Calcium buffering of resting, voltage-dependent Ca2+ influx by sarcoplasmic reticulum in femoral arteries from spontaneously hypertensive rats at prehypertensive stage.

We examined the Ca2+-buffering function of the sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents that inhibit SR Ca2+-ATPase, and of ryanodine, which depletes SR Ca2+, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 4-week-old SHR and normotensive Wistar-Kyoto rats (WKY). Addition of CPA, thapsigargin or ryanodine to the resting state of the strips caused an elevation of cytosolic Ca2+ level and a contraction in both WKY and SHR. These responses were larger in SHR than in WKY. The contractions were inhibited strongly by 100 nM nifedipine or 3 microM verapamil and were abolished by Ca2+-free solution. Nifedipine, verapamil or Ca2+-free solution itself caused a relaxation from the resting state of SHR strips, but not from that of WKY strips. The resting Ca2+ influx in arteries measured by a 5-min incubation with 45Ca was significantly larger in SHR than in WKY. This influx was decreased by 10 microM CPA or 10 microM ryanodine in both WKY and SHR. These results suggest that in the resting state of the femoral artery from 4-week-old SHR, the greater part of the increased Ca2+ influx via L-type Ca2+ channels is buffered by Ca2+ uptake into the SR, while some Ca2+ reaches the myofilaments, resulting in the maintenance of resting tone.     

Alteration in sarcoplasmic reticulum-dependent contraction of tail arteries in response to caffeine and noradrenaline in spontaneously hypertensive rats

Since the membrane Ca2+ handling properties of the arterial smooth muscle sarcoplasmic reticulum may be altered in genetic hypertension, we studied caffeine- and noradrenaline-induced contractions in tail arteries from spontaneously hypertensive rats (SHR) at the prehypertensive stage (4 weeks old) and from age-matched Wistar-Kyoto rats (WKY). After the sarcoplasmic reticulum had been loaded with Ca2+ by pretreatment with physiological Ca2+ solution, caffeine- and noradrenaline-induced contractions of the tail arteries, measured in a Ca2(+)-free solution [containing 0.1 mmol/l ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid], were smaller in SHR than in WKY. After caffeine-releasable Ca2+ in the sarcoplasmic reticulum had been depleted by pretreatment with the Ca2(+)-free solution, the caffeine-induced arterial contractions in a low-Ca2+ (0.5 mmol/l) solution were smaller in SHR than in WKY. The Ca2+ concentration-tension relationship in skinned arterial fibres was similar in WKY and SHR. These data suggest that the ability of the sarcoplasmic reticulum to take up and store caffeine- and noradrenaline-releasable Ca2+ is decreased in SHR. The development of hypertension in SHR may be explained by an impaired function of the sarcoplasmic reticulum in arterial smooth muscle.     

Contractile response of aorta to alpha 1-adrenergic stimulation in Ca(2+)-free medium is reduced in spontaneous hypertension.

Vascular responses of aortic rings to alpha 1-adrenergic stimulation by phenylephrine (Phe) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were studied in Ca(2+)-containing medium and Ca(2+)-free medium plus 50 mumol/L EGTA. Although there was no difference in the sustained force development between SHR and WKY vessels in response to 100 mmol/L KCl or 10 mumol/L Phe in Ca(2+)-containing medium, the transient contractile response to 10 mumol/L Phe in Ca(2+)-free medium was substantially smaller in SHR compared to that in WKY. Subsequent addition of 2.5 mmol/L Ca2+ restored the sustained contractile response to a similar level in both SHR and WKY vessels. The transient contractile response to Phe in Ca(2+)-free medium containing EGTA, presumably due to the release of intracellular Ca2+, decreased progressively with preincubation time in Ca(2+)-free medium, indicating intracellular Ca2+ depletion. Such a temporal change of aortic response was more pronounced in SHR than in WKY. The subsequent response to Ca2+ repletion in the presence of Phe, on the other hand, increased progressively with Ca(2+)-depletion period and was higher in SHR than in WKY. The rate of relaxation after washout of Phe was slower in SHR aorta compared to WKY aorta. These results, together with our earlier findings, collectively suggest that the previous known deficiency in Ca2+ pumping mechanisms of vascular muscle microsomes leading to a reduced functional size of intracellular Ca2+ pool may account for the smaller contractile response of SHR aorta to alpha 1-adrenergic stimulation in Ca(2+)-free medium and the slower rate of relaxation.     

Ca2+ buffering function of the sarcoplasmic reticulum is increased in the carotid artery from spontaneously hypertensive rats.

To clarify whether the Ca2+ uptake function of the sarcoplasmic reticulum (SR) during arterial contraction is altered in hypertension, the effects of cyclopiazonic acid (CPA) and thapsigargin, which inhibit SR Ca2+-ATPase, on the contractile responses to Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of carotid arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of Bay k 8644 (1-300 nM) to the strips caused a concentration-dependent contraction that was larger in SHR than in WKY. The contractile responses to Bay k 8644 were augmented by CPA (10 microM) or thapsigargin (100 nM) in both strains. This augmentation was greater in SHR. Each of CPA and thapsigargin induced a relatively transient contraction, and both of these contractions were larger in SHR than in WKY. The basal 45Ca influx in this artery was larger in SHR than in WKY. The addition of caffeine (1-20 mM) caused a transient contraction that was larger in SHR than in WKY. Our results indicate that 1) the large Ca2+ influx during rest in the SHR carotid artery is strongly buffered by Ca2+ uptake into the superficial SR; and 2) the Ca2+ uptake function of the SR during the contraction with Bay k 8644 was increased in SHR compared with WKY. We conclude that the SHR carotid artery has an increased total capacity of SR for Ca2+ storage as an attempt to compensate for the large Ca2+ influx.     

Possible role of phosphorylation-dephosphorylation in the regulation of calcium metabolism in cardiovascular tissues of SHR

Spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) were compared for phosphorylation-dephosphorylation mechanism(s) in aorta, caudal artery, inferior vena cava, and right and left ventricles. Reduction of cAMP-induced phosphorylation of microsomes and cAMP-dependent protein kinase activity was significant in the aorta and caudal artery of SHR compared with WKY. These changes were not observed in the vena cava of SHR. Phosphoprotein phosphatase activity was significantly increased (p less than 0.05) in the soluble fraction of arterial smooth muscle. No changes were observed, however, in the myocardium or vein. Furthermore, the extent of phosphorylation, and Ca2+ uptake ability and the protein kinase activity in the soluble and the microsomal fractions were not reduced in the myocardium of SHR compared with WKY. These data suggest that phosphorylation-dephosphorylation mechanisms are altered in the microsomal fraction of the aorta and caudal artery of SHR, which may result in reduced Ca2+ uptake by the intracellular organelle. The changes observed could have a significant effect on vasodilatation of arteries in the hypertensive state. The lesion appears specific to the arterial smooth muscle in the cardiovascular tissues.     

Effect of 5-lipoxygenase blockade on blood pressure and acetylcholine-evoked endothelium-dependent contraction in aorta from spontaneously hypertensive rats

OBJECTIVE: Cysteinyl leukotrienes (cysLT) are pro-inflammatory and vasoactive products suspected to be involved in the regulation of vascular tone and blood pressure in hypertension. DESIGN: We investigated, in spontaneously hypertensive rats (SHR), the involvement of cysLT in the in-vivo regulation of blood pressure and the in-vitro endothelium-dependent contraction to acetylcholine in isolated aorta. METHODS: SHR and Wistar-Kyoto rats (WKY) were orally treated for 3 weeks with either the cysLT biosynthesis inhibitor MK-886 (0.1 mg/ml) or vehicle. After mean arterial blood pressure (MABP) measurement, aortic ring preparations were removed from all groups of animals, and contractions and relaxations were monitored subsequent to stimulation with acetylcholine. RESULTS: MABP was higher in SHR. Chronic treatment with MK-886 did not alter MABP in either SHR or WKY. In the presence of the N-nitro-L-arginine (L-NA, 100 micromol/l), and on prostaglandin F2alpha (PGF2alpha)-induced tone, acetylcholine evoked concentration-dependent contractions in intact aortic rings from SHR only. Pretreatment with either MK-886 (10 micromol/l), the 5-lipoxygenase (5-LO) inhibitor AA861 (10 micromol/l), or the cysLT1 receptor antagonist MK571 (1 micromol/l) reduced (P < 0.05) acetylcholine-induced contractions in intact aortic rings from SHR only. Acetylcholine-induced contractions were weaker (P < 0.01) in SHR chronically treated with MK-886 than in SHR. In the presence of L-NA, leukotriene (LT) D4 induced greater (P < 0.05) concentration-dependent contractions in aortic rings from SHR than from WKY. MK571 abolished LTD4-evoked contractions. CONCLUSION: These data suggested that 5-LO-derived products, through the activation of cysLT1 receptors, could be involved in the endothelium-dependent contraction to acetylcholine in aorta from SHR but not in the regulation of MABP in SHR.     

Alterations in the release of norepinephrine at the vascular neuroeffector junction in hypertension

The field stimulation induced release of 3H-norepinephrine (NE) from the isolated portal vein and endogenous NE from the isolated caudal artery and perfused mesenteric arterial bed of spontaneously hypertensive rats (SHR) and age-matched normotensive rats (Wistar-Kyoto or Sprague-Dawley) was studied. There was a significantly greater release of NE from all three preparations obtained from 10- to 12-week-old SHR compared to normotensive animals. In addition, there was a greater release of NE from the caudal artery of 5- to 6-week-old SHR compared to controls. No differences were seen in the evoked release of NE from portal vein or caudal artery obtained from renal or DOCA salt hypertensives compared to vessels obtained from sham controls. Neuropeptide Y (NPY) produced a concentration-dependent decrease in the field stimulation induced release of NE from the perfused mesenteric artery. Low concentrations of NPY decreased while higher concentrations potentiated the increase in perfusion pressure. The NPY induced inhibition of evoked NE release was not altered by alpha 1- or alpha 2-adrenoceptor antagonists while the alpha 1-adrenoceptor antagonist, prazosin, prevented the postjunctional response. These results are consistent with there being an alteration of NE release at the vascular neuroeffector junction in SHR which may contribute to the development or maintenance of hypertension. NPY exerts a modulatory role in noradrenergic transmission at the vascular neuroeffector junction.     

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats

OBJECTIVE : Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. METHODS : Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. RESULTS : ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. CONCLUSIONS : We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.     

Enhanced neuropeptide Y immunoreactivity and vasoconstriction in mesenteric small arteries from spontaneously hypertensive rats

Enhanced sympathetic nerve activity is thought to play a role in the pathogenesis of hypertension. The purpose of the present study was to investigate the mechanisms underlying the enhanced vasocontractile response to perivascular stimulation of mesenteric arteries isolated from female spontaneously hypertensive rats (SHR). Innervation of mesenteric small arteries was evaluated by immunohistochemistry and confocal microscopy while functional studies were conducted in a microvascular myograph. The distribution of nerve terminals immunoreactive for tyrosine hydroxylase (TH) and neuropeptide Y (NPY) was similar in mesenteric small arteries from Wistar-Kyoto (WKY) and SHR rats. However, immunointensity of TH or NPY immunoreactivities were much higher in small arteries from SHR compared to WKY. Expressed as percentage of contractions elicited by 124 mM K(+), concentration-response curves for noradrenaline (NA) and NPY were shifted leftward in SHR compared with WKY rats. The combination of noradrenaline (1 microM) and NPY (10 nM) contracted mesenteric arteries from WKY and SHR to higher levels than compared to either contractile agent added alone. The NPY Y(1) receptor antagonist, BIBP 3226, inhibited these contractions with 87 +/- 0.7 and 80 +/- 1.3% (p < 0.05, n = 6) in arteries from WKY and SHR rats, respectively. In arteries incubated with the alpha(1)-adrenoceptor antagonist, prazosin, and preactivated with vasopressin, electrical field stimulation evoked contractions which were more pronounced in mesenteric arteries from SHR compared to WKY rats. BIBP 3226 partially inhibited these contractions. In vasopressin-activated arteries BIBP 3226 caused rightward shifts of the concentration-response curves for NPY in mesenteric arteries from SHR rats, but in addition it also abolished the maximal NPY contraction in arteries from WKY rats. In the presence of BIBP 3226, low concentrations (1 pM to 10 nM) of NPY caused relaxations in arteries from WKY, but not in segments from SHR rats. Mechanical removal of the endothelium abolished NPY relaxation in arteries from WKY. In arteries activated with vasopressin and exposed to either forskolin or sodium nitroprusside, the addition of NPY evoked contractions which were more pronounced in arteries from SHR compared to WKY arteries. The present study suggests that enhanced NPY content and vasoconstriction to NPY in arteries from hypertensive rats can contribute to the enhanced sympathetic nerve activity and vascular resistance in female hypertensive rats. Endothelial cell dysfunction as well as alterations in smooth muscle response to NPY seem to contribute to the enhanced vasoconstriction in arteries from hypertensive animals.     

Influence of cold-induced increases in sympathetic nerve activity on norepinephrine content in the vasculature of the spontaneously hypertensive rat

In spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY), we have examined both the endogenous norepinephrine (NE) contents of caudal arteries, mesenteric arteries and cardiac tissue as well as the rates of decline of NE in these tissues after inhibition of NE synthesis. The endogenous NE contents of caudal and mesenteric arteries from SHR rats were greater than those from WKY rats. In contrast, the NE contents of hearts from SHR and WKY rats were similar. After synthesis inhibition with alpha-methyl-p-tyrosine (300 mg/kg i.p.), the NE contents of hearts and mesenteric arteries decreased in a monoexponential fashion. The rates of decline of NE were similar for corresponding tissues from SHR and WKY rats. Cold stress, reported to selectively activate sympathetic discharge, did not influence the rates of decline of NE in mesenteric arteries of either SHR or WKY animals. In contrast, cold exposure dramatically accelerated the rate of decline of NE in cardiac tissue from both SHR and WKY rats. It is concluded that in mesenteric arteries from SHR rats there is a larger pool of NE with turnover characteristics not dissimilar from that prevailing in vessels from normotensive animals. The failure of cold stress to modify the rates of decline of NE in mesenteric and caudal arteries of SHR and WKY rats suggests that these arteries are under considerable sympathetic influence at ambient temperature. The results support the view that the hypernoradrenergic innervation found in SHR blood vessels, together with normal functioning of the sympathetic nervous system, may have the potential for producing a heightened peripheral vascular resistance in this model.     

Prostaglandin H2 may be the endothelium-derived contracting factor released by acetylcholine in the aorta of the rat

The present experiment was performed to identify endothelium-derived contracting factor produced by acetylcholine stimulation in the aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The rings of the thoracic aorta were obtained from age-matched SHR and WKY rats, and changes in isometric tension were recorded. The relaxant responses to acetylcholine in the aortic rings from SHR were significantly weaker than those from WKY rats. The relaxant responses to acetylcholine were significantly enhanced by pretreatment with a cyclooxygenase inhibitor (indomethacin) or thromboxane A2/prostaglandin H2 receptor antagonist (ONO-3708) in aortic rings from both SHR and WKY rats. A thromboxane A2 synthetase inhibitor (OKY-046) did not affect the acetylcholine-induced relaxation in the aortic rings from SHR or WKY rats. In the organ bath solution, after acetylcholine stimulation, prostaglandin E2 and 6-keto-prostaglandin F1 alpha concentrations increased but not prostaglandin F2 alpha and thromboxane B2 concentrations. Exogenous prostaglandin H2, a stable analogue of thromboxane A2, and prostaglandin F2 alpha induced contractions of the SHR rings at a lower concentration than prostaglandin E2, prostaglandin D2, and prostaglandin I2. These contractile responses to various prostaglandins were markedly inhibited by pretreatment with ONO-3708. A prostacyclin synthetase inhibitor did not affect the relaxant responses to acetylcholine in the SHR rings. These results show that endothelium-derived contracting factor is produced and released by acetylcholine stimulation not only in the aorta of SHR but also in those of WKY rats and suggest that prostaglandin H2, a precursor of the released prostaglandins, is a strong candidate for endothelium-derived contracting factor produced by acetylcholine stimulation.     

Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat

To study the mechanism of decreased endothelium-dependent relaxations in spontaneously hypertensive rats (SHR), rings of thoracic aorta with and without endothelium were taken from age-matched male SHR and normotensive Wistar-Kyoto rats (WKY) and suspended for isometric tension recording. Acetylcholine caused endothelium-dependent contractions in quiescent rings from SHR but not in those from WKY. These contractions were inhibited by atropine but not by hexamethonium and were prevented by inhibitors of phospholipase A2 or cyclooxygenase but not by inhibitors of prostacyclin synthetase, thromboxane synthetase, or leukotriene synthetase. Prostaglandin D2, E1, E2, and F2 alpha caused concentration-dependent contractions in rings without endothelium from both SHR and WKY; the responses to the highest concentration (10(-5) M) of the individual prostaglandins were comparable in both strains. Endothelium-dependent relaxations evoked by high but not by low concentrations of acetylcholine were significantly depressed in SHR as compared with those in WKY (p less than 0.05). Indomethacin normalized endothelium-dependent relaxations in SHR. Thus, acetylcholine can activate muscarinic receptors that evoke endothelium-dependent contractions in the aorta of SHR but not in that of WKY. The contraction probably is mediated by a cyclooxygenase product(s) other than prostacyclin or thromboxane A2. The reduced endothelium-dependent relaxations to acetylcholine in the SHR probably are not due to a decreased release of endothelium-derived relaxing factor(s) but to the simultaneous release of endothelium-derived contracting substance(s).     

Increased Ca2+ buffering function of sarcoplasmic reticulum in small mesenteric arteries from spontaneously hypertensive rats.

We compared the Ca2+ buffering function of the superficial sarcoplasmic reticulum (SR) during rest and during contraction in endothelium-denuded strips of small mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of caffeine (1-20 mM) caused a transient contraction in both strains, and the contraction was significantly larger in SHR. When the SR Ca2+ buffering function was eliminated by cyclopiazonic acid (CPA; 10 microM) or thapsigargin (100 nM), both of which inhibit SR Ca2+-ATPase, or by ryanodine (10 microM), which depletes the SR Ca2+, there was a larger contraction in SHR than in WKY, suggesting that the Ca2+ buffering function of the SR during rest is more important in SHR than in WKY. Judging from the augmenting effects of these three agents on the contractile responses to Bay k 8644 (1-300 nM), an agonist of L-type Ca2+ channels, or norepinephrine (10(-9)-10(-4) M), an alpha-adrenoceptor agonist, the effects were significantly greater in SHR than in WKY. We conclude that 1) the Ca2+ influx during rest and during stimulation with Bay k 8644 or norepinephrine is strongly buffered by Ca2+ uptake into the superficial SR in the small mesenteric arteries from SHR and WKY; and 2) these Ca2+ buffering functions are increased in SHR because of the larger capacity of SR for Ca2+ storage.     

Magnesium removal impairs the regulatory role of rat endothelium.

Vascular endothelium has been shown to play an important role in the regulation of vascular tone and, hence, impairment of the endothelium may induce hypertension. Although magnesium (Mg) deficiency could induce hypertension, the role of Mg on the endothelium is unclear. We examined the effects of Mg removal on endothelium-dependent and -independent responses using ring preparations of femoral arteries obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Norepinephrine (10(-9)-10(-4) M) evoked concentration-dependent contractions in arteries with endothelium. The maximal response was greater in SHR than in WKY. Removal of external Mg augmented the contraction in WKY but not in SHR. As a result, the contraction obtained in arteries with endothelium was identical in the two groups. Removal of the endothelium enhanced the contraction in both strains, with a greater response occurring in WKY than in SHR in Krebs, but not in Mg-free, solution. As a result, in arteries without endothelium, the contractions were identical in WKY and SHR both in Krebs and Mg-free solutions. Acetylcholine (10(-9)-10(-4) M) evoked concentration-dependent relaxation in arteries with, but not in those without, endothelium obtained from WKY and SHR. The relaxation did not differ between the two strains, nor was it altered by Mg removal. Thus, Mg removal impairs inhibitory function of the endothelium against contraction induced by norepinephrine, without affecting endothelium-dependent relaxation in response to acetylcholine, in the rat femoral artery. The effect of Mg removal is not apparent in SHR. The fact that after removal of external Mg the contraction in response to norepinephrine in arteries with endothelium is identical in WKY and SHR suggests that a normotensive artery with Mg deficiency may mimic a hypertensive artery through endothelial impairment.     

Eicosanoids and membrane properties in arteries of aged spontaneously hypertensive rats

OBJECTIVE: The arteries of aged spontaneously hypertensive rats (SHR) exhibit spontaneous electrical activity together with membrane depolarization. Vascular eicosanoid production is increased in SHR, which is further accelerated with aging. We tested the hypothesis that eicosanoids are involved in spontaneous electrical activity, membrane depolarization or both in mesenteric arteries of aged SHR. DESIGN AND METHODS: Membrane potentials were recorded with microelectrodes from the mesenteric arteries of aged (24 months and older) SHR, aged Wistar-Kyoto (WKY) rats and adult (6- to 8-month-old) SHR. RESULTS: The membrane potential was less negative in aged SHR (-38.5 +/- 0.9 mV) than in either aged WKY rats or adult SHR (-49.8 +/- 0.5 and -47.2 +/- 0.6 mV, respectively; P < 0.05 for both). Spontaneous electrical activity (5-20 mV, 1-7/min) was present only in arteries of aged SHR. Spontaneous electrical activity was not affected by phentolamine, atropine or tetrodotoxin, but was abolished by indomethacin, a cyclooxygenase inhibitor, and ONO-3708, a thromboxane A2/prostaglandin H2 receptor antagonist. Furthermore, indomethacin and ONO-3708 hyperpolarized the membrane by about 5 mV in aged SHR but not in the other two groups. Spontaneous electrical activity was enhanced by a thromboxane A2 analog and prostaglandin H2, and was abolished by a Ca2+ antagonist, nicardipine, and Ca(2+)-free solution. CONCLUSIONS: These findings suggest that cyclooxygenase-dependent eicosanoids contribute importantly to both spontaneous electrical activity and membrane depolarization, presumably through activation of the thromboxane A2/prostaglandin H2 receptor, in mesenteric arteries of aged SHR, and that spontaneous electrical activity is mediated by a Ca2+ influx through voltage-dependent Ca2+ channels.     

Abnormal contractile response of aortas from spontaneously hypertensive rats to Ca2+ after depletion of Ca2+ in Ca2+-free medium

Vascular responses of aortic rings from spontaneously hypertensive rats (SHR) were compared to those of the normotensive Wistar-Kyoto rats (WKY) in three sets of experimental protocols. The responses to cumulative doses of KCl indicated that SHR aortic rings were hyperresponsive to low but not high doses of KCl compared to WKY aortic rings. After Ca depletion by prolonged incubation of the rat aortic rings with Ca2+-free, EGTA containing solution, Ca repletion resulted in contraction. The magnitude of such a contraction was dependent on the period of Ca depletion and was highly sensitive to dihydropyridine Ca channel blocker, nifedipine. Although the Ca-depleted aortic rings eventually developed to the same level of maximum tension development upon Ca repletion, it took a considerably shorter period of Ca depletion for SHR than for WKY aortic rings to reach the maximum contraction upon Ca repletion. Our findings support the view that cell membranes of vascular smooth muscle in hypertension are more excitable and more susceptible to membrane destabilization by Ca removal.     

The role of endothelium-derived contracting factor (EDCF) and endothelium-derived relaxing factor (EDRF) in the aorta of the rat: identification of EDCF

The present experiment was performed to identify endothelium-derived contracting factor produced by acetylcholine stimulation in the aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The rings of the thoracic aorta were obtained from age-matched SHR and WKY, and changes in isometric tension were recorded. The relaxant responses to acetylcholine in the rings from SHR were significantly weaker than those obtained in WKY. The relaxant responses to acetylcholine were significantly enhanced by pretreatment with a cyclooxygenase-inhibitor (indomethacin) or thromboxane A2/prostaglandin H2 receptor antagonist (ONO-3708) both in the SHR and WKY rings. A thromboxane A2 synthetase inhibitor (OKY-046) did not affect the acetylcholine-induced relaxation in the rings from SHR or WKY. In the organ bath solution, following acetylcholine stimulation, prostaglandin E2 and 6-keto-prostaglandin F1 alpha concentrations increased, but prostaglandin F2 alpha and thromboxane B2 concentrations did not increase. Exogenous prostaglandin H2, a stable analogue of thromboxane A2 (STA2) and prostaglandin F2 alpha induced contractions of the SHR rings at a lower concentration than prostaglandin E2, prostaglandin D2 and prostaglandin I2. These contractile responses to various prostaglandins were markedly inhibited by pretreatment with ONO-3708. A prostacyclin synthetase inhibitor did not affect the relaxant responses to acetylcholine in the SHR rings. These results show that endothelium-derived contracting factor is produced and released by acetylcholine stimulation not only in the aorta of SHR but also in that of WKY. The results also suggest that prostaglandin H2, a precursor of the released prostaglandins, is a strong candidate for endothelium-derived contracting factor produced by acetylcholine stimulation.     

Increased oxidative stress impairs endothelial modulation of contractions in arteries from spontaneously hypertensive rats

OBJECTIVES: The endothelium modulates vascular contractions. We investigated the effects of oxidative stress on endothelial modulation of contractions in hypertension. METHODS: Changes in isometric tension of femoral arterial rings from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were recorded. RESULTS: The contractile response to norepinephrine of arteries with endothelium was greater in SHR than in WKY rats (P < 0.0001). Endothelium removal augmented the norepinephrine-induced contraction (P < 0.05). The augmentation was more pronounced in WKY than in SHR, which resulted in comparable contraction of arteries without endothelium in both strains. Nomega-nitro-L-arginine methyl ester (100 micromol/l) mimicked the effect of endothelium removal. Production of nitric oxide (NO, assessed by measuring nitrite/nitrate concentrations) during the contraction was not different between SHR and WKY. Vitamin C suppressed the contraction of arteries with endothelium from SHR but not from WKY (P < 0.05). Diphenyleneiodonium and apocynin, inhibitors of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase, attenuated the contraction of arteries with endothelium from SHR (P < 0.001) but not WKY, but did not affect contractions induced by serotonin. Superoxide generated by xanthine oxidase/hypoxanthine enhanced the norepinephrine-induced contraction of arteries with endothelium from WKY (P < 0.0001), and this effect was reversed by vitamin C. CONCLUSIONS: In rat femoral arteries, NO released from the endothelium modulates vascular contraction. In SHR, production of superoxide by NADH/NADPH oxidase, which may be activated by norepinephrine, is enhanced, resulting in the inactivation of NO and impairment of endothelial modulation of vascular contractions. Vascular oxidative stress may contribute to the altered circulation in hypertension by impairing endothelial modulation of vascular contractions.     

Enhanced 5-hydroxytryptamine release from vascular adrenergic nerves in spontaneously hypertensive rats

The release of 5-hydroxytryptamine from the vascular adrenergic nerve by periarterial nerve stimulation in spontaneously hypertensive rats (SHR) was compared with that in normotensive Wistar-Kyoto rats (WKY). The isolated mesenteric vascular bed was perfused at a constant flow rate of 5 ml/min. Vasoconstrictor responses to periarterial nerve stimulation (4, 8, 12, and 16 Hz for 30 seconds) and 5-hydroxytryptamine (1 microM), but not norepinephrine (1 nmol), were significantly greater in SHR than in WKY. After treatment with 5-hydroxytryptamine (1 microM) for 15 minutes, vasoconstrictor responses to periarterial nerve stimulation previously reduced by prazosin (50 nM) were restored and a frequency-dependent pressor response reappeared. However, 5-HT treatment did not significantly affect the pressor response to exogenously administered norepinephrine (1 nmol), which was previously inhibited by prazosin. The degree of the restoration in SHR was significantly greater than that in WKY at all frequencies used. The restoration of the pressor response to periarterial nerve stimulation after 5-hydroxytryptamine treatment did not occur in the presence of the selective 5-hydroxytryptamine2 receptor antagonists ketanserin (10 nM) or LY53857 (10 nM). In the perfused mesenteric vascular bed of both WKY and SHR prelabeled with [3H]5-hydroxytryptamine, periarterial nerve stimulation (4-16 Hz) evoked a frequency-dependent increase in tritium efflux that was abolished by Ca2+-free Krebs-Ringer solution or tetrodotoxin (100 nM) and treatment with 6-hydroxydopamine. The tritium efflux evoked by periarterial nerve stimulation was significantly greater in SHR than in WKY at all frequencies used. These results suggest that the release of 5-hydroxytryptamine from adrenergic nerve endings by periarterial nerve stimulation is enhanced in the mesenteric vascular bed of the SHR.     

Pre- and postsynaptic effects of angiotensins in the femoral artery of spontaneously hypertensive and Wistar-Kyoto rats

The effects of angiotensin I (AI) and angiotensin II (AII) on ring segments of femoral arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were studied. AI and AII elicited significantly greater direct contractile response in arteries from SHR than those from WKY. These peptides also potentiated the contractile response to transmural adrenergic nerve stimulation (TNS) in both preparations, but to a greater extent in those of WKY than SHR, without potentiating the contractile response to exogenous norepinephrine (NE). The potentiation of the TNS response and direct contraction caused by AI were markedly attenuated by captopril, an AI-converting enzyme inhibitor. Destruction of endothelium failed to alter the contractile response to AI in both WKY and SHR but augmented that to AII in WKY. Isoproterenol and salbutamol produced significant potentiation of TNS response only in arteries of SHR. Yohimbine and prostaglandin F2 alpha potentiated TNS response to a similar extent in arteries of WKY and SHR. These results suggest that AII locally generated from AI can act postsynaptically to cause contraction and presynaptically to promote adrenergic neurotransmission in the isolated rat femoral artery. The AI to AII conversion appears to take place mainly at sites other than endothelial cells. The postsynaptic effect of AII is greater in SHR than WKY, but its presynaptic effect is diminished in SHR unlike some other agents which facilitate adrenergic neurotransmission, and unlike that in mesenteric arteries of SHR.