Norepinephrine Contraction Research Articles

Differential effects of fatty acids on glycolysis and glycogen metabolism in vascular smooth muscle

The effects of fatty acids of different chaing lengths on aerobic glycolysis, lactic acid production, glycogen metabolism and contractile function of vascular smooth muscle were investigated. Porcine carotid artery segments were treated with 50 μM idoacetate and perchloric acid tissue extracts were then analyzed by 31P-NMR spectroscopy to observe the accumulation of phosphorylated glycolytic intermediates so that the activity of the Embden-Myerhof pathway could be tracked under various experimental paradigms. Aerobic glycolysis and lactate production in resting arteries were almost completely inhibited with 0.5 mM octanoate, partially inhibited with 0.5 mM acetate and unaffected by 0.5 mM palmitate. Inhibition of glycolysis by octanoate was not attributable to inhibition of glucose uptake or glucose phosphorylation. Basal glycogen synthesis was unchanged with palmitate and acetate, but was inhibited by 52% with octanoate incubation. The characteristic glycogenolysis which occurs upon isometric contraction with 80 mM KCl in the absence of fatty acid in the medium was not demonstrable in the presence of any of the fatty acids tested. Glycogen sparing was also demonstrable in norepinephrine contractions with octanoate and acetate, but not with palmitate. Additionally, norepinephrine-stimulated isometric contraction was associated with enhanced synthesis of glycogen amounting to 6-times the basal rate in medium containing octanoate. Contractile responses to norepinephrine were attenuated by 20% in media containing fatty acids. Thus, fatty acids significantly alter metabolism and contractility of vascular smooth muscle. Fatty acids of different chain lengths affect smooth muscle differentially; the pattern of substrate utilization during contraction depends on the contractile agonist and the fatty acid present in the medium.

Nicorandil-Induced Vasorelaxation

Using a series of functional criteria, we wished to evaluate the K+ conductance mechanism and the cyclic GMP mechanism implicated in the actions of nicorandil (NIC) as a vasodilator. In rabbit isolated superior mesenteric artery, NIC exhibited two relaxation dose-response curves (DRCs): one with a lower IC50 of 4.8 x 10(-6) M for norepinephrine (NE 5 microM) contraction, and another with a higher IC50 of 1.4 x 10(-4) M for 80 mM K+ contraction. K+ channel blockers (TEA 1-10 mM), Ba2+ (0.1-0.5 mM), glyburide (1 microM), and increased [K+]ex (20 mM), all caused significant attenuations in the ability of NIC to relax NE contraction, but did not influence the ability of NIC to relax high-K+ contraction. Pretreatment with 5 microM methylene blue, a guanylate cyclase inhibitor, produced a pronounced inhibition of nitroglycerine (NTG) relaxation, but only a marginal inhibitory effect on the NIC relaxation DRC for NE contraction. Functional studies demonstrated that the inhibitory effect of NIC on NE-sensitive intracellular Ca2+ release occurred in the same concentration range as that required for relaxation of 80 mM K+ contractions (10(-5)-10(-3) M). Furthermore, NIC also caused increases in cellular cyclic GMP levels at this higher concentration range. Finally, NIC relaxation of NE contraction was not prone either to self-tolerance (30 mM NIC preexposure) or cross-tolerance (0.55 mM NTG preexposure) development. In contrast, a modest but significant degree of self-tolerance to NIC could be demonstrated under high-K+ contraction condition. These studies thus show the existence of both cellular mechanisms for NIC in the same vascular preparation and further show that these two mechanistic components are separate and independent. The K+ channel-dependent component occurs at lower concentrations, is blocked by K+ channel blockers, is not inhibited by methylene blue, is not associated with increases in cyclic GMP, and is not prone to tolerance development. In this, NIC resembles other K+ channel openers. The cyclic GMP-dependent component is evident at relatively higher concentrations, is associated with inhibition of [Ca2+]i release, is associated with increases in cyclic GMP levels, and is prone to tolerance development. In this, NIC resembles other nitrovasodilators. A combination of these characteristics of the actions of NIC may contribute to the differences in the acute versus chronic hemodynamic profile of NIC.

Evidence that neuropeptide Y and norepinephrine mediate electrical field-stimulated vasoconstriction of rabbit middle cerebral artery.

We investigated the contractile response of isolated rabbit middle cerebral artery (MCA) segments to electrical field stimulation (EFS). The dynamics of the EFS contraction were compared with a similar-sized branch of rabbit ear artery. In comparison with the ear artery, the EFS contractions of the MCA displayed a longer latency and a higher stimulus frequency threshold. Greater stimulation train lengths were required to attain equilibrium, and the time course of EFS response--including force development, plateau, and return to rest tension--was significantly slower than in the ear artery. Morphological and pharmacological studies of the MCA showed that it receives sympathetic adrenergic innervation: whole-mount preparations displayed catecholamine histofluorescence; electron micrographs of MCA sections revealed a population of varicosities containing chromaffin positive large and small vesicles; and EFS contractions were blocked by tetrodotoxin (30 nM) and guanethidine (5 microM) and by chronic surgical sympathectomy. Exposure to prazosin (10 microM) or phenoxybenzamine (1 microM) blocked norepinephrine contractions but did not significantly influence the EFS contraction. Procedures and drugs that antagonized the responses to neuropeptide Y, serotonin, or histamine were also ineffective in blocking the EFS contraction. The involvement of ATP could not be assessed, since the purinergic P2 agonist alpha,beta-methylene ATP was ineffective in blocking ATP-mediated contractions. The EFS contraction, however, could be blocked by a combination of neuropeptide Y desensitization and phenoxybenzamine (30 nM) or prazosin (0.1 microM). These results suggest that norepinephrine and neuropeptide Y are released from sympathetic nerves and mediate EFS contraction by occupation of postjunctional alpha-adrenoceptor and neuropeptide Y receptors. Since the blockade of only one of these components does not diminish the response to EFS, the adrenergic neuroeffector system in this artery may involve complex prejunctional regulatory mechanisms.

Response of Perfused Lung and Isolated Pulmonary Artery to Adenosine

Adenosine (AD) has been reported to induce both pulmonary arterial constriction and dilation. We investigated the effect of AD using two complementary techniques. The isolated rat lung perfused with Earle's balanced salt solution containing albumin was used to measure pulmonary arterial (Ppa), venous, and double occlusion (microvascular; Pmv) pressure, and resistance changes. AD alone had no effect on Ppa, Pmv, or resistance at any dose tested (5 x 10(-7) through 10(-3) M). However, when Ppa was elevated by pretreatment with 5 x 10(-7) M norepinephrine (NE), then 10(-4) M AD lowered Ppa by 19.5 +/- 3.2% and Pmv by 6.0 +/- 6.1% and attenuated the increase in upstream resistance caused by NE. Higher doses of AD (10(-3) M) resulted in greater relaxation. In isolated segments from rat and guinea pig pulmonary lobar arteries, isometric force production in response to AD was measured as a percentage of the active isometric force produced in response to 10(-5) M NE (% NE contraction). No response was observed in rat pulmonary arterial rings for doses of AD less than 10(-6) M. In vessels with intact endothelium, 10(-6) M AD caused a slight increase in isometric tension (2.3 +/- 1.2% NE contraction; p less than 0.05), but 10(-4) M AD caused relaxation (-17.2 +/- 2.2% NE contraction; p less than 0.05), and 10(-3) M caused further relaxation (-61.5 +/- 5.0% NE contraction; p less than 0.05). In vessels without endothelium, only relaxation was observed. Isolated guinea pig arterial rings responded to AD with vasodilation similar to the results in the rat arterial rings. Results of this study show that AD primarily causes a direct dose-dependent relaxation of pulmonary arterial smooth muscle in both the isolated perfused lung and isolated arterial ring preparation.

A sensitive in vitro functional assay to detect K+ — channel-dependent vasodilators

This study describes a sensitive in vitro relaxation assay using isolated rabbit mesenteric artery to detect the activity of a vasodilator as a K+-channel activator. Thus, comparison of several known K+-channel activators was made with other vasodilators known to work via various cellular mechanisms. The vasodilators used were minoxidil sulfate (MNXS; 5 μM), BRL-34915 (cromakalim, 0.1 μM), nicorandil (10 μM), pinacidil (1 μM), diazoxide (100 μM), sodium nitroprusside (10 μM), forskolin (1 μM), D600 (0.5 and 10 μM), hydralazine (10 μM), and viprostal (PGE1 analog, 5 μM). The concentrations chosen were equipotent to produce greater than 80% relaxation of the maximal norepinephrine (NE) (5 μM) contraction. At these concentrations, MNXS, cromakalim, pinacidil, nicorandil, and diazoxide were found to be ineffective in producing relaxation of 80 mM K+-contractions. Subsequently, pretreatment of tissues with 20 mM K+ before NE contraction was found to attenuate relaxation significantly by these agents, but had no effect on the relaxations by forskolin or D600. These initial criteria helped to establish cromakalim, pinacidil, nicorandil, and diazoxide as compounds acting similarly to MNXS as K+-channel-dependent. In another set of experiments, the effects of tetraethylammonium (TEA) (10 mM), Ba2+ (0.5 mM), and glyburide (1 μM) as K+-channel blockers were examined. Again it was found that these blockers had the most inhibitory effect on the class of compounds identified as K+ channel activators. Additionally, it was found that these K+-channel activators were without any significant effect on the NE-sensitive intracellular Ca2+ release as studied by contraction in a Ca2+-free solution. Thus, this series of functional criteria clearly show that the profile of these K+-channel activators is distinctly different from the vasodilators working via other mechanisms such as cyclic AMP (cAMP) (forskolin), cyclic GMP (cCMP) (nitroprusside), and Ca2+ antagonists (D600). It is suggested that appropriately defined, systematic functional studies, such as the one described here, can provide a sensitive and reproducible vascular model to discover and delineate the role of pharmacologically relevant mechanisms for vasodilation.

Two distinct α1‐adrenoceptor subtypes involved in noradrenaline contraction of the rabbit thoracic aorta

1. Recently, alpha 1-adrenoceptors in blood vessels have been classified into three subtypes (alpha 1H, alpha 1L and alpha 1N). We examined which subtype (or subtypes) is involved in the noradrenaline-induced contraction of rabbit thoracic aorta. 2. Noradrenaline produced a concentration-dependent contraction in the rabbit isolated thoracic aorta. Prazosin antagonized the contractions to noradrenaline, resulting in a rightward displacement of the concentration-response curve. However, the shift was not proportional to the concentration of prazosin; Schild plots showed that the inhibition by prazosin was biphasic, implying that noradrenaline acted through two receptor populations. Two affinity constants (pKB values of 10.02 and 8.83) were determined for prazosin at these sites. 3. However, under continuous treatment with 1 nM prazosin, or in strips pretreated with chlorethylclonidine (CEC; an alpha 1H inactivating agent) to remove the contribution of one receptor population, prazosin showed a single pKB or pA2 value of approximately 8.3. 4. Yohimbine also produced biphasic antagonism of noradrenaline-induced contractions, resulting in two affinity constants (pKB = 6.52 and 6.17). However, a monophasic Schild plot was obtained for yohimbine either in the presence of 1 nM prazosin (pA2 = 6.08) or in strips pretreated with CEC (pA2 = 6.03). 5. The Schild plot for HV723 (a selective alpha 1N-antagonist) yielded a monophasic slope (pKB = 8.47) and the inhibition was not affected by 1 nM prazosin or CEC-pretreatment. 6. [3H]-prazosin bound to alpha 1-adrenoceptors of the aortic membrane preparations with two different affinities (pKD = 9.94 and 8.37). The high but not the low affinity site was completely masked by 1 nM prazosin and inactivated by pretreatment with CEC. 7. These results strongly suggest that noradrenaline-induced contraction of the rabbit thoracic aorta is mediated through two distinct alpha l-adrenoceptor subtypes, designated alpha 1H and (alpha lL*

Effects of angiotensin converting enzyme inhibitors and of hydralazine on endothelial function in hypertensive rats.

The function of the endothelium is impaired in hypertension. In spontaneously hypertensive rats (SHR), acetylcholine-induced relaxation is decreased and serotonin-induced constriction is increased. The goal of our study was to evaluate the effect of a long-term treatment with cilazapril, a new angiotensin converting enzyme inhibitor, or hydralazine, a vasodilator, on the endothelium-dependent responses in aorta of SHR. Wistar-Kyoto rats were used as normotensive reference. Isolated aortic rings with or without endothelium were suspended in organ chambers. The rings with intact endothelium were contracted with norepinephrine. Acetylcholine-induced relaxation was markedly enhanced by cilazapril treatment. The tension achieved at maximal relaxation was 8 +/- 4% of norepinephrine contraction in the cilazapril-treated SHR versus 55 +/- 5% in the untreated SHR (p less than 0.001). Hydralazine had no significant effect. The effect of serotonin was also markedly modified by cilazapril. In untreated SHR, serotonin induced the release of a vasoconstrictor substance by the endothelium as assessed by the ratio of maximal tension induced by serotonin in rings with endothelium over maximal tension in rings without endothelium, which was greater than 1. This ratio was reversed in cilazapril-treated SHR but not in hydralazine-treated SHR. Captopril had effects similar to cilazapril. Finally, evaluation of carotid arteries showed that cilazapril also prevented morphological changes of the intima in SHR (i.e., infiltration by mononuclear cells). We conclude that angiotensin converting enzyme inhibitors prevent the functional and morphological alterations in endothelium that are found in hypertension and speculate that this action might participate in their antihypertensive effect.

Protein Kinase C Influences Rat Pulmonary Vascular Reactivity

To test the role of protein kinase C in the control of pulmonary vascular tone and reactivity, we examined the effects of the activators, phorbol 12-myristate-13-acetate (PMA), mezerein, and 1,2-dioctanyl-rac-glycerol (1,2-DOG), in rat isolated large pulmonary arteries and salt solution-perfused lungs. PMA (500 nM) and mezerein (100 nM) induced slow, sustained contractions of isolated pulmonary arteries. These contractions were not inhibited by nifedipine (1 microM), the intracellular Ca2+ blocker TMB-8 (50 microM), a 0 Ca2+ and EGTA (2 mM) bath, or endothelial denudation. They were reduced by amiloride (500 microM), an inhibitor of Na+/H+ exchange, and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); (50 microM), an inhibitor of protein kinase C. PMA caused concentration-dependent (5 to 500 nM) potentiation of KCl, serotonin, and A23187 contractions of isolated pulmonary arteries. The effect of PMA on norepinephrine contraction was dependent on both concentration and time. Whereas PMA potentiated norepinephrine contraction after 5 min, it inhibited the response after 60 min. PMA and mezerein also caused concentration-dependent (0.5 to 500 nM) vasoconstriction in isolated lungs. The PMA vasoconstriction was unaltered by meclofenamate (1.6 microM) but was attenuated by nifedipine (1 microM). Low concentrations of PMA (5 nM) and 1,2-DOG (50 microM) potentiated both hypoxic and KCl vasoconstriction in isolated lungs. In contrast, the hypoxic and KCl responses were blunted by H-7 (30 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

An agonist action at 5-HT 1A receptors functionally antagonises opioid antinociception in rodents

The rat testicular capsule is a thin tissue surrounding the testis, whose precise function is still unknown. We have studied the contractile effects of electrical field stimulation, noradrenaline, and the blockade by antagonists of adrenergic receptors, in order to characterize sympathetic neurotransmission, and adrenoceptor subtypes. In addition, reverse transcription polymerase chain reaction (RT-PCR) assays were made to check for the expression of the three known subtypes of α1-adrenoceptors. The effects of electrical field stimulation (2 to 20 Hz, 1 ms, 60 V) were almost totally abolished by depletion of neuronal noradrenaline storage with reserpine (10 mg/Kg), but not by the purinergic receptor antagonist suramin (10− 5 M), indicating that noradrenaline, but not ATP, was involved in contractions. The selective α1-adrenoceptor antagonist prazosin (10− 7 M) was more effective than the selective α2-adrenoceptor antagonist idazoxan (10− 7 M) to inhibit contractions induced by electrical field stimulation, pointing out a major involvement of α1-adrenoceptor. When noradrenaline was used instead of electrical field stimulation, it showed a high potency (pD2 = 7.9). Noradrenaline-induced contractions were competitively blocked by the selective α1A-adrenoceptor antagonists WB 4101 (pA2 = 8.88), phentolamine (pA2 = 8.39) and by the α1B-adrenoceptor antagonist spiperone (pA2 = 8.57), indicating the presence of functional α1A- and α1B-adrenoceptors. In addition, contractions were not blocked by the selective α1D-adrenoceptor antagonist BMY 7378 (up to 10− 6 M), while selective α2-adrenoceptor antagonists showed low pA2 values (yohimbine, 7.25 and idazoxan, 7.49), suggesting a minor role, if any, for α1D- and α2-adrenoceptors. To check the proportionate role of α1A- and α1B-adrenoceptors, we blocked α1B-adrenoceptors with chloroethylclonidine (CEC, 30 μM, 45 min), that reduced the maximal effect of noradrenaline by about 60%. The remnant CEC-insensitive noradrenaline contraction was assumed to be unrelated to α1B-adrenoceptor, and was inhibited by 5-methyl-urapidil (pA2 = 8.94) and by the Ca2+ channel blocker nifedipine (3 μM), confirming the involvement of α1A-adrenoceptors. The presence of mRNA encoding α1A- and α1B-adrenoceptor was also shown on RT-PCR assays. Unexpectedly, α1D-transcripts were also detected in these assays. Taken together, our results show that ATP co-transmission could not be detected, and that neurotransmission involves the interaction of noradrenaline with both α1A- and α1B-, but not with α1D- or α2-adrenoceptor. The fact that the functional α1D-adrenoceptor could not be detected in spite of the presence of the corresponding mRNA, remains to be investigated.

Pharmacological characterization of postjunctional α‐adrenoceptors in cerebral arteries from the sheep

1. The responsiveness to noradrenaline was characterized in cerebral arteries from the sheep, since this species was large enough to permit a comparison of arteries from different parts of the cerebral vasculature. 2. Noradrenaline caused contraction of the basilar artery, middle cerebral artery and small pial arteries by stimulation of alpha 1-adrenoceptors. 3. The maximum contraction to noradrenaline was small in the basilar artery (28% of the 5-hydroxytryptamine (5-HT) maximum) but larger in the middle cerebral artery (78% of the 5-HT maximum) and pial artery (92% of the 5-HT maximum) of the sheep. 4. Cocaine (10 microM) potentiated noradrenaline-induced contractions in the sheep middle cerebral artery but not in the sheep basilar artery. 5. The noradrenaline contraction, relative to the 5-HT contraction, was not affected by removal of the endothelium in either the sheep basilar or middle cerebral artery. 6. The results showed a variation within the sheep cerebral vasculature in the response to noradrenaline which cannot be explained by regional differences in alpha-adrenoceptor subtypes, noradrenaline uptake mechanisms or endothelial function.

Potassium-induced relaxation of arteries in hypertension: modulation by extracellular calcium

Smooth muscle relaxation due to activation of Na+-K+-adenosinetriphosphatase (ATPase) (K+ relaxation) was studied in isolated tail arteries from stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats. Exposure to K+ (7 mM) relaxed a norepinephrine (1 microM) contraction induced in K+-free physiological salt solution (PSS) by 46 +/- 3% in WKY and by 81 +/- 3% in SHRSP. Incubation with monensin (10 microM) augmented the K+-relaxation in WKY to 61 +/- 5%. Incubation with amiloride (10 microM) or with low-Na+ (10 mM) PSS attenuated the K+ relaxation in SHRSP to 34 +/- 3 and 5 +/- 2%, respectively. Incubation with high-Ca2+ (6.6 mM) PSS attenuated the K+ relaxation in WKY to 25 +/- 3% but resulted in only a slight decrease in the relaxation in SHRSP (to 73 +/- 2%). We conclude 1) that a greater Na+ leakiness of the plasma membrane in SHRSP than in WKY while the Na+ pump is inactive in K+-free PSS can explain the greater relaxation observed when the Na+ pump is reactivated and 2) that the Na+ leakiness of the membrane is more attenuated by Ca2+ in the WKY than in the SHRSP. We hypothesize that this Ca2+ effect reflects a normally large amount of this ion that can be bound to and thereby stabilize the membrane of the WKY, decreasing its permeability to Na+. The membrane of the SHRSP is deficient in its Ca2+-binding sites. Hence the added Ca2+ does not further stabilize its membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of pulmonary artery contraction by endothelium-derived relaxing factor

In isolated rat pulmonary artery rings, both endothelial denudation and treatment with 10 −5 M hemoglobin inhibited relaxation to acetylcholine and increased contractile sensitivities, i.e. decreased the EC 50s, to KCl, angiotensin II and norepinephrine. Denudation caused similar inhibition of acetylcholine relaxation and potentiation of KCl and norepinephrine contractions in isolated bovine pulmonary arteries. These results indicate that endothelium-derived relaxing factor plays a significant role in modulating the contractile sensitivity of isolated pulmonary arteries to at least some agonists.

Glucose dependence of sequential norepinephrine contractions of vascular smooth muscle.

The effects of successive, norepinephrine (NE)-stimulated, contractions of porcine carotid artery intima-media strips as a function of recovery medium were studied. Recovery following NE stimulation required the presence of glucose in the bathing medium for subsequent force production in response to NE stimulation. Potassium stimulation following failed NE contractions produced maximal contractions. Phosphorous nuclear magnetic resonance studies under similar conditions indicated that the energy state of the tissue was not impaired during and after recovery from NE stimulation without glucose. This study shows that the phasic phase of norepinephrine-stimulated contractions is dependent on the availability of extracellular glucose during the poststimulation recovery period for successive NE contractions.

Sodium-calcium exchange in vascular smooth muscle of Wistar-Kyoto and stroke-prone spontaneously hypertensive rats.

The role of the sodium-calcium (Na-Ca) exchange in vascular smooth muscle contraction was examined in tail artery rings isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and in normotensive Wistar-Kyoto rats (WKY). The rings were repeatedly stimulated with noradrenaline (1 microM) in physiological salt solution (Na = 130 mM), until two successive contractions were of the same magnitude. The rings were then placed in physiological salt solution with reduced sodium concentrations (65 mM or 0 mM, replaced isosmotically with sucrose), and the noradrenaline stimulations continued. In WKY rings, the reduction of sodium concentration produced an increase in the response to noradrenaline, which was significant in sodium-free physiological salt solution. In SHRSP rings, however, the same reductions in sodium concentration produced significantly less potentiation of the noradrenaline contraction, even in sodium-free physiological salt solution. We conclude that (1) in WKY, the reduced and reversed activity of the Na-Ca exchange produced by the reductions in sodium concentration makes more calcium available for contraction when the smooth muscle is stimulated with noradrenaline; and (2) the failure of sodium reductions to produce a normal potentiation of the response to noradrenaline in SHRSP indicates a depressed activity of the Na-Ca exchange in this tissue.

Effects of BRL 34915, a putative K channel opener, on transmembrane 45Ca movements in rabbit aortic smooth muscle

The effects of BRL 34915, a putative K channel opener, on transmembrane Ca 2+ movements were studied in isolated rabbit aortic strips, using the 45Ca flux techniques. BRL 34915 (1 and 10 μM) inhibited the contraction and Ca entry in response to norepinephrine (NE) and angiotensin II, each at 0.3 μM. BRL 34915 differed from Na nitroprusside in that it demonstrated little or no inhibition on the contraction and 45Ca efflux provoked by NE in Ca 2+-free buffer, suggesting that the effect of BRL 34915 on intracellular Ca release plays a minor role in inhibiting the vasoconstrictor responses to receptor agonists. Moreover, BRL 34915 (100 μM) did not stimulate cyclic GMP formation in rabbit aortic slices. BRL 34915 (10 μM) failed to inhibit the vascular responses to KCl (20 and 40 mM). Tetraethylammonium (20 mM), a K channel blocker, abolished the vasorelaxant action of BRL 34915 against NE. However, Na nitroprusside inhibition of NE contraction was similarly affected. It is concluded that reduction of Ca entry appears to be the predominant mechanism mediating the inhibitory effect of BRL 34915 on the contractile responses to vasoconstrictor stimuli.

Pinacidil opens K+-selective channels causing hyperpolarization and relaxation of noradrenaline contractions in rat mesenteric resistance vessels.

1. The effects of pinacidil on noradrenaline-induced tone, smooth muscle membrane potential and 42K- and 86Rb-efflux from isolated mesenteric resistance vessels (internal diameter 200 microns) of the rat have been studied. 2. Pinacidil (0.3-10 microM) produced concentration-dependent suppression of noradrenaline-induced tone. 3. Pinacidil (0.3-10 microM) caused concentration-dependent hyperpolarization of the smooth muscle. 4. In rat resistance vessels loaded with 42K, pinacidil (1-10 microM) significantly increased the 42K-efflux rate constant. 5. With the use of 86Rb as a marker for K+, 1 microM pinacidil did not affect the 86Rb-efflux rate constant, while 10 microM pinacidil transiently increased the 86Rb rate constant. 6. The results indicate that the relaxant action of pinacidil in these vessels is due to the opening of K+-channels and consequent hyperpolarization. The K+-channels opened are selective for 42K over 86Rb.

Prolonged Inhibitory Effects of Nilvadipine (FR34235) Following Washout in Isolated Rabbit Aorta and Mesenteric Artery

Duration of the inhibitory effects of nilvadipine and some other Ca2+ entry blockers following washout was studied in isolated rabbit aorta and distal portion of mesenteric artery. The inhibitory effects of nilvadipine on KCl-induced contraction of aorta lasted longer than those of diltiazem, nifedipine, and nicardipine. Durations of nilvadipine were dose-dependently prolonged. The 50% recovery time, a parameter of the duration of action following washout, of nilvadipine at 10(-9) and 3 X 10(-9) M was 2.5 and 3.2 h, respectively. The long-lasting inhibitory effects of nilvadipine were pronounced at low [Ca]o. Similar prolonged actions of nilvadipine, compared with those of diltiazem and nifedipine, were observed in norepinephrine (NE)-induced contraction of the distal portion of mesenteric artery, of which tonic contractions were highly sensitive to Ca2+ entry blockers. Despite the long action of nilvadipine after washout in mesenteric artery, the onset of the inhibition of NE contraction was prompt. These pharmacological properties of nilvadipine, especially those seen in mesenteric artery, may be consistent with its long action as an antihypertensive agent.

The influence of magnesium on the release of calcium from intracellular depots in vascular smooth muscle cells.

Vasoconstrictor responses to prostaglandin F2 alpha and noradrenaline were investigated in ring segments of feline femoral, coronary and cerebral arteries incubated in calcium-free solutions containing different concentrations of magnesium (1.2, 4.4 and 13.2 mM). Contractions produced by prostaglandin F2 alpha and noradrenaline were depressed when calcium was omitted from the incubation solution. The presence of raised concentrations of magnesium (4.4 or 13.2 mM) in the tissue bath further depressed the prostaglandin F2 alpha and noradrenaline contractions in calcium-free medium. In a separate set of experiments the vessel wall contents of cAMP and cGMP were measured before and after the additions of 4.4 or 13.2 mM magnesium; stable relaxations by magnesium were noted but there was no parallel change in the vessel content of cAMP or cGMP. The results indicate that magnesium may interfere with the release of calcium from intracellular depots, and that neither adenylate cyclase, nor guanylate cyclase are involved in the dilator activity of magnesium in isolated arteries.

Vasodilatation with pinacidil. Mode of action in rat resistance vessels.

Pinacidil is a newly developed antihypertensive vasodilator, proposed to belong to the new group of smooth muscle relaxants, the K+ channel openers. The in vitro effects of pinacidil on induced tone, smooth muscle membrane potential and 86Rb and 42K efflux from rat resistance vessels (internal diameter about 200 microns) were studied. Tone induced with noradrenaline was concentration-dependently inhibited by pinacidil. Responses to electrical field stimulation were also inhibited. However, tone induced with high K+ depolarisation, noradrenaline in the presence of high K+, caffeine-induced contractions and noradrenaline contractions in the presence of felodipine were little affected by pinacidil. Pinacidil caused concentration-dependent hyperpolarisation of the resting smooth muscle. Pinacidil caused only a small and transient increase of the 86Rb efflux rate constant, while the same concentrations of pinacidil produced a significant increase in the 42K efflux rate constant. Our results seem to indicate that the relaxant effect of pinacidil is the result of an increase in K+ permeability, thus causing hyperpolarisation and relaxation. The opened K+ channels appear to be selective for K+ over Rb+.

Comparison of Inhibitory Effects of Calcium Channel Blockers and That of a Calmodulin Antagonist in Strips of Mesenteric Arteries from Spontaneously Hypertensive and Normotensive Rats

Effects of calcium channel blockers and of calmodulin antagonist on the contractile responses to norepinephrine (NE) were compared between strips of mesenteric arteries from 6- and 14-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wistar-Kyoto rats (WKY). The ratio of the maximum contraction developed by NE to that by 60 mM KCI was significantly increased in strips from 14-week-old SHR. Niludipine, verapamil and diltiazem antagonized the maximum NE contraction to a greater extent in strips from 14-week-old SHR than in those from the WKY. However, the antagonism by niludipine of the KCI- or caffeine-induced contraction was not significantly different between the strips from 14-week-old SHR and those from WKY. In strips from 6-week-old rats, there was no difference in the antagonism by niludipine of the maximum NE contraction. On the other hand, the effect of W-7 on the maximum NE contraction was not significantly different between the strips from 14-week-old SHR and those from WKY. Schild plot analyses demonstrated that α1-adrenoceptors were the same for the strips from SHR and WKY. These results suggest that the enhanced maximum NE contraction in the mesenteric artery from 14-week-old SHR reflects the increased transmembrane influx of calcium, and the activity of calmodulin seems to be the same for the two strains.