Responses Of Arterial Smooth Muscle Research Articles

Abnormal myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization contribute to impaired myogenic regulation of cerebral arterial diameter in the type 2 diabetic Goto-Kakizaki rat

The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain phosphatase by phosphorylating its targeting subunit myosin phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8–10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18–20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.

CaV3.2 knockout mice display enhanced myogenic tone due to reduced BKCa‐mediated feedback (1077.3)

In resistance arteries, there is an emerging view that CaV3.2 channel restrains vascular constriction through a feedback response that involves ryanodine receptors (RyR), Ca2+ spark generation and the modulation of large‐conductance Ca2+ activated K+ channel (BKCa). In the present study, we tested this hypothesis by examining the vasomotor, electrophysiological and Ca2+ responses of mesenteric arterial smooth muscle isolated from wild type (WT) and CaV3.2 knockout (KO) mice. Using vessel myography, we first observed enhanced myogenic tone in KO arteries, a finding consistent with the loss of feedback response. Control myography and electrophysiological experiments subsequently confirmed the inability of Ni2+ (CaV3.2 blocker) to either constrict arteries or suppress T‐type currents in KO tissues. Line scan analysis, performed on opened arteries loaded with fluo‐4, illustrated the presence of Ca2+ sparks in all arteries with the subsequent application of Ni2+ only affecting WT arteries. Consistent with Ca2+ imaging, the frequency of BKCa‐induced spontaneous transient outward currents (STOC) dropped in WT but not KO cells upon the pharmacological suppression of CaV3.2 channel. Overall, our findings support the view that CaV3.2 channels drive a local Ca2+‐induced Ca2+ release‐like process that restrains arterial constriction through the induction of Ca2+ sparks and the activation of BKCa channels.

Symphony of Vascular Contraction

The principle functions of arterial smooth muscle cells include contraction, relaxation, and growth. Calcium signaling mechanisms govern the main functions of arterial smooth muscle and trigger specific responses.1–3 Cytosolic calcium concentrations and calcium signals are finely tuned by intracellular sources such as the sarcoplasmic reticulum, calcium-binding proteins, and plasma membrane calcium permeable channels. In hypertension, these mechanisms are substantially modified, promoting a hypercontractile state and arterial wall remodeling. In this review, we will discuss various elements that are central to intracellular calcium handling and signaling in arterial smooth muscle cells. Emphasis will be given to most recent discoveries of components that link intracellular calcium stores to plasma membrane calcium entry channels. In addition, we will propose a novel paradigm, suggesting that in hypertension, alarm signals generated by chronic innate immune system activation and transduced by pattern recognition receptors modulate calcium signaling mechanisms in arterial smooth muscle, promoting vascular dysfunction. Finally, new research directions in the context of calcium signaling in hypertension will be addressed. Arterial smooth muscle contraction is regulated by receptor or mechanical activation of the contractile proteins actin and myosin.4 Changes in the membrane potential can also initiate contraction. The phosphorylation state of the light chain of myosin determines the contractile activity of arterial smooth muscle. Specifically, for contraction to occur, myosin light chain (MLC) kinase must phosphorylate Ser 19 of the 20-kDa regulatory MLC, enabling the interaction between myosin and actin.5,6 The cycling of the myosin cross-bridges with actin is promoted by energy released from ATP by myosin ATPase activity.7,8 In some arteries, MLC is in a phosphorylated state in the absence of any external stimuli (ie, vascular smooth muscle tone). An increase in cytosolic calcium concentration is the trigger for vascular contraction.9 Hypertensive patients and …

Agonist‐dependent modulation of arterial endothelinA receptor function

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) causes long-lasting vasoconstrictions. These can be prevented by ET(A) receptor antagonists but are only poorly reversed by these drugs. We tested the hypothesis that endothelin ET(A) receptors are susceptible to allosteric modulation by endogenous agonists and exogenous ligands. EXPERIMENTAL APPROACH Rat isolated mesenteric resistance arteries were pretreated with capsaicin and studied in wire myographs, in the presence of L-NAME and indomethacin to concentrate on arterial smooth muscle responses. KEY RESULTS Endothelins caused contractions with equal maximum but differing potency (ET-1 = ET-2 > ET-3). ET-1(1-15) neither mimicked nor antagonized these effects in the absence and presence of ET(16-21). 4(Ala) ET-1 (ET(B) agonist) and BQ788 (ET(B) antagonist) were without effects. BQ123 (peptide ET(A) antagonist) reduced the sensitivity and relaxed the contractile responses to endothelins. Both effects depended on the agonist (pK(B): ET-3 = ET-1 > ET-2; % relaxation: ET-3 = ET-2 > ET-1). Also, with PD156707 (non-peptide ET(A) antagonist) agonist-dependence and a discrepancy between preventive and inhibitory effects were observed. The latter was even more marked with bulky analogues of BQ123 and PD156707. CONCLUSIONS AND IMPLICATIONS These findings indicate allosteric modulation of arterial smooth muscle ET(A) receptor function by endogenous agonists and by exogenous endothelin receptor antagonists. This may have consequences for the diagnosis and pharmacotherapy of diseases involving endothelins.

Interactions between calcium and reactive oxygen species in pulmonary arterial smooth muscle responses to hypoxia

In contrast to the systemic vasculature, where hypoxia causes vasodilation, pulmonary arteries constrict in response to hypoxia. The mechanisms underlying this unique response have been the subject of investigation for over 50 years, and still remain a topic of great debate. Over the last 20 years, there has emerged a general consensus that both increases in intracellular calcium concentration and changes in reactive oxygen species (ROS) generation play key roles in the pulmonary vascular response to hypoxia. Controversy exists, however, regarding whether ROS increase or decrease during hypoxia, the source of ROS, and the mechanisms by which changes in ROS might impact intracellular calcium, and vice versa. This review will discuss the mechanisms regulating [Ca2+]i and ROS in PASMCs, and the interaction between ROS and Ca2+ signaling during exposure to acute hypoxia.

Adaptive response of pulmonary arterial smooth muscle to length change

Hypervasoconstriction is associated with pulmonary hypertension and dysfunction of the pulmonary arterial smooth muscle (PASM) is implicated. However, relatively little is known about the mechanical properties of PASM. Recent advances in our understanding of plastic adaptation in smooth muscle may shed light on the disease mechanism. In this study, we determined whether PASM is capable of adapting to length changes (especially shortening) and regain its contractile force. We examined the time course of length adaptation in PASM in response to step changes in length and to length oscillations mimicking the periodic stretches due to pulsatile arterial pressure. Rings from sheep pulmonary artery were mounted on myograph and stimulated using electrical field stimulation (12-16 s, 20 V, 60 Hz). The length-force relationship was determined at L(ref) to 0.6 L(ref), where L(ref) was a reference length close to the in situ length of PASM. The response to length oscillations was determined at L(ref), after the muscle was subjected to length oscillation of various amplitudes for 200 s at 1.5 Hz. Release (or stretch) of resting PASM from L(ref) to 0.6 (and vice versa) was followed by a significant force recovery (73 and 63%, respectively), characteristic of length adaptation. All recoveries of force followed a monoexponential time course. Length oscillations with amplitudes ranging from 5 to 20% L(ref) caused no significant change in force generation in subsequent contractions. It is concluded that, like many smooth muscles, PASM possesses substantial capability to adapt to changes in length. Under pathological conditions, this could contribute to hypervasoconstriction in pulmonary hypertension.

High Glucose Activates Nuclear Factor of Activated T Cells in Native Vascular Smooth Muscle

Hyperglycemia has been suggested to play a role in the development of vascular disease associated with diabetes. Atypical Ca2+ signaling and gene expression are characteristic of vascular dysfunction; however, little is known regarding the effects of high glucose on Ca2+-dependent transcription in the vascular wall. Using confocal immunofluorescence, we show that modest elevation of extracellular glucose (ie, from 2 to 11.5 mmol/L) increased [Ca2+]i, leading to nuclear accumulation of nuclear factor of activated T cells (NFAT) in intact cerebral arteries from mouse. This was accompanied by increased NFAT-dependent transcriptional activity. Both the increase in Ca2+ and NFAT activation were prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. We provide evidence that the potent vasoconstrictors and growth stimulators UTP and UDP mediate glucose-induced NFAT activation via P2Y receptors. NFAT nuclear accumulation was inhibited by the voltage-dependent Ca2+ channel blockers verapamil and nifedipine, the calcineurin inhibitor cyclosporine A, and the novel NFAT blocker A-285222. High glucose also regulated glycogen synthase kinase 3beta and c-Jun N-terminal kinase activity, yielding decreased kinase activity and reduced export of NFAT from the nucleus, providing additional mechanisms underlying the glucose-induced NFAT activation. Our results identify the calcineurin/NFAT signaling pathway as a potential metabolic sensor for the arterial smooth muscle response to high glucose.

Efficacy of different vasodilators on human umbilical arterial smooth muscle under normal and reduced oxygen conditions

The ability of the smooth muscle of the human umbilical artery to relax may vary under physiological and pathological conditions. We investigated the responsiveness of that preparation to relaxation, as well as the influence of reduced oxygen condition on these responses. Rings of human umbilical arteries from full-term Caesarian deliveries were suspended in modified Krebs–Henseleit solutions bubbled with a gas mixture of 95% O 2 : 5% CO 2 (normal oxygen condition) or 2.5% O 2 : 8% CO 2 balanced with N 2 (reduced oxygen condition). These rings were contracted with potassium chloride, serotonin or the thromboxane A 2 mimetic U46619, before being exposed to either the nitrovasodilator sodium nitroprusside, the potassium channel opener levcromakalim or the calcium channel antagonist amlodipine. While sodium nitroprusside elicited relaxation in this blood vessel, the maximal relaxation to the nitric oxide donor was significantly smaller than that induced by levcromakalim and amlodipine. The nature of the constrictor agent used, or changes of oxygen conditions did not significantly affect the relaxation profile of this human blood vessel. These data suggest that the smooth muscle of the human umbilical artery may be less responsive to vasodilators that act via the nitric oxide pathway. Moreover, vascular responses of umbilical arterial smooth muscle to relaxing agents do not alter under hypoxic or different vasoconstricting conditions.

Relation of Brachial Artery Reactivity to Nitroglycerin and Heart Rate Recovery Following Exercise in Healthy Male Volunteers

The parasympathetic nervous system facilitates peripheral arterial vasodilation and is also responsible for a decrease in heart rate immediately after exercise (heart rate recovery [HRR]). The relation among parasympathetic tone measured by HRR after exercise, endothelium-mediated vasodilation, and nitroglycerin-mediated vasodilation (determined with brachial artery ultrasound) was assessed in 25 healthy young men. One-minute HRR was nonsignificantly related to endothelium-mediated vasodilation (r = -0.35, p = 0.08) but was significantly related to nitroglycerin-mediated vasodilation (r = -0.63, p = 0.0008), a finding that persisted after adjustment for heart rate at rest, insulin resistance, lipid variables, and blood pressure. This suggests that parasympathetic tone may be inversely related to the responsiveness of arterial smooth muscle to nitrates in healthy humans.

Cardiovascular hypertrophy in one-kidney, one-clip renal hypertension is resistant to heparin.

Heparin inhibits vascular hypertrophy in angiotensin-induced hypertension, in addition to its well-known role in inhibiting injury-induced vascular smooth muscle proliferation. We tested whether hypertension and vascular hypertrophy could be reduced by heparin independently from the renin-angiotensin system. Rats were made hypertensive with a one-kidney, one-clip (1K1C) procedure and received heparin from osmotic minipumps (0.3 mg/h per kg i.v.) or saline vehicle for 2 weeks. Blood pressure was measured by the tail-cuff method and vessel cross-sectional area was measured by morphometry in the aorta and mesenteric arteries. Proliferation was assessed with bromodeoxyuridine labelling. Blood pressure elevation and cardiovascular hypertrophy were evident in 1K1C rats. The media of mesenteric arteries was increased by 25%, and the media : lumen ratio by 35%, in hypertensive rats. DNA synthesis by smooth muscle cells in the mesenteric arteries was increased sevenfold in renal hypertension. Heparin treatment did not influence either the increase in blood pressure, the cardiovascular hypertrophy response or hypertension-mediated proliferation of arterial smooth muscle cells. These data suggest that the vascular hypertrophy mechanisms operating in 1K1C renal hypertension are not inhibited by heparin and thus are different from those in angiotensin-mediated hypertension. Identifying such mechanisms in the future will be important for devising appropriate intervention strategies in angiotensin-independent forms of vascular hypertrophy.

Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane.

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.

Effect of puberty on coronary arteries from female pigs.

Vascular function changes following loss of ovarian hormones in women at menopause and in experimental animals following surgical ovariectomy. Little is known about changes in vascular function during hormonal transition from sexual immaturity (juvenile) to sexual maturity. Therefore, experiments were designed to determine effects of natural puberty on vascular function in female pigs. Tissue was studied from eight juvenile (2-3 mo) and eight adult (5-6 mo) female pigs. Plasma nitric oxide (NO) was measured, and mRNA for endothelium-derived NO synthase (eNOS) and eNOS protein were determined in aortic endothelial cells. Rings of coronary arteries were suspended for measurement of isometric force in organ chambers. Serum 17beta-estradiol levels were comparable in the two groups, whereas the arithmetic mean of progesterone levels was about two-thirds lower in adults compared with juvenile pigs. Plasma NO was significantly higher in juveniles compared with adults, but mRNA and protein for eNOS were comparable. In coronary arteries, an alpha2-adrenergic agonist caused greater endothelium-dependent relaxations in rings from juvenile compared with adult pigs. Relaxations to bradykinin were similar in arteries from both groups, but inhibition of NO reduced relaxations only in arteries from juvenile pigs. Relaxations from NO were greater in arteries from adult compared with juvenile female pigs. In conclusion, coronary arterial endothelial and smooth muscle responses are selectively modulated at puberty in female pigs. At maturity, plasma NO is reduced and sensitivity of the smooth muscle to exogenous NO is increased. Posttranscriptional regulation of eNOS protein may explain differences in NO bioavailability in juvenile pigs.

Mechanical and Electrophysiological Effects of Isoprostanes on Bronchial Arterial Smooth Muscle

We investigated the mechanical and electrophysiological responses of human and porcine bronchial arterial smooth muscle to isoprostanes (metabolites of membrane lipid peroxidation). These evoked a constrictor response which was sensitive to blockade of thromboxane receptors, as well as to a non-specific tyrosine kinase inhibitor and an inhibitor of Rho-kinase. The patch clamp technique was used to characterize the K+ and Ca2+ currents in these tissues, and to show that isoprostanes caused a brief enhancement of K+ currents followed by prolonged and marked suppression of the same.

Response of arterial smooth muscle to length perturbation.

The ability of arterial smooth muscle to generate tension is influenced by muscle length. An unsettled question is whether the length-tension relationship is a simple reflection of the contractile filament overlap, as it is in skeletal muscle. There are several factors that could potentially affect tension generation in arterial smooth muscle; these include stretch-induced myogenic response and length-oscillation-induced disruption of the contractile filament organization. In this study, in which rabbit carotid arterial preparations were used, we found that different length-tension curves could be obtained at different times after a length change. In addition, length oscillation at a frequency of normal pulse rate and with small to moderate oscillation amplitude was found to potentiate tension generation but reduced tension at large amplitudes. The observed response could be attributed to adaptation of the muscle to length change over time and to myogenic potentiation associated with stretching of the muscle.

The influence of the wall tension on the contractile responses of arteries.

The present study aimed at determining the influence of tension on the responses of arterial smooth muscle to noradrenaline, phenylephrine and angiotensin II. Concentration-response curves to these agonists were obtained in the rat aorta, at two levels of tension: 3 g (29.4 mN) and 0.5 g (4.9 mN). The results obtained show that the maximal responses to the agonists used (in percent of the maximal response to noradrenaline) were significantly larger (P < 0.05) at 3 g than at 0.5 g: 113% versus 66%, respectively, for noradrenaline; 95% versus 59%, respectively, for phenylephrine; 60% versus 24%, respectively, for angiotensin II. In the presence of gadolinium (100 mumol/L)--a mechanogated ion channel blocker--the responses to noradrenaline at 3 g were still significantly larger than responses at 0.5 g: 103% versus 67%, respectively. The compound H-7 (20 mumol/L)--a protein kinase C inhibitor--caused a marked decrease in the maximal responses to noradrenaline at both levels of tension, the responses being reduced to 44% at 3 g and to 20% at 0.5 g. Isradipine (1 mumol/L)--a calcium channel blocker--caused a slight decrease in the responses to noradrenaline at both levels of tension, the responses being reduced to 86% (3 g) and to 50% (0.5 g). In endothelium-free arterial rings, the responses to noradrenaline at 3 g and 0.5 g were also significantly different: 118% versus 80%, respectively. It is concluded that the tension of the arterial wall is a major factor influencing the effects of vasoconstrictor agents; however the mechanisms underlying this supersensitivity at higher tension remain unknown.

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+.

Reduced responsiveness of rat mesenteric resistance artery smooth muscle to phenylephrine and calcium following myocardial infarction.

1. We evaluated responses of peripheral resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation in a rat model of heart failure in relation to neurohumoral changes, wall structure, receptor density and cellular calcium handling. 2. Plasma samples and third order mesenteric artery side-branches were obtained from Wistar rats after induction of left ventricular infarction (M1) or sham surgery. Vessels were denuded of endothelium, sympathectomized, depleted of neuropeptides, and mounted in a myograph for recording of isometric force development in response to calcium, agonist and high potassium. Also, the morphology of these preparations was determined. Separate vessel segments were used in radioligand binding assays with [1H]-prazosin. 3. At 1 week after MI, circulating plasma levels of adrenaline, angiotensin II, atrial natriuretic factor (ANF) and vasopressin were significantly elevated. At 5 weeks only a significant elevation of ANF persisted. 4. At 5 weeks after MI, the structure of the vessels and responsiveness to high potassium or Bay K 8466 (10(6) mol l-1) were not modified. Yet, at this stage, sensitivity to phenylephrine was increased (pD2: 6.24 +/- 0.04 vs 5.98 +/- 0.04 for controls) while maximal contractile responses to phenylephrine in the presence of 2.5 mmol l-1 calcium (2.26 +/- 0.28 vs 3.53 +/- 0.34 N m-1) and the sensitivity to calcium in the presence of phenylephrine (pD2: 2.81 +/- 0.22 vs 3.74 +/- 0.16) were reduced. Responses to the agonist in calcium-free solution and the calcium sensitivity in the presence of 125 mmol l-1 potassium or of phorbol myristate acetate (PMA, 10(-6) mol l-1) were not altered. 5. At 5 weeks after MI, the density of prazosin binding sites was not reduced (4.04 +/- 1.40 vs 2.29 +/- 0.21 fmol microgram-1 DNA in controls). 6. In conclusion, myocardial infarction leads in the rat to a reduction of contractile responses of mesenteric resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation. This seems to involve impaired agonist-stimulated calcium influx.

Increased responsiveness of human pulmonary arteries in patients with positive bronchodilator response.

1. The effects of noradrenaline, endothelin-1, acetylcholine and sodium nitroprusside were studied in isolated pulmonary arteries obtained from 14 patients undergoing lobectomy for lung carcinoma. Seven patients had shown increased response to a bronchodilator test prior to operation. In the remaining patients (control) the bronchodilator test was negative. 2. Artery rings from patients with a positive bronchodilator response showed greater contraction to noradrenaline (pD2 = 6.44 +/- 0.1; Emax = 93 +/- 9% of response to 100 mM KCl) and endothelin-1 (pD2 = 8.92 +/- 0.1; Emax = 130 +/- 16%) than the rings from control patients (pD2 = 6.04 +/- 0.08; Emax = 56 +/- 8% for noradrenaline; pD2 = 8.29 +/- 0.1; Emax = 78 +/- 10% for endothelin-1). There was no significant difference in the contractile responses to 100 mM KCl between arteries from either group of patients. 3. Arterial rings from patients with a positive bronchodilator test achieved 96 +/- 3% of maximal relaxation in response to acetylcholine, whereas rings from control patients achieved a maximal relaxation of 72 +/- 5%. Rings from both the controls and the patients with a positive bronchodilator test achieved complete relaxation in response to sodium nitroprusside but pD2 values were significantly higher in patients with a positive bronchodilator test. 4. Removal of endothelium or treatment with NG-nitro-L-arginine methyl ester of artery rings from both the control and the patients with a positive bronchodilator test reduced the relaxation to acetylcholine (P < 0.05) but did not modify relaxation to sodium nitroprusside. 5. It is concluded that responsiveness of pulmonary arterial smooth muscle to dilator and constrictor agents is increased in patients showing reversibility of airway constriction. Thus hyperresponsiveness of airway smooth muscle may be associated with a similar phenomenon in the surrounding vascular smooth muscle.

Effects of chronic oral administration of levcromakalim on in vitro contractile responses of arterial smooth muscle

It has been shown that oral administration of 0.038–0.15 mg/kg levcromakalim elicits a dose-related antihypertensive response in spontaneously hypertensive rats (Clapham et al., Arzneim. Forsch. 41 (1991) 385). In the present study, we examined the effects of long term administration of a high dose of levcromakalim on in vitro vascular contractility. Levcromakalim (2.25 mg/kg/day) was administered to the rats for 2 weeks and the thoracic aorta was then isolated. The levcromakalim treatment markedly reduced the relaxant effect of levcromakalim itself on norepinephrine-induced contraction. Relaxant effects of sodium nitroprusside and 8-bromo-cGMP were also attenuated by the levcromakalim treatment, although the relaxant effects of verapamil and forskolin were unchanged. The levcromakalim treatment decreased the threshold concentration for KCl and norepinephrine to induce contraction. The chronic levcromakalim treatment did not affect the cGMP production due to 3-isobutyl-1-methylxanthine and/or sodium nitroprusside. The aorta isolated from spontaneous hypertensive rats did not exhibit spontaneous activity in normal solution. After treatment with levcromakalim, however, the aorta showed spontaneous rhythmic contractions. Verapamil (10 μM) completely suppressed the spontaneous activity and decreased the basal tension below the original level. Similar to the effects of chronic treatment with levcromakalim, high-K + solution (15.4 mM) augmented the contractile response to norepinephrine in the aorta of normotensive rats and induced rhythmic contractions in the aorta of spontaneously hypertensive rats. These results suggest that chronic treatment with a high dose of levcromakalim attenuates not only the effects of levcromakalim itself but also the cGMP-mediated relaxation, possibly by desensitizing the K + channel.

Ca(2+)‐activated Cl‐ and K+ channels and their modulation by endothelin‐1 in rat pulmonary arterial smooth muscle cells

Using the patch-clamp recording technique, we observed that endothelin-1 (ET-1; 0.8-16 nM) enhanced a voltage-activated outward current (Iout) and induced periodic oscillations of inward current in smooth muscle cells isolated from small pulmonary arteries (200-400 microns in diameter). Anion substitution experiments revealed that the ET-1-induced inward current was carried by Cl- ions. Application of bosentan (10 microM; and ETA and ETB receptor antagonist) and FR 139317 (1-10 microM; a selective ETA receptor antagonist) prevented initiation of inward currents or enhancement of Iout by ET-1. The ETB receptor agonist tetra-Ala-endothelin-1 (1-20 nM) failed to evoke these responses. Caffeine (10 mM) induced a single transient inward current and prevented any further activation of inward current, or enhancement of Iout, by subsequent application of 16 nM ET-1, suggesting that these currents were mediated by Ca2+ release from internal stores. Rapid intracellular release of Ca2+ by photolysis of nitr-5 activated an inward Cl- current and increased the magnitude of Iout. These results demonstrate the existence of Ca(2+)-activated Cl- and K+ channels in pulmonary arterial smooth muscle. The physiological role of these channels is at present uncertain, although their activation may be involved in the contractile responses of pulmonary arterial smooth muscle to ET-1.