Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes.

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

The balloon catheter induces an increase in contralateral carotid artery reactivity to angiotensin II and phenylephrine.

1. The effects of balloon injury on the reactivity of ipsilateral and contralateral carotid arteries were compared to those observed in arteries from intact animals (control arteries). 2. Carotid arteries were obtained from Wistar rats 2, 4, 7, 15, 30 or 45 days after injury and mounted in an isolated organ bath. Reactivity to angiotensin II (Ang II), phenylephrine (Phe) and bradykinin (BK) was studied. Curves were constructed in the absence or presence of endothelium or after incubation with 10 microm indomethacin, 500 microm valeryl salicylate or 0.1 microm celecoxib. 3. Phe, Ang II and BK maximum effects (Emax) were decreased in ipsilateral arteries when compared to control arteries. No differences were observed among pD2 or Hill coefficient. 4. Emax to Phe (4 and 7 days) and to Ang II (15 and 30 days) increased in the contralateral artery. In addition, Phe or Ang II reactivity was not significantly different in aorta rings from control or carotid-injured animals. 5. The increased responsiveness of contralateral artery was not due to changes in carotid blood flow or resting membrane potential. The endothelium-dependent inhibitory component is not present in the contraction of contralateral arteries and it is not related to superoxide anion production. 6. Indomethacin decreased contralateral artery responsiveness to Phe and Ang II. Valeryl salicylate reduced the Ang II response in contralateral and control arteries. Celecoxib decreased the Phe Emax of contralateral artery. 7. In conclusion, decreased endothelium-derived factors and increased prostanoids appear to be responsible for the increased reactivity of contralateral arteries after injury.

Hydrochlorothiazide abolishes the anti-atherosclerotic effect of quinapril.

1. Antihypertensive treatment has been demonstrated to result in persistent reductions in morbidity and mortality due to stroke. However, the coronary risk attributable to hypertension has been only partially reversed. We hypothesized that diuretics could have unfavourable effects on atherosclerosis. 2. New Zealand rabbits were fed a 0.5% cholesterol-enriched diet for 12 weeks, followed by a 0.1% cholesterol diet for another 12 weeks. During the last 12 week period, 40 animals were randomly assigned to one of four groups: (i) group I was the control group; (ii) group II received hydrochlorothiazide (10 mg/day); (iii) group III received quinapril (30 mg/day); and (iv) group IV was treated with hydrochlorothiazide (10 mg/day) plus quinapril (30 mg/day). 3. The treatments did not affect either the lipid profile or serum electrolytes and oxidative stress. However, endothelium-dependent vasorelaxation in isolated aortic rings was significantly improved with quinapril (group III) treatment (P < 0.001 vs other groups). In addition, therapy with quinapril promoted a significant reduction in atherosclerosis (intima area, intima/media ratio and perimeter of vessel with plaque; P < 0.05 vs other groups), as well as in cholesterol content of the aorta (P < 0.05 vs groups II and IV). 4. In conclusion, hydrochlorothiazide did not modify atherosclerosis and, when added to quinapril treatment, impaired the anti-atherosclerotic effect seen with quinapril alone.

Angiotensin actions on the isolated rat uterus during the estrous cycle: influence of resting membrane potential and uterine morphology.

The involvement of AT1 and AT2 receptor subtypes in the response of the isolated rat uterus to angiotensin II (AngII) was studied throughout the estrous cycle. The AngII potency varied during the different estrous cycle phases, as indicated by significantly different pD2 values. No significant differences were observed in AngII metabolism among different estrous phases. Morphological analysis indicated that external and internal myometrium layers were thicker during estrus. In addition, the highest resting membrane potential was also observed during this phase, when compared with the proestrus and diestrus phases. The AngII-induced uterine contractions were blocked by losartan. Different losartan pD2 values were observed. PD123319 had no effect on the contractile response to AngII. The results also indicate that estrous cycle-dependent changes in AngII potency are correlated with uterine morphological and/or membrane potential changes.

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs.

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50 degrees C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.

Role of smooth muscle cell membrane potential in neointima formation in arteries of spontaneously hypertensive rats.

Based on observations that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) have altered resting potentials as well as abnormal cell proliferation rates, neointima formation after controlled balloon injury was compared in arteries from SHR and Wistar Kyoto rats (WKY). SHR aortic VSMC showed hyperpolarized resting membrane potentials (-93+/-8 mV) when compared to those from WKY (-61+/-6 mV). Histomorphometric analysis of cross sections from aortic segments submitted to balloon injury showed reduced neointima formation in SHR (neointima/media ratio: 0.04+/-0.03) as compared to WKY (0.2+/-0.1). On the other hand, in injured carotid arteries, neointima formation was more extensive in SHR (neointima/media ratio 5.0+/-0.9) than in WKY (0.8+/-0.7), leading in most cases to luminal occlusion. Measurements of VSMC resting potential showed that carotid artery cells from SHR were depolarized with respect to those from WKY (-46+/-4 vs. -69+/-5 mV, respectively). The results demonstrate an inverse relationship between VSMC membrane polarization and neointima formation in SHR arteries, suggesting that genetic modifications in SHR determine a dysfunctional cellular physiology that may influence cell proliferation subsequent to injury.

Alpha-2 adrenoceptors are present in rat aorta smooth muscle cells, and their action is mediated by ATP-sensitive K(+) channels.

The role of alpha(2)-adrenoceptors in the response of aorta smooth muscle rings to the alpha(2)-adrenoceptors agonists UK 14,304 and clonidine was studied. Stimulation by 1 - 10 nM UK 14,304 caused dose-dependent relaxant responses in BaCl(2)-contracted endothelium-denuded aorta rings, and hyperpolarization in rings with or without endothelium, which were inhibited by yohimbine and glibenclamide, but not affected by prazosin, propranolol, apamin or iberiotoxin. At higher concentrations (10 nM - 10 microM) UK 14,304 also induced a depolarizing effect which was potentiated by yohimbine and inhibited by prazosin. These results indicate that UK 14,304 acts on alpha(2)-adrenoceptors at lower concentrations and on both alpha(1)- and alpha(2)-adrenoceptors above 10 nM. In rings, with or without endothelium, noradrenaline had a depolarizing effect which was inhibited by prazosin. Adrenaline did not affect the membrane potential but in the presence of prazosin caused hyperpolarization, which was inhibited by yohimbine and glibenclamide. These results indicate that noradrenaline is more selective for alpha(1)-, whereas adrenaline has similar affinities for alpha(1)- and alpha(2)-adrenoceptors. In aortae with endothelium, L-NNA caused a small depolarization but did not affect the hyperpolarization induced by UK 14,304, indicating that NO is not involved in that response. Glibenclamide induced a small depolarization in aortae, with or without endothelium, indicating that ATP-sensitive K(+) channels may play a role in maintaining the smooth muscle's membrane potential. Our results indicate that, in rat aorta, alpha(2)-adrenoceptors are also present in the smooth muscle, and that these receptors act through small-conductance ATP-sensitive K(+) channels.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells.

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 +/- 1.9 mV) compared to NWR (-69 +/- 1.4 mV), NWR 1K-1C (-60.8 +/- 1.6 mV), WKY (-67.1 +/- 3.2 mV) and WKY 1K-1C (-56.9 +/- 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 +/- 0.86) when compared to arteries of all the other groups (NWR 0.93 +/- 0.65, NWR 1K-1C 1.24 +/- 0.45, WKY 1.22 +/- 0.32, WKY 1K-1C 1.15 +/- 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 + or - 1.9 mV) compared to NWR (-69 + or - 1.4 mV), NWR 1K-1C (-60.8 + or - 1.6 mV), WKY (-67.1 + or - 3.2 mV) and WKY 1K-1C (-56.9 + or - 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 + or - 0.86) when compared to arteries of all the other groups (NWR 0.93 + or - 0.65, NWR 1K-1C 1.24 + or - 0.45, WKY 1.22 + or - 0.32, WKY 1K-1C 1.15 + or - 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state

Pharmacological characterization of RMP-7, a novel bradykinin agonist in smooth muscle.

RMP-7 is a bradykinin (BK) agonist designed to be resistant to kininases such as angiotensin-converting enzyme (ACE). Pharmacological assays were performed with RMP-7 in isolated guinea-pig ileum and rat mesenteric artery. RMP-7 induced contractile responses in the guinea-pig ileum, where the apparent affinity of the peptide (pD2) was significantly lower than that determined for BK (7.3 +/- 0.07 vs. 8.3 +/- 0.05, respectively). HOE-140 blocked this effect indicating that B2 receptor was involved. Captopril (1 microM) had no potentiating effect on RMP-7 but increased pD2 value determined for BK (8.8 +/- 0.1), confirming a high resistance of RMP-7 to the ACE. In rat mesenteric artery, RMP-7 induced endothelium-dependent relaxation (7.8 +/- 0.4), with a higher affinity than that of BK which induced vasodilatation only in the presence of 1 microM captopril (6.9 +/- 0.36). Nevertheless, the maximum effect induced by RMP-7 was lower than that of BK in contrast to that observed in guinea-pig ileum although B2 receptor was involved in both cases. We concluded that: RMP-7 is greatly resistant to the ACE and that the receptor sites activated by RMP-7 and BK show important differences in vascular and non-vascular preparations probably due to the different sensitivity of the B2 receptor to RMP-7.

Effect of cholecalciferol treatment on the relaxant responses of spontaneously hypertensive rat arteries to acetylcholine.

We studied the effect of oral cholecalciferol treatment on the endothelium-dependent vascular relaxation and hyperpolarization induced by acetylcholine (ACh), which is impaired in spontaneously hypertensive rats (SHR). Adult female SHR and normotensive Wistar-Kyoto rat (WKY) controls received 125 microg of cholecalciferol per kilogram body weight per day for 6 weeks. The responses to ACh of the isolated mesenteric vascular bed and mesenteric artery rings were measured, as well as the smooth muscle cell membrane potential. After cholecalciferol treatment, the systolic blood pressure and basal perfusion pressure of the mesenteric vascular bed of the SHR fell to control levels. The relaxant and hyperpolarizing effects of ACh, which are reduced in SHR, were also brought to control levels after cholecalciferol treatment. These effects of ACh were inhibited by N(omega)-nitro-L-arginine in SHR and by apamin in WKY. After cholecalciferol treatment, SHR hyperpolarizing responses showed the same inhibition pattern as those of WKY. This indicates that, after cholecalciferol treatment, SHR vascular mesenteric preparation responses to ACh are mediated by endothelium-derived hyperpolarizing factor, which induces activation of Ca(2+)-dependent K(+) channels, as in WKY. In untreated SHR, the ACh-mediated response is entirely due to ACh acting via the release of nitric oxide.

Recovery of impaired K+ channels in mesenteric arteries from spontaneously hypertensive rats by prolonged treatment with cholecalciferol.

1. The mechanism responsible for blood pressure reduction in spontaneously hypertensive rats (SHR) after prolonged cholecalciferol treatment was studied. Two-week treatment of SHR with 0.125 mg cholecalciferol kg-1 body weight per day orally caused significant reductions of systolic blood pressure and of the resting perfusion pressure of the mesenteric vascular bed at constant flow. 2. In addition, the treated animals presented a normalization of the maximum vasoconstriction response to noradrenaline and a reduction of the maximum effect of the adrenaline concentration-response curves. This latter effect probably was due to recovery of the impaired Ca(2+)-dependent K+ channels coupled to alpha 2-adrenoceptors since it was prevented by apamin. 3. The treatment with cholecalciferol also normalized the smooth muscle cell membrane potential of de-endothelialized mesenteric arteries of SHR and their hyperpolarizing responses to alpha 2-adrenergic agonists, which were depressed in untreated SHR. 4. In mesenteric rings with endothelium, alpha 2-adrenergic agonists caused similar hyperpolarizing responses in the SHR and in normotensive Wistar (NWR) and Wistar Kyoto (WKY). In non cholecalciferol-treated SHR the hyperpolarizing mediator involved in this effect was NO, while in NWR it was the endothelium-derived hyperpolarizing factor (EDHF). After cholecalciferol treatment, the hyperpolarization induced by alpha 2-adrenergic agonists in SHR smooth muscle cells was mediated by EDHF, as in NWR. 5. Our results indicate that the hypotensive effect of cholecalciferol in the SHR is probably due to the normalization of vascular reactivity, by restoring the functioning of apamin- and ATP-sensitive K+ channels located in the vascular smooth muscle cell membrane, which are impaired in the SHR.

Different pathways for Ca2+ mobilization by angiotensin II and carbachol in the circular muscle of the guinea-pig ileum.

Ca2+ pathways activated by angiotensin II and carbachol were evaluated in the circular muscle of the guinea-pig ileum by recording mechanical and electrical activities. Transient contractions induced by angiotensin II were greatly reduced by Ca2+ removal from the medium whereas carbachol-induced responses were not significantly altered. Nifedipine had no effect on the responses to both agonists. A high concentration of tetrodotoxin (0.1 microM) inhibited angiotensin II-induced contractile responses without affecting the depolarization, whereas 1 mM Ni2+ inhibited the mechanical and electrical effects. Neither tetrodotoxin nor Ni2+ affected carbachol-induced effects. These results indicate that angiotensin II-induced phasic contractions depend on extracellular Ca2+ but not on voltage-dependent L-type Ca2+ channels. It is suggested that angiotensin II activates Ni2+-sensitive Na+ and non-specific cationic channels, whereas the responses to carbachol are dependent on receptor-activated Ca2+ release. Furthermore the different response of the longitudinal and circular muscles to the inhibitory effects of tetrodotoxin and Ni2+ on the angiotensin II- and carbachol-induced contractions indicates that these agonists exert their own myogenic effects on each layer and are able to trigger different Ca2+ mobilization pathways.

Impaired function of alpha-2 adrenoceptors in smooth muscle of mesenteric arteries from spontaneously hypertensive rats.

The alpha2-adrenoceptor function in mesenteric arteries of spontaneously hypertensive rats (SHR) was investigated by comparing membrane potential changes in response to adrenergic agonists in preparations from female SHR, Wistar-Kyoto (WKY) and normotensive Wistar rats (NWR). Resting membrane potential was found to be less negative in mesenteric arteries from SHR than in those from NWR and WKY. Apamin induced a decrease in the membrane potential of mesenteric artery rings without endothelium from NWR and WKY, but had no effects in those from SHR. Both UK 14,304 and adrenaline, in the presence of prazosin, induced a hyperpolarization that was significantly lower in de-endothelialized mesenteric rings from SHR than in those from NWR and WKY. In mesenteric rings with endothelium, however, similar hyperpolarization was observed in the three strains. In NWR mesenteric rings with endothelium the hyperpolarization induced by activation of alpha2-adrenoceptors was abolished by apamin, whereas in intact SHR mesenteric rings this hyperpolarization was slightly reduced by apamin and more efficiently reduced by Nomega-nitro-L-arginine. It is concluded that the activity of potassium channels coupled to alpha2-adrenoceptors is altered in the smooth muscle cells of SHR mesenteric arteries, contributing to their less negative membrane potential. On the other hand, the endothelial alpha2-receptors are functioning in mesenteric vessels from SHR and their stimulation induces a hyperpolarization mainly through the release of nitric oxide.

Dietary magnesium improves endothelial dependent relaxation of balloon injured arteries in rats.

The purpose of the present study was to examine the importance of magnesium in endothelial function after arterial balloon injury. Male Wistar rats were fed normal, high or low concentrations of magnesium. Three weeks later the animals underwent endothelial injury of the thoracic aorta by a balloon catheter or a sham operation. Biochemical, histological and endothelial function analysis were performed 15 days after the surgical treatment. The animals fed a low magnesium diet presented the lowest level of serum magnesium and the highest ionized blood calcium levels. Histomorphometric analysis revealed no differences among groups neither regarding the magnitude of intimal thickening nor the recovery of endothelial coverage. However, when vasoreactivity responses were compared in the balloon-injured group, those animals fed a high magnesium diet had the better endothelium-dependent vascular relaxation. In conclusion, a higher magnesium level in the diet was beneficial to vessels that underwent endothelial injury by balloon catheter.

Effect of treatment with cholecalciferol on the membrane potential and contractility of aortae from spontaneously hypertensive rats.

1. The diet of spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NWR) was supplemented with 12.5 micrograms cholecalciferol per 100 g body weight daily, by gavage, for 4 weeks. 2. The amplitude of the contractile responses of aortic rings from SHR to potassium and adrenaline, which was smaller than in NWR aortae, was increased after treatment with cholecalciferol. No further changes were observed in the responses of NWR and SHR aortae in the presence of 100 nM apamin. 3. The membrane potentials of aortae from SHR, which were higher than those of aortae from NWR, decreased after treatment with cholecalciferol. Further depolarization was observed in aortic rings from NWR, but not in aortic rings from SHR, after their preincubation with 100 nM apamin. 4. It is concluded that cholecalciferol normalizes the membrane potential and contractility of aortae from SHR, probably through an effect on lipid composition and structure of the plasma membrane.

Dual effect of clonidine on mesenteric artery adrenoceptors: agonistic (alpha-2) and antagonistic (alpha-1).

The effect of clonidine on the mesenteric vascular bed and the isolated mesenteric artery was examined in preparations in which tonus was induced by norepinephrine or endothelin. In preparations precontracted by norepinephrine, clonidine caused a relaxation which was not inhibited by the alpha-2 antagonists yohimbine and idazoxan or by the K+ channel blockers apamine, tetraethylammonium and glibenclamide. In preparations precontracted with endothelin, clonidine increased the depolarization and induced a contraction. Both these effects were inhibited by prazosin. In isolated mesenteric arteries, norepinephrine cause a significant depolarization that was inhibited by clonidine or prazosin. On the other hand, clonidine caused a hyperpolarization which was inhibited by idazoxan or yohimbine, but not by prazosin. This hyperpolarization was also abolished by apamine, tetraethylammonium and glibenclamide. It is concluded that clonidine acts on alpha-1 adrenoceptors as a partial agonist, causing relaxation of the mesenteric artery precontracted with norepinephrine or contraction of preparations precontracted with endothelin. Moreover, clonidine can open K+ channels and hyperpolarize the plasma membrane of mesenteric artery by acting on alpha-2 adrenoceptors.

Angiotensin II tachyphylaxis in the guinea pig ileum and its prevention: a pharmacological and biochemical study.

Angiotensin II (AII) tachyphylaxis occurs in the guinea pig ileum, but is not induced by analogs lacking the N-terminal amino group or the Arg2 guanidino group. Both AII and Lys2AII increased cell inositol trisphoshate content in cultured intestinal smooth muscle cells. Protein kinase C inhibition by staurosporine or downregulation by prolonged incubation with phorbol reverted tachyphylaxis of the inositol trisphoshate response, but not that of the Na+ uptake response, indicating that the uncoupling of the phosphoinositide signal system by protein kinase C did not involve all processes distal to receptor activation. Tachyphylaxis of the Na+ uptake response was prevented when receptor internalization was blocked by reduction of the temperature (4 degrees C) or by pretreatment of the cells with phenylarsine oxide. Acid washings, which prevented tachyphylaxis of the 24Na+ influx response, also prevented tachyphylaxis of the contractile response of the guinea pig ileum to AII. Although these findings suggest that sequestration or internalization of the AII receptor might be involved in AII tachyphylaxis, binding of [125I]AII and of [125I]Lys2AII to the cells was equally unaffected by repeated administrations of the peptides. The results suggest that conformational change of the AII-receptor complex within the plasma membrane, but not internalization, is the most important factor responsible for tachyphylaxis.

Role of Ca(+)-dependent K-channels in the membrane potential and contractility of aorta from spontaneously hypertensive rats.

1. Contractile responses to KCl and membrane potentials were determined in aortic rings from spontaneously hypertensive rats (SHR), normotensive Wistar rats (NWR) and Wistar Kyoto rats (WKY) both in the absence and in the presence of the Ca(2+)-dependent K-channel blockers, apamin and tetraethylammonium (TEA). 2. Compared to NWR, aortic rings from WKY and SHR were less reactive and their Ca2+ uptake after stimulation with K+ was decreased. 3. Smooth muscle cell membrane potentials were higher in aortae from SHR and WKY than in NWR aortae, whereas SHR had higher K+ and lower Na+ intracellular activities than WKY and NWR, suggesting overactivity of the Na+/K+ pump in the hypertensive animals. 4. Treatment with apamin caused depolarization of WKY and SHR aortae, and increased their contractile responses to the same level as those of the NWR. Treatment with TEA also caused depolarization of aortae from WKY and SHR, but in the SHR the depolarization induced by TEA was smaller than that produced by apamin and the contractile responses to KCl did not reach the level of those of aortae from NWR. 5. It is concluded that overactivity of Ca(2+)-dependent K-channels in aortae of WKY and SHR contributes to their higher membrane potentials and lower responsiveness to vasoconstrictor stimuli. In SHR, an overactive Na+/K+ pump is also present, and the contribution of apamin-sensitive Ca(2+)-dependent K-channels to the membrane potential and reactivity appears to be more relevant than that of TEA-sensitive channels.