Enhancement of arterial relaxation by long-term atenolol treatment in spontaneously hypertensive rats.

1. The effects of long-term atenolol (25 mg kg-1 day-1) therapy on arterial function were studied in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. The 14-week treatment attenuated the increase in blood pressure by approximately 30 mmHg in SHR, but did not affect blood pressure in WKY rats. 2. Responses of mesenteric arterial rings in vitro were examined at the end of the study. The relaxation to acetylcholine was similar in WKY rats and atenolol-treated SHR and more pronounced than in untreated SHR, whereas the relaxation to the nitric oxide donor 3-morpholinosydnonimine (SIN-1) was comparable in all study groups. Moreover, after maximal relaxations to acetylcholine, marked recontractions developed in untreated SHR but not in the other groups. Vasorelaxation to isoprenaline was also attenuated in SHR and was moderately improved by the atenolol therapy. 3. Arterial relaxation induced by return of potassium to the organ bath upon precontractions elicited by potassium-free solution were used to evaluate vascular smooth muscle Na+, K+-ATPase. The rate of potassium relaxation was fastest in WKY rats and was also faster in atenolol-treated than in untreated SHR. 4. The ability of vascular smooth muscle to sequester calcium was evaluated by eliciting responses to caffeine or noradrenaline after loading periods in different organ bath calcium concentrations. The subsequent contractions were lower in untreated SHR than in WKY rats, and augmented in SHR by the atenolol treatment. 5. Smooth muscle contractions to noradrenaline were comparable in SHR and WKY rats, while atenolol treatment slightly increased the maximal response to this agonist in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular calcium regulatory machinery of vasorelaxation elicited by petasin

1. The present study examined the cytosolic Ca²⁺ regulatory machinery involved in the vasorelaxation produced by petasin, a sesquiterpene isolated from Petasites formosanus. 2. Aortic rings isolated from Sprague‐Dawley rats were exposed to petasin (0.01–100 μmol/L) to elucidate its vascular effects on isometric contraction elicited by vasoconstrictors, as well as the contribution of the endothelium and Ca²⁺ to the responses observed. In addition, L‐type voltage‐dependent Ca²⁺ channel (VDCC) activity and [Ca²⁺]_i were determined in cultured vascular smooth muscle cells (VSMCs) from Sprague‐Dawley rats in the presence of 1–100 μmol/L petasin using whole‐cell patch‐clamp recording and the fluorescent probe fura‐2/AM. The effects of petasin on vascular responses were compared between aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto (WKY) rats. 3. Petasin reduced isometric contraction elicited by KCl or the L‐type Ca²⁺ channel opener BayK 8644 (IC₅₀ 3.0 ± 0.4 and 4.1 ± 1.1 μmol/L, respectively) in aortic rings isolated from Sprague‐Dawley rats, independent of the endothelium. In addition, petasin triggered a rightward shift in the concentration–response curve to KCl while reducing the maximal response by 82%. In Ca²⁺‐depleted and high K⁺‐depolarized aortic rings, 1–100 μmol/L petasin pretreatment attenuated the Ca²⁺‐induced contraction in a concentration‐dependent manner. 4. In cultured VSMCs, whole‐cell patch‐clamp recording revealed that petasin inhibited VDCC activity. Measurement of [Ca²⁺]_i using fura‐2/AM fluorescence indicated that petasin suppressed the KCl‐induced increase in [Ca²⁺]_i. However, receptor binding assays failed to identify any significant interaction between petasin and the dihydropyridine binding sites of the L‐type VDCC. 5. In aortic rings from SHR and WKY rats, petasin inhibited Ca²⁺‐induced contractions in Ca²⁺‐depleted and high K⁺‐depolarized solution with a more pronounced effect in rings from SHR. 6. Together, the results suggest that direct Ca²⁺ antagonism of L‐type VDCC in vascular smooth muscle may account, at least in part, for petasin‐induced vasorelaxation. The more pronounced effect of the sesquiterpene in blood vessels from SHR suggests its possible therapeutic potential in the mangement of hypertension.     

Increased calcium sensitivity in isolated resistance arteries from spontaneously hypertensive rats: effects of dihydropyridines.

We recorded the contractile responses to calcium in mesenteric resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) during depolarization or stimulation with noradrenaline. The effects of Bay-K8644 and nimodipine on these responses were evaluated. Calcium sensitivity was greater in noradrenaline-stimulated than in depolarized vessels. Nimodipine decreased and Bay-K8644 increased calcium sensitivity. Both substances were more potent in the presence of potassium than in the presence of noradrenaline. Calcium sensitivity was greater in SHR than in WKY vessels only during stimulation with noradrenaline. The rhythmic responses of SHR vessels during stimulation with noradrenaline were abolished by nimodipine. Rhythmicity could be induced in WKY vessels by Bay-K8644. Modulation of calcium sensitivity by dihydropyridines during electrochemical as well as pharmacological stimulation suggests that, in resistance arterial smooth muscle, the function of potential-operated calcium channels can be modulated by noradrenaline. This modulation could differ quantitatively between mesenteric resistance arteries of SHR and WKY.     

Comparative Study of Calcium Ion Dynamics and Contractile Response in Rat Middle Cerebral and Basilar Arteries

The objective of this study was to compare intracellular calcium concentration ([Ca²⁺]_i) and contractile responses in isolated rat middle cerebral artery (MCA) with those in basilar artery (BA) employing real-time confocal laser microscopy. KCl elicited transient [Ca²⁺]_i elevation and sustained contraction in both arteries; moreover, nearly equal responses were evident in both arteries. Application of 5-hydroxytryptamine (5-HT), vasopressin (VP), and α,β-methylene adenosine 5’-triphosphate (α,β-me ATP) also induced elevation of [Ca²⁺]_i and contraction in both arteries. The maximum response of 5-HT and VP necessary to increase [Ca²⁺]_i and to constrict the MCA was less in comparison to the BA; however, a linear relationship emerged between the maximum response of [Ca²⁺]_i and that of contraction. Additionally, the slope of the correlation regression line of MCA was nearly identical to that of BA. On the other hand, cyclopiazonic acid (CPA)-induced Ca²⁺ release from store sites following contraction of MCA was distinct from that of BA. In MCA, velocity of [Ca²⁺]_i elevation in smooth muscle cells and Ca²⁺-wave propagation along smooth muscle cells induced by 5-HT were slower than those in BA. These observations revealed that different regions of arteries along the same cerebral tissue may display distinct [Ca²⁺]_i response; moreover, this difference may be one reason for the distinct contractile response.     

Arterial contractions induced by cumulative addition of calcium in hypertensive and normotensive rats: influence of endothelium

Responses to cumulative addition of Ca²⁺ (0.2–2.5 mM) after precontraction with potassium chloride (KCl) and noradrenaline in Ca²⁺-free medium were studied in isolated mesenteric arterial rings from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The Ca²⁺ contractions in 125 mM KCl-stimulated endothelium-denuded rings in the presence of atenolol (10 M) and phentolamine (10 M) were less marked in SHR than WKY, although the contractions to high concentrations of KCl in normal organ bath Ca²⁺ (1.6 mM) were similar in these strains. The difference in Ca²⁺ contractions between SHR and WKY during KCl stimulation was also present after 10-min pretreatment with 1 mM ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) in Ca²⁺-free medium. However, when noradrenaline (1 M) was used as the agonist the Ca²⁺ contractions of endothelium-denuded rings in the two strains were comparable, while exposure to EGTA reduced these responses more effectively in SHR than WKY. Nifedipine (0.5 nM and 10 nM in KCl- and noradrenaline-stimulated rings, respectively) more efficiently inhibited the Ca²⁺ contractions in hypertensive than in normotensive rats.The presence of intact vascular endothelium attenuated the contractions to Ca²⁺ addition comparably (during KCl stimulation) or even more (during noradrenaline) in SHR when compared with WKY NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) counteracted this attenuation correspondingly in WKY and SHR, and L-arginine (1 mM) restored it in both strains, whereas indomethacin (10 mM) was without effect on the response. However, mesenteric arterial relaxations induced by the endothelium-dependent agonists acetylcholine and ADP in noradrenaline-precontracted (1 M) rings were clearly impaired in SHR, and also L-NAME (0.1 mM) reduced the responses to acetylcholine more efficiently in SHR. In contrast, the relaxations to acetylcholine and ADP in KCl-precontracted (60 mM) rings in the absence and presence of L-NAME were comparable between the two strains.In conclusion, attenuated contractile response to cumulative Ca²⁺ addition during stimulation with KCl clearly differentiated arterial smooth muscle of hypertensive and normotensive rats, suggesting altered function of cell membrane in SHR. The more pronounced effect of nifedipine on the response indicates abnormal function of voltage-dependent Ca²⁺ channels, and higher diminishing effect of EGTA on the contraction during noradrenaline suggests exaggerated action of the chelator on membrane-bound Ca²⁺ in SHR. Interestingly, the depressant effect of intact endothelium on the Ca²⁺ contraction response, mediated largely via nitric oxide, was not attenuated in SHR. Furthermore, impaired endothelium-dependent agonist-induced relaxations can be attributed to reduced release of endothelium-derived hyperpolarizing factor in this type of genetic hypertension. Correspondence to: M. Kähönen at the above address     

Augmented sphingosylphosphorylcholine-induced Ca2+-sensitization of mesenteric artery contraction in spontaneously hypertensive rat

Sphingosylphosphorylcholine (SPC) is a vasoconstricting lysosphingolipid, and the RhoA/Rho-kinase pathway plays an important role in SPC-induced contraction. Since RhoA/Rho-kinase-mediated signaling is involved in the generation and/or maintenance of hypertension, we compared the effect of SPC on the contractility of endothelium-denuded small mesenteric arteries in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Fura-2 Ca²⁺ signals, contractile responses, and phosphorylation of 20-kDa myosin light chains (MLC₂₀) were measured. Ten μM SPC induced a gradual and sustained vasoconstriction, which was greater in arteries of the SHR (82.5±4.3%, n=9) than in those of the WKY (26.7±4.5%, n=10). In Ca²⁺-free media, SPC gradually increased vascular tone in the SHR, but caused little vasoconstriction in the WKY. In the SHR and WKY, SPC evoked a greater vasoconstriction than did high K⁺depolarization at a given Ca²⁺ ratio, and the Ca²⁺ ratio–tension curve induced by SPC was significantly shifted to the left compared with that induced by high K⁺ depolarization. However, the magnitude of shift to the left was greater in the SHR than in the WKY. The Rho-kinase inhibitor Y-27632 significantly inhibited SPC-induced contractions, but neither the protein kinase C inhibitor calphostin-C nor PD98059, which inhibits activation of some mitogen-activated protein kinases, had any effect on the SHR or the WKY. SPC significantly increased the phosphorylation of MLC₂₀ in both the SHR and the WKY, and Y-27632 inhibited the SPC-induced increase in MLC₂₀ phosphorylation in the SHR. Our results suggest that SPC induces greater vascular tone in the SHR than in the WKY. Furthermore, our results indicate that activation of the Rho-kinase pathway plays an important role in the SPC-induced Ca²⁺ sensitization in the SHR.     

The Effect of High Calcium Intake on Intracellular Free [Ca2+] and Na+-H+ Exchange in DOC-NaCl-Hypertensive Rats

Abstract: The effects of calcium supplementation on blood pressure, intracellular free calcium concentration ([Ca²⁺],) and rate of Na⁺-H⁺ exchange were studied in DOC-NaCl-hypertensive rats. All the animals were uninephrectomized and divided into two main groups: the first group received deoxycorticosterone (DOC) (25 mg/kg, s.c.) once a week and had 0.7% NaCl as drinking fluid while the other received equal volumes of saline and tap water to drink. The animals were further divided according to dietary calcium intake: in the Control and DOC groups the chow contained 1.1% calcium, in the Calcium and DOC + Calcium groups, 2.5%. After 6 and 8 weeks, blood pressure in the DOC group was higher than in the Control group; on the other hand, the development of hypertension was attenuated in the DOC + Calcium compared with the DOC group. The Control and Calcium groups did not differ from each other. Platelets and lymphocytes were used as experimental models to study changes in the regulation of [Ca²⁺]ⁱ, evaluated by fluorescent indicators indo-1 and quin-2. In lymphocytes, basal [Ca²⁺]_i was highest in the DOC group, but similar in DOC + Calcium and Control groups. In platelets, both basal and thrombin-stimulated [Ca²⁺]_i were higher in the DOC and DOC + Calcium groups than in the Control group. In both cell types [Ca²⁺]_i was similar in Control and Calcium groups. In addition, platelets were used to study the ability of the cells to recover from intracellular acidification by first blocking the Na⁺ -H⁺ exchange in a Na⁺-free medium and then restarting the exchange mechanism by increasing the extracellular Na⁺ concentration at constant speed. Changes in [pH]_i were monitored by fluorescent indicator BCECF. The rate of Na⁺-H⁺ exchange in the recovery phase did not reveal differences between the experimental groups. Vascular smooth muscle function was examined by determining concentration-response curves for noradrenaline in mesenteric arterial rings and a shift to the left was observed in the DOC and DOC + Calcium groups compared with the Control group. The present data indicate that high calcium intake attenuates the development of mineralocorticoid-salt hypertension. The DOC-induced elevation of blood pressure is associated with higher [Ca²⁺]_i in platelets and lymphocytes, evidencing disturbances in cellular calcium handling. However, possibly due to differing characteristics in regulatory mechanisms between these cell types, supplementary calcium reduced [Ca²⁺]_i only in lymphocytes. The assumed elevation in intracellular sodium did not cause detectable changes in the Na⁺-H⁺ exchange rate in platelets. Enhanced vascular responses to noradrenaline observed in the DOC group were not altered by high calcium intake.     

(−)-Epigallocatechin-3-gallate induces contraction of the rat aorta by a calcium influx-dependent mechanism

Although the consumption of tea has been associated with beneficial cardiovascular effects, (–)-epigallocatechin-3-gallate (EGCG), the most abundant catechin in this beverage has shown seemingly contradictory actions on vascular tissues, for example vasorelaxant activity that could contribute favourably to prevention of cardiovascular disease, and contractile activity that could act in the opposite direction. The purpose of the present work was to study the contractile effects of EGCG on isolated rat thoracic aorta rings and its effects on the cytosolic free [Ca²⁺] ([Ca²⁺]_i) measured with fura-2 in cultured rat aortic smooth muscle cell line.In partially depolarised (15 mM KCl) aortic rings EGCG (30–300 µM), (±)-BAY K 8644 (0.1 µM) and thapsigargin (1 µM) induced a Ca²⁺-dependent, endothelium-independent contraction associated with [Ca²⁺]_i elevation in RASMC. EGCG enhanced the responses elicited by (±)-BAY K 8644 and thapsigargin both in aortic rings and in RASMC. Nifedipine totally inhibited the (±)-BAY K 8644-induced contraction, but only partially blocked the contractile responses to EGCG and thapsigargin, while SKF 96365 abolished both responses. The effects of these channel blockers were associated with a decrease in [Ca²⁺]_i in RASMC. Re-introduction of Ca²⁺ in the medium after depletion of intracellular Ca²⁺ stores with thapsigargin in a Ca²⁺-free solution elicited a contraction of aortic rings and an increase in [Ca²⁺]_i in RASMC. In both cases, this response was partially sensitive to nifedipine, abolished by SKF 96365 and clearly enhanced by EGCG.These results suggest that EGCG induces a transient endothelium-independent contraction in the rat aorta, probably by increasing smooth vascular cell membrane permeability to Ca²⁺ through both non-specific and dihydropyridine-sensitive Ca²⁺ channels.     

马尾松花粉硫酸酯化多糖对大鼠主动脉平滑肌细胞 [Ca2+]i调控及增殖的影响

目的 研究马尾松花粉多糖PPM60-A及其硫酸酯化物SPPM60-A对大鼠动脉平滑肌细胞 [Ca²⁺]_i调控及增殖的影响。方法 常规水提醇沉法制备马尾松花粉多糖,Sephacryl S-400HR色谱分离得PPM60-A,氯磺酸-吡啶法得硫酸酯化物SPPM60-A,酯化度为1.28。酶解法分离制备大鼠动脉平滑肌细胞,测定酯化前后多糖对其胞内 [Ca²⁺]_i和细胞增殖的影响。结果 PPM60-A和SPPM60-A均可以降低 [Ca²⁺]_i,抑制高K⁺和去甲肾上腺素（NE）诱导的钙离子升高,降低高K⁺引起的钙离子水平上升,对NE诱导的血管主动脉平滑肌细胞增殖具有显著的抑制作用。PPM60-A作用效果好于SPPM60-A。结论 PPM60-A及SPPM60-A均能抑制细胞外Ca²⁺内流,抑制血管平滑肌细胞增殖。     

Methanol-Induced Contraction of Canine Cerebral Artery and Its Possible Mechanism of Action

In the present report, we investigated the effects of methanol on canine basilar cerebral arterial rings. Our data indicate that acute methanol exposure (5–675 mM) induces potent contractile responses of cerebral arteries in a concentration-dependent manner. Pharmacological antagonists, such as propranolol, phentolamine, haloperidol, methysergide, naloxone, diphenhydramine, and cimetidine, did not exert any effects on these methanol-induced contractions. Likewise, a potent antagonist of cyclo-oxygenase, and subsequent synthesis of prostanoids (i.e., indomethacin), failed to exert any effect on methanol-induced contractions. No differences in responsiveness to methanol in canine cerebral arteries were found in vessel segments with or without endothelial cells. Removal of extracellular Ca²⁺([Ca²⁺]_o) partially attenuated methanol-induced contractions, while withdrawal of extracellular Mg²⁺([Mg²⁺]_o) potentiated the contractions. In the complete absence of [Ca²⁺]_o, 10 mM caffeine and 400 mM methanol induced similar, transient contractions followed by relaxation in K⁺-depolarized cerebral vascular tissues. Methanol-induced contractions were, however, completely abolished by pretreatment of tissue with 10 mM caffeine. Our results indicate that (1) methanol causes contractile responses of cerebral arterial smooth muscle (independent of amine, prostanoid, or opioid mediation; (2) in addition to a need for [Ca²⁺]_o, an intracellular release of Ca²⁺is required for methanol-induced contractions; and (3) Mg deficiency potentiates the contractile responses of methanol on these brain vessels. The data presented in the study suggest that methanol-induced contractions occur via an sarcoplasmic reticulum-releasable store of [Ca²⁺]_i; via mediation of either ryanodine–caffeine type receptors or a caffeine-releasable intracellular store of Ca²⁺.     

BRL 38227 (levcromakalim)-induced hyperpolarization reduces the sensitivity to Ca2+ of contractile elements in canine coronary artery

Summary Potassium (K⁺) channel openers decrease intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) by hyperpolarizing the membrane and deactivating the Ca²⁺-channels. To examine whether the hyperpolarization produced by K⁺-channel openers has other effects on the mechanical activity of vascular smooth muscle, we investigated the effects of levcromakalim (BRL 38227) on membrane potential, [Ca²⁺]_i, as measured with fura-2, and force of contraction induced by 30 mmol/l KCl-physiological salt solution (PSS), in canine coronary arteries. BRL 38227 hyperpolarized the membrane and reduced increases in [Ca²⁺]_i and in contractile force induced by 30 mmol/l KCl-PSS. The [Ca²⁺]_i-contractile force curve, determined in the presence of BRL 38227, was located to the right of the control curve determined by decreasing extracellular Ca²⁺ concentrations ([Ca²⁺]_o) in 30 mmol/l KCl-PSS. The [Ca²⁺ _i-contractile force curve, determined by decreasing extracellular K⁺ concentrations ([K⁺]_o), was also located to the right of that determined by decreasing [Ca²⁺]_o in 30 mmol/l KCl-PSS. The effect of BRL 38227, a reduction in the Ca ²⁺-sensitivity of contractile elements, was antagonized by the ATP-sensitive K⁺-channel blocker, glibenclamide (10^–6 or 10^–5 mol/1). These results suggest that the membrane hyperpolarization induced by BRL 38227, or the repolarization caused by reducing ([K⁺]_o), decreases the Ca²⁺-sensitivity of contractile elements of vascular smooth muscle.Correspondence to T. Yanagisawa at the above address     

Mechanisms of reduced contractility in an animal model of hypertensive heart failure

1. Alterations in intracellular Ca²⁺ homeostasis have frequently been implicated as underlying the contractile dysfunction of failing hearts. Contraction in cardiac muscle is due to a balance between sarcolemmal (SL) and sarcoplasmic reticulum (SR) Ca²⁺ transport, which has been studied in single cells and small tissue samples. However, many studies have not used physiological temperatures and pacing rates, and this could be problematic given different temperature dependencies and kinetics for transport processes. 2. Spontaneously‐hypertensive rats (SHR) and their age‐matched Wistar Kyoto controls (WKY) provide an animal model of hypertensive failure with many features in common to heart failure in humans. Steady‐state measurements of Ca²⁺ and force showed that peak stress was reduced in trabeculae from failing SHR hearts in comparison to WKY, although the Ca²⁺ transients were bigger and decayed more slowly. 3. Dynamic Ca²⁺ cycling was investigated by determining the recirculation fraction (RF) of activator Ca²⁺ through the SR between beats during recovery from experimental protocols that potentiated twitch force. No difference in RF between rat strains was found, although the RF was dependent on the potentiation protocol used. 4. Superfusion with 10 mmol/L caffeine and 0 mmol/L [Ca²⁺]_o was used to measure SL Ca²⁺ extrusion. The caffeine‐induced [Ca²⁺]_i transient decayed more slowly in SHR trabeculae, suggesting that SL Ca²⁺ extrusion was slower in SHR. 5. An ultrastructural immunohistochemical analysis of left ventricular free wall sections using confocal microscopy showed that t‐tubule organization was disrupted in myocytes from SHR, with reduced labelling of the SR Ca²⁺‐ATPase and Na⁺–Ca²⁺ exchanger in comparison to WKY, with the latter possibly related to a lower fraction of t‐tubules per unit cell volume. 6. We suggest that although Ca²⁺ transport is altered in the progression to heart failure, force development is not limited by the amplitude of the Ca²⁺ transient. Despite slower SR Ca²⁺ transport, the recirculation fraction and dynamic response to a change of inotropic state minimally altered changes in the SHR model because there was a similar slowing in Ca²⁺ extrusion across the surface membrane.     

Intraplatelet Free Calcium and Calcium-Regulating Hormones in Plasma are Not Related to the Antihypertensive Effect of Nifedipine in Hypertensive Pregnancy

Abstract: Intracellular free calcium regulates contraction-relaxation processes in vascular smooth muscle. We compared intraplatelet free calcium ([Ca²⁺]_i) and pH ([pH]_i) in hypertensive pregnant women to those in normotensive pregnant and non-pregnant women. Plasma parathormone and vitamin D metabolite were simultaneously assessed. In hypertensive pregnancy, [Ca²⁺]_i tended to be lower than in normotensive pregnant (P=0.08) and non-pregnant subjects (P=0.06). In hypertensive pregnancy, 1,25 (OH)₂ vitamin D in plasma was in the same range as in non-pregnant women and significantly lower than in normotensive pregnancy (p<0.01). The other two vitamin D metabolites, parathormone and [pH]_i were equal in the three groups. A five-day nifedipine treatment (10 mg t.i.d.) increased [Ca²⁺]_i in hypertensive pregnant (P<0.05) and normotensive non-pregnant subjects (P=0.06), whereas [pH]_i (P<0.05) and 25 (OH) vitamin D (P<0.05) decreased in the former and 24,25 (OH)₂ vitamin D increased in the latter group (P<0.05). Initial [Ca²⁺]_i did not correlate with blood pressure in any group. The antihypertensive effect of nifedipine did not correlate with any variable measured. In conclusion, [Ca²⁺]_i and calcium-regulating hormones seem not to be related to the antihypertensive effect of nifedipine in hypertensive pregnancy. In this type of hypertension, intraplatelet calcium may not reflect calcium balance in smooth muscle cells regulating vascular tone and blood pressure.     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.     

Exposure of piglet coronary arterial muscle cells to low concentrations of Mg2+ found in blood of ischemic heart disease patients result in rapid elevation of cytosolic Ca2+: Relevance to sudden infant death syndrome

Exposure of cultured piglet primary neonatal coronary arterial smooth muscle cells to concentrations of ionized Mg²⁺ ([Mg²⁺]_o (i.e., 0.48, 0.3, 0.15 mM) found in blood of patients presenting with ischemic heart disease and in hypoxic neonates resulted in concentration-dependent elevation in intracellular free Ca²⁺ ions ([Ca²⁺]_i; the lower the [Mg²⁺]_o, the higher the [Ca²⁺]_i rise. The lowest concentration of [Mg²⁺]_o tested, i.e., 0.15 mM, resulted in a clear rounding-up (i.e., contraction) of many of the coronary smooth muscle cells; reintroduction of normal 1.2 mM [Mg²⁺]_o failed to restore either normal [Ca²⁺]_i or cell shape.     

Three levels of dietary calcium-effects on blood pressure and electrolyte balance in spontaneously hypertensive rats

Summary The effects of three levels of calcium intake on blood pressure (BP) and electrolyte balance were studied for 12 weeks in spontaneously hypertensive rats (SHR): the chow of the SHR-1 group contained 1.1% calcium, and that of the supplemented groups 2.1% (SHR-2) and 3.1% (SHR-3) calcium. Wistar-Kyoto rats on a 1.1% calcium diet (WKY-1) served as normotensive controls. After 10 and 12 weeks BP was significantly lower in both calcium-supplemented groups than in the SHR-1 group, the SHR-2 and SHR-3 groups not deviating from each other. Platelets and lymphocytes were used as experimental cell models to study the effects of the calcium diets on intracellular free calcium ([Ca²⁺]_i) level, which was measured by the fluorescent indicator quin-2. At the end of the study [Ca²⁺]_i was lower in both cell types in SHR-2 and SHR-3 than in SHR-1, the supplemented groups being comparable to each other. In platelets [Ca²⁺]_i still remained higher in the calcium-treated than the WKY-1 group, while in lymphocytes the levels were similar between SHR-2, SHR-3 and WKY-1. Plasma sodium, calcium and magnesium levels did not differ in the SHR groups, but plasma potassium was higher in both supplemented groups than in SHR-1. Plasma renin activity was comparable in SHR-1, SHR-2 and WKY-1, but was suppressed in the SHR-3 group. Creatinine clearance in the SHR-3 group was higher than in SHR-1 and SHR-2, but still remained lower than in WKY 1. High calcium intake was associated with a dose-dependent increase in urinary magnesium excretion, while the excretions of sodium and potassium were proportional to the intakes. The tissue Na⁺ :K⁺ ratio in abdominal aorta and tail artery was reduced in SHR-2,.but only a nonsignificant tendency was observed in SHR-3 when compared with the SHR-1 group.In summary, high calcium intake reduces [Ca²⁺]_i in both platelets and lymphocytes in SHR, suggesting that alterations in cellular calcium regulation may explain the BP-lowering effect of calcium supplementation. Elevation of dietary calcium level from 1.1% to 2.1% is associated with a lowering of the Na⁺ :K⁺ ratio in the arterial wall. A further increase in calcium content from 2.1% to 3.1% appears to have a favourable effect on renal function in SHR, but also aggravates magnesium loss into the urine. During the higher supplemented calcium intake, suppression of the renin-angiotensin system seems to be involved in the lowering of BP. Correspondence to H. Wuorela at the above address     

Mechanisms of 5-hydroxytryptamine-induced inhibition in the porcine myometrium

1 The present experiments were designed to clarify the mechanisms of the inhibitory response of 5-hydroxytryptamine (5-HT) in the porcine uterine circular muscle. 2 Inhibitory responses induced by 5-HT (1 n m –1 μm ) were not affected by apamin (1 μm ), charybdotoxin (100 n m ) or glibenclamide (20 μm ) but were significantly attenuated by 4-aminopyridine (3 m m ) and tetraethylammonium (3 m m ). 3 Imidazole (100 μm ) decreased but 3-isobutyl-1-methylxanthine (30 μm ), milrinone (30 μm ) and Ro 20–1724 (10 and 30 μm ) potentiated the 5-HT-induced inhibition. On the other hand, zaprinast (3–30 μm ) had no significant effect on the inhibitory response of 5-HT. 4 5-HT caused a time (0–5 min)-and concentration (1 n m –1 μm )-dependent increase in the tissue cyclic AMP level, but had no effect on the tissue cyclic GMP level. A significant correlation (P < 0.05) was observed between the inhibition of contraction and tissue cyclic AMP level. 5 The effect of 5-HT on contractile force and cytosolic Ca²⁺ level ([Ca²⁺]_i) was investigated using fura-PE3-loaded myometrial strips. A low concentration of 5-HT (≤ 10 n m ) inhibited the spontaneous contraction without changing the amplitude of the spontaneous [Ca²⁺]_i increase, but a higher concentration of 5-HT (≥ 100 n m ) decreased the resting [Ca²⁺]_i and inhibited both the spontaneous [Ca²⁺]_i increase and spontaneous contraction. 6 High-K⁺ (50 m m ) caused increases in muscle contractile force and [Ca²⁺]_i. 5-HT concentration-dependently inhibited the high-K⁺-induced contraction (EC₅₀, 45 n m ) with only a small decrease in [Ca²⁺]_i increase. 7 Carbachol also caused increases in muscle contractile force and [Ca²⁺]_i. 5-HT significantly decreased both the carbachol-induced contraction and [Ca²⁺]_i increase, but was more potent at inhibition of contractile force than [Ca²⁺]_i. 8 In Ca²⁺ -loaded myometrial strips, carbachol, but not caffeine, caused a transient increase in [Ca²⁺]_i and contraction in the absence of external Ca²⁺ (EGTA, 1 m m ). 5-HT inhibited both the carbachol-induced increases in [Ca²⁺]_i release and contractile force. 9 In the β-escin permeabilized myometrium, 5-HT significantly inhibited the Ca²⁺-induced contraction. 10 The present results indicate that 5-HT stimulates tissue cyclic AMP production, and inhibits the porcine uterine muscle contractility by a reduction in [Ca²⁺]_i and in Ca²⁺ sensitivity of the contractile elements. Activation of K⁺ channels might be partially involved in 5-HT-induced inhibition of the myometrial contractility.     

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+-activated K+ channels

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca²⁺-activated K⁺ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K⁺ released from the endothelium facilitates EDH by increasing inward rectifier K⁺ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger I_Kir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller I_Kir in SHR than WKY. While the treatment with I_Kir inhibitor (0.1 mmol/L Ba²⁺) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher I_Kir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1 mmol/L Ba²⁺ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of I_Kir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.     

NS11021, a novel opener of large-conductance Ca2+-activated K+ channels,enhances erectile responses in rats

Background and purpose:

Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1-(3,5-Bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK_Ca channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5.

Experimental approach:

Patch clamp was used to record whole cell current in rat isolated corpus cavernosum smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs). Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from rats and men, and simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tension were performed in intracavernous arteries. Erectile response was measured in anaesthetized rats.

Key results:

In patch clamp recordings, NS11021 increased currents sensitive to the selective BK_Ca channel blocker, iberiotoxin (IbTX) in SMCs, but did not modulate K⁺ current in HUVECs. NS11021 reduced [Ca²⁺]_i and tension in penile arteries. IbTX inhibited the vasorelaxation induced by NS11021 and sildenafil in human erectile tissue. NS11021 and sildenafil but not vehicle increased erectile responses in anaesthetized rats, an effect which was abolished after pretreatment with tetraethylammonium.

Conclusions and implications:

NS11021 leads to relaxation of both intracavernous arteries and corpus cavernosum strips primarily through opening of BK_Ca channels. It is also effective in facilitating erectile responses in anaesthetized rats. These results suggest a potential for use of BK_Ca openers in the treatment of ED.     

Impairment of α1‐adrenoceptor‐mediated contractile activity in caudal arterial smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. In the present study, we compared the responsiveness of de‐endothelialized caudal artery smooth muscle strips, isolated from Type 2 diabetic Goto‐Kakizaki (GK) and normal Wistar rats, to α₁‐adrenoceptor stimulation (cirazoline) and membrane depolarization (K⁺). 2. The contractile and myosin 20 kDa light chain (LC₂₀) phosphorylation responses to 0.3 μmol/L cirazoline of caudal artery strips isolated from 12‐week‐old GK rats were significantly reduced compared with those of age‐matched Wistar rats, whereas the contractile and LC₂₀ phosphorylation responses to 60 mmol/L K⁺ were unaltered. 3. Stimulation of fura 2‐AM‐loaded strips from GK rats with 0.3 μmol/L cirazoline induced a significantly smaller rise in [Ca²⁺]_i (by ～20%) compared with that in strips from Wistar rats, whereas comparable Ca²⁺ transients were evoked by K⁺ in both. 4. Using quantitative polymerase chain reaction, no significant differences were detected in the mRNA expression of α_1A‐, α_1B‐ and α_1D‐adrenoceptor subtypes between GK and Wistar rats. 5. Cirazoline (1 μmol/L)‐ and caffeine (20 mmol/L)‐induced contractions in the absence of extracellular Ca²⁺ were unaltered in GK rats, suggesting that the release of Ca²⁺ from the sarcoplasmic reticulum in response to cirazoline does not differ between GK and Wistar rats. 6. The results of the present study suggest that Ca²⁺ entry from the extracellular space via α₁‐adrenoceptor‐activated, Ca²⁺‐permeable channels, but not via membrane depolarization and voltage‐gated L‐type Ca²⁺ channels, is impaired in caudal artery smooth muscle of GK rats.