCl increased 45Ca2+ uptake at 20 min by 94 +/- 17 (SE) % in SHR and 38 +/- 6% in WKY. The activated 45Ca2+ uptake was significantly greater in SHR than in WKY (p < 0.01). There was no significant difference in 45Ca2+ uptake at 20 min in the presence of 5 x 10(-8)M AVP between SHR and WKY. These results suggest that calcium uptake, at least through VSCC, is increased in VSMC of SHR. This enhanced activity may be implicated in the hypertensive mechanisms in this model of hypertension.

Transforming growth factor beta 1 expression and effect in aortic smooth muscle cells from spontaneously hypertensive rats.

Previous studies demonstrated that in addition to an increased response to growth factors, cultured vascular smooth muscle cells derived from spontaneously hypertensive rats (SHRs) grow to a greater density than cells from normotensive Wistar-Kyoto (WKY) rats. Transforming growth factor beta 1 (TGF-beta 1) has a bimodal effect on vascular smooth muscle cell growth, depending on cell density. The present study investigated the relation between cell density and expression of the proto-oncogene c-fos and TGF-beta 1 in cells from WKY rats and SHRs. The results demonstrate an increased accumulation of c-fos mRNA in calf serum-stimulated SHR cells but only at a high cell density. The expression of TGF-beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 hours after inoculation, with a further increase at later times. The effect of exogenous TGF-beta 1 on new DNA synthesis was evaluated by [3H]thymidine incorporation. At a low cell density, TGF-beta 1 had no effect on DNA synthesis in either WKY or SHR vascular smooth muscle cells. At a high cell density, there was a significant increase of DNA synthesis in response to TGF-beta 1 in SHR cells without any effect in WKY cells. In conclusion, contact inhibition of vascular smooth muscle cells from SHRs at a higher cell density is accompanied by an earlier expression of the marker gene c-fos and preceded by an exaggerated expression of TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Effects of Antihypertensive Agents on Proliferation of Vascular Smooth Muscle Cells from Spontaneously hypertensive Rats.

We have previously shown that Ca-antagonists and alpha-blockers substantially inhibit the cellular proliferation of cultured rat vascular smooth muscle cells (VSMC). This study explored whether these inhibitory effects on cellular proliferation differ between cultured VSMC from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). SHR VSMC proliferated much faster than WKY VSMC in 10% FCS. Cellular proliferation, determined by both cell number count and 3H-thymidine incorporation, was significantly blunted in the presence of either nifedipine (Nif) or bunazosin (Bun). The magnitude of these inhibitory effects was more pronounced for SHR cells than WKY cells (% reduction of 3H-thymidine uptake with Nif: 62.1 +/- 7.8% for SHR vs 75.3 +/- 10.2% for WKY, n = 6, p less than 0.05, and with Bun: 70.2 +/- 7.8% for SHR vs 82.1 +/- 9.9% for WKY, n = 6, p less than 0.05). In contrast, the intracellular water volume was unaffected by these antihypertensive agents based on equilibrium distribution of 3-O-methyl-D-glucose14C. It is concluded that SHR VSMC grow much faster than WKY VSMC and that this abnormality is innate to the SHR cells. It is also concluded that both Ca-antagonists and alpha-blockers exerted a substantial inhibitory effect on cellular proliferation of the cultured VSMC of either SHR or WKY. Furthermore, the greater inhibition of proliferation in the SHR VSMC suggests that Ca mediated- and/or alpha-receptor mediated processes of cellular proliferation of SHR could differ from that of WKY and that these abnormalities may contribute to the hyperproliferative changes of VSMC in this model.

Lower Na+- H+ antiport activity in vascular smooth muscle cells of Wistar???Kyoto rats than spontaneously hypertensive and Wistar rats

To determine whether increased Na(+)-H+ antiport activity in vascular smooth muscle cells may relate to the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR), we monitored Na(+)-dependent alkalinization of acidified cells from the hypertensive strain and two normotensive controls, the Wistar-Kyoto rat (WKY) and the Wistar rat. Changes in intracellular pH (pHi) of cultured aortic cells were measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The initial maximal reaction velocity of Na(+)-dependent alkalinization was significantly higher in SHR and Wistar than WKY cells. Similar results were obtained for the maximal velocity of the proton equivalent efflux: SHR, 7.51 +/- 0.71; Wistar, 9.14 +/- 0.85; WKY, 4.38 +/- 0.55 mmol H+/liter x 10 s. There were no differences in the basal pHi or cellular buffering power among the three rat strains. These findings indicate that the activity of the Na(+)-H+ antiport is higher in SHR vascular smooth muscle cells than in WKY cells. However, by itself, this difference cannot explain the hypertensive process in the SHR, since this transport system is also higher in vascular cells of the Wistar rat.

Abnormal calcium handling in vascular smooth muscle cells of spontaneously hypertensive rats

We have developed a system for measuring the dynamic changes in the cytoplasmic free calcium concentration [( Ca2+]i) in single vascular smooth muscle cells (VSMC) that is highly sensitive and does not cause cellular damage. Marked increases in [Ca2+]i in response to stimulation with caffeine and angiotensin II occurred in some VSMC of 4-week-old spontaneously hypertensive rats (SHR), in which the blood pressure and basal [Ca2+]i levels are not yet elevated. In 8-week-old rats, the basal [Ca2+]i level in VSMC was higher in SHR than in Wistar-Kyoto rats. Although the effects of high blood pressure on [Ca2+]i in vivo were expected to disappear during the passage culture, the [Ca2+]i in the fifth-passage cells was similar to that in the primary cells. These results suggest that the maintenance of high [Ca2+]i levels in VSMC of SHR is genetically regulated and is one of the mechanisms of hypertension in this strain, and that abnormal calcium regulation in VSMC of SHR is expressed even before overt hypertension.

Roles of endogenous vasodepressor prostaglandins in growth of vascular smooth muscle cells in spontaneously hypertensive rats.

We designed experiments to investigate the roles of endogenous prostaglandins (PG) for the rapid proliferation of vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR). Both the basal and arachidonate-stimulated vasodepressor PG generations were significantly enhanced in the VSMC of SHR when they were at the 1st or 2nd passage. Conversely, the generating capacity was significantly lowered in the VSMC of SHR when the cells reached the 4th or older generation. Based on the (3H)thymidine uptake and doubling time of VSMC, the decline of PG generating capacity seen in the VSMC of SHR was markedly associated with the increased VSMC growth. Indeed, the stimulation of endogenous vasodepressor PG by arachidonate produced a decrease in (3H)thymidine uptake in the VSMC of Wistar-Kyoto rats, whereas the dose was not sufficient to retard the uptake in SHR. On the other hand, the PG synthesis inhibition by indomethacin, a cyclooxygenase inhibitor, significantly enhanced the uptake by the VSMC of SHR. Thus, these data indicate that the impaired vasodepressor PG system is at least partly responsible for the rapid VSMC growth in SHR.

Cytosolic Ca2+ Regulation Attenuates ANP-Induced cGMP Response in Vascular Smooth Muscle Cells of the SHR

Cultured vascular smooth muscle cells (VSMCs) of the spontaneously hypertensive rat (SHR) show a higher density (Bmax) of ANP receptors and a blunted cyclic (c) GMP response to the hormone. To explore the idea that a higher cytosolic free Ca2+ (Cai2+) may be responsible for a blunted cGMP response to ANP, we examined the effect of raising Cai2+ by the Ca2+-ionophore A23187 or K+ depolarization on the ANP-induced cGMP response. Treatment of VSMCs from the SHR, Wistar-Kyoto rats (WKY), or American Wistar (WIS) rats with A23187 or high K+ resulted in a uniform reduction of the ANP-cGMP response in 1.8 mmol/L Ca2+ medium but not Ca2+-free medium. We conclude that a higher level of Cai2+, probably at the domain adjacent to the membrane, may inhibit ANP-induced activation of the particulate guanylate cyclase, and that this may be the cause of the blunted cGMP response of SHR VSMCs to ANP.

Inhibition of epidermal growth factor-mediated DNA synthesis by a specific tyrosine kinase inhibitor in vascular smooth muscle cells of the spontaneously hypertensive rat

Aortic vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) grow nearly twice as fast in vitro as cells isolated from several normotensive control strains of rats. We have previously shown that DNA synthesis in SHR cells from both young and adult animals in response to epidermal growth factor is selectively enhanced compared with normotensive controls, suggesting that epidermal growth factor may be at least partly responsible for the enhanced growth rate. To determine whether the enhanced DNA synthesis in response to epidermal growth factor in SHR cells is mediated via an enhanced epidermal growth factor receptor tyrosine kinase, we measured thymidine incorporation in epidermal growth factor-stimulated vascular smooth muscle cells in the presence of the highly specific tyrosine kinase inhibitor genistein. The 50% inhibitory dose (IC50) of genistein was higher for the SHR vascular smooth muscle cells than for the normotensive Wistar rat (NBR; National Institutes of Health Black rat). This suggests that the increased DNA synthesis in response to epidermal growth factor in SHR cells is a result of higher receptor tyrosine kinase activity initiating further intracellular signals.

Blunted cGMP response to ANF in vascular smooth muscle cells of SHR

Abnormalities in the coupling of atrial natriuretic factor (ANF) receptors with the guanosine 5'-cyclic monophosphate (cGMP) system in vascular smooth muscle cells (VSMCs) may play a role in the pathophysiology of hypertension in the spontaneously hypertensive rat (SHR). This concept was examined in cultured, aortic VSMCs (passages 6-10) from SHR, Wistar-Kyoto (WKY), and American Wistar (Wis) rats. Quiescent VSMCs of the SHR (serum deprived for 24 h) had higher ANF receptor density (Bmax) and lower affinity [i.e., increased equilibrium dissociation constant (Kd)] than cells from normotensive controls. Maximal binding (Bmax) (specific binding sites/cell) values for these cells were SHR 112,855 +/- 6,951, WKY 48,650 +/- 3,607, and Wis 36,122 +/- 2,607 (means +/- SE; P less than 0.001 for SHR vs. both WKY and Wis). The Kd values were (in nM) SHR 1.20 +/- 0.098, WKY 0.657 +/- 0.065, and Wis 0.37 +/- 0.037 (P less than 0.001 for SHR vs. both WKY and Wis). Despite their higher Bmax, VSMCs of the SHR showed a substantially lower maximal stimulation of cGMP accumulation in response to ANF: 987 +/- 29.3, 1,992 +/- 574.2, and 2,019 +/- 273.8 fmol.4 min-1.10(6) cells-1 for SHR, WKY, and Wis, respectively (P less than 0.01 for SHR vs. Wis and P less than 0.02 for SHR vs. WKY). Further experiments demonstrated that the poor response of SHR VSMCs to the ANF was not due to a population of receptors that did not couple to the particulate guanylate cyclase. Such findings demonstrate a dissociation of the cGMP response to ANF from the binding of the hormone to its receptors in VSMCs of the SHR compared with controls. This appears to represent a primary and innate defect in these cells that may contribute to the hypertensive process in the SHR.

Reduced Na-K-Cl cotransport in vascular smooth muscle cells from spontaneously hypertensive rats.

We have previously demonstrated the presence of a prominent, cyclic nucleotide-sensitive Na-K-Cl cotransport in vascular smooth muscle cells (VSMC). Others have observed that Na-K-Cl cotransport levels are reduced in erythrocytes of patients with essential hypertension and have proposed that a defect in this Na transport system may play a role in the pathogenesis of the disease. However, such a defect has not been demonstrated in the putative target tissue for essential hypertension, i.e., the VSMC. In the present study, we compared Na-K-Cl cotransport of VSMC from spontaneously hypertensive rats (SHR) with Na-K-Cl cotransport of VSMC from normotensive Wistar-Kyoto rats (WKY). We found that Na-K-Cl cotransport of SHR VSMC is significantly reduced relative to that of WKY VSMC (3.09 vs. 4.39 mumol K.g protein-1.min-1). The apparent ion affinities for Na-K-Cl cotransport of SHR VSMC did not differ from those determined for WKY VSMC. Furthermore, cyclic nucleotide regulation of cotransport also appeared to be the same for the two types of VSMC. In contrast, maximal saturable binding of [3H]bumetanide observed in SHR VSMC was markedly reduced compared with that of WKY VSMC, but the Kd values were similar. Our data suggest that the reduction in cotransport observed in SHR VSMC is the result of a decrease in the number of available cotransport sites.

Enhanced generation of vascular thromboxane A2 in spontaneously hypertensive rats and its role in the rapid proliferation of vascular smooth muscle cells.

We examined vascular thromboxane A2 (TXA2) generation and its relation to a proliferation of vascular smooth muscle cells (VSMCs) in spontaneously hypertensive rats (SHRs). Aortic TXA2 release was significantly enhanced in 5-week-old SHRs, as compared with Wistar-Kyoto strain rats (WKY). The cultured VSMCs of SHRs exhibited a shorter doubling time and a greater [3H]thymidine uptake than those of WKY rats. OKY 046 a thromboxane synthetase inhibitor, tempered proliferation of VSMCs in SHRs, but not in WKY rats. STA2, a stable analog of TXA2, stimulated VSMC growth in WKY rats more than in SHRs. It is indicated that vascular TXA2 generation is enhanced, and partially participates in the rapid proliferation of VSMCs in SHRs.

22Na+ and 86Rb+ Transport in Vascular Smooth Muscle of SHR, Wistar Kyoto, and Wistar Rats

To gain further insight into differences in cellular Na+ and K+ regulation between the spontaneously hypertensive rat (SHR), Wistar Kyoto (WKY), and American Wistar (W) rats, 22Na+ and 86Rb+ washouts were performed under steady-state conditions in cultured vascular smooth muscle cells from the three rat strains. SHR vascular smooth muscle cells showed significantly higher bumetanide sensitive 86Rb+ washout rate constant (x 10(-4)/min; mean +/- SEM) than WKY cells (-38.6 +/- 2.84 and -23.8 +/- 3.58, respectively; p less than 0.005). SHR vascular smooth muscle cells also exhibited significantly higher values than WKY cells in the total 22Na+ washout rate constant (x 10(-2)/min) (-61.0 +/- 1.57 vs. -53.8 +/- 1.24; p less than 0.005). The amiloride sensitive component of the 22Na+ washout rate constant accounted for these differences (-18.6 +/- 1.04 for SHR and -12.1 +/- 2.00 for WKY; p less than 0.05). There were no apparent differences in cellular Na+ concentrations between WKY and SHR cells. In general, the 86Rb+ and 22Na+ washout parameters of W rat cells were quite similar to those of cells from SHR. We conclude that the bumetanide-sensitive 86Rb+ washout (the Na+ K+-cotransport), the overall, and the amiloride-sensitive 22Na+ washout (the latter primarily represents the Na+/H+ antiport) are higher in SHR than WKY rat vascular smooth muscle cells. These findings indicate innate differences in cellular Na+ and K+ transport in vascular smooth muscle cells of the SHR and WKY rat. The mechanisms responsible for these differences are yet to be determined.

Increased atrial natriuretic factor receptor density in cultured vascular smooth muscle cells of the spontaneously hypertensive rat.

To explore the role of the atrial natriuretic factor (ANF) system in the pathophysiology of hypertension we examined the binding kinetics of synthetic ANF to cultured vascular smooth muscle cells (VSMCs) derived from the spontaneously hypertensive rat (SHR) and two normotensive controls-the Wistar Kyoto (WKY) and American Wistar (W). The number of maximal binding sites (Bmax) per cell (mean +/- SEM; X10(3] were: SHR = 278.0 +/- 33.0, WKY = 28.3 +/- 7.1 and W = 26.6 +/- 4.2. The differences between the SHR and normotensive strains were significant at p less than 0.001. The equilibrium dissociation constant (Kd; X 10(-9)M) was higher in SHR VSMCs (0.94 +/- 0.14) than in WKY (0.22 +/- 0.09; p less than 0.01) and W (0.39 +/- 0.14; p less than 0.02) cells. The plasma levels of the immunoreactive ANF were higher in SHR than the normotensive controls. We suggest that the relatively greater ANF receptor density in cultured VSMCs of the SHR represents a response to the in vitro environment which is relatively more deficient in ANF for VSMCs of the SHR as compared with the normotensive rats. Thus, the capacity of the SHR VSMC to regulate ANF receptor density appears to be independent of the blood pressure level.

Ouabain binding to cultured vascular smooth muscle cells of the spontaneously hypertensive rat

The binding of ouabain and K+ to the Na+ pump were analyzed in serially passed cultured vascular smooth muscle cells (VSMCs) originating from spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and American Wistar (W) rats. Our techniques have utilized analyses of displacement of [3H]ouabain by both unlabeled ouabain and K+ from specific binding sites on the VSMCs. We have found that each of the VSMC preparations from the three rat strains appeared to demonstrate one population of specific ouabain receptors (Na+ pumps); the number of Na+ pump units (mean +/- SE, expressed as 10(5) units/cell; number of observations indicated in parentheses) of both the SH and WKY rats was significantly lower than the number of Na+ pump units of W rat VSMCs [SH: 3.00 +/- 0.02 (231), WKY: 2.87 +/- 0.05 (245), and W: 3.62 +/- 0.04 (225)]; the equilibrium dissociation constant values (microM) for ouabain in VSMCs of SH and WKY rats were similar but were significantly higher than that of VSMCs derived from W rats [SH: 4.69 +/- 0.09 (231), WKY: 4.57 +/- 0.12 (245), and W: 3.69 +/- 0.17 (225)]; and among the VSMCs originating from the three rat strains, the apparent equilibrium dissociation constant value for K+ (mM) was the lowest in those of the SH rat [1.04 +/- 0.003 (143), compared with VSMCs of the WKY rat [1.54 +/- 0.006 (135)] and W rat [1.19 +/- 0.003 (136)]. Our previous studies have demonstrated increased passive Na+ and K+ transport rate constants of SH rat VSMCs compared with either W or WKY rat cells. These findings suggest the possibility of higher permeabilities of the SH cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated vascular reactivity in the timolol-treated spontaneously hypertensive rat.

Spontaneously hypertensive rats (SHR) treated in utero with a low dose, and from weaning with 6 mg/kg per day of oral timolol, a noncardioselective beta-adrenergic blocker, did not develop hypertension. Isolated aortic rings from these animals showed increased reactivity to raised extracellular K+, when compared with tissues from timolol-treated Kyoto-Wistar (WKY) control rats. The normotensive SHR aorta also showed significant responsiveness to H+ and to high Ca2+ concentrations without previously depolarizing the tissue with high K+. These observations suggest that the increased reactivity seen in vascular smooth muscle from this animal does not develop secondary to elevated peripheral resistance and subsequent hypertrophy, and that the initiation of hypertension in this animal may be related to a membrane defect and (or) cellular defect which results in a facilitation of Ca2+ availability for the contractile proteins of the SHR vascular smooth muscle cell.