Dissociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1-receptors in guinea-pig left atria

Summary The present study was undertaken to determine whether the phosphoinositide hydrolysis is responsible for the positive inotropic effect of histamine in guinea-pig left atria. Histamine induced hydrolysis of phosphoinositides and a positive inotropic effect in a concentration-dependent manner. These effects were antagonized by chlorpheniramine (0.1 mol/l) but not by cimetidine (10 mol/l). At a concentration of 1 mol/l histamine produced a dual-component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Histamine (10 mol/l) caused a gradual increase in the formation of [³H]inositol trisphosphate (IP3) and a significant increase in the [³H]IP₃ level was detected 10 min after the stimulation. Thus, the increase in IP₃ did not precede the increase in force of contraction. The phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (100 mol/l) and neomycin (100 mol/l) significantly reduced the histamine-induced [³H]inositol monophosphate accumulation. However, pretreatment with the phospholipase C inhibitors did not affect the positive inotropic effect of histamine, either in its extent or in its pattern. The phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nmol/l) and phorbol-12,13-dibutyrate (PDBu) (100 nmol/l) also significantly inhibited the phosphoinositide hydrolysis induced by histamine. The inhibitory effect of the phorbol esters on the phosphoinositide response was completely abolished in the presence of 10 mol/l 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor. TPA significantly attenuated the positive inotropic effect of histamine without changing the dual-component pattern, whereas PDBu merged two distinct components of the histamine inotropic response into one and potentiated the early part of the positive inotropic effect. However, neither of the changes which the phorbol esters produced in the positive inotropic response to histamine was blocked by H-7. In addition, H-7 itself failed to modify the positive inotropic effect of histamine. These results indicate that histamine induces hydrolysis of phosphoinositides in guinea-pig left atria that is mediated by H₁-receptors, but this biochemical event does not appear to contribute to the H₁-receptor-mediated positive inotropic action. Send offprint requests to Y. Hattori at the above address     

Effect of some new histamine H2-receptor antagonists on the guinea-pig papillary muscle

Summary Several histamine H₂-receptor antagonists (cimetidine, ranitidine, oxmetidine and tiotidine) were tested for their activity on the papillary muscle of the guinea pig stimulated by histamine. All of the compounds exerted a competitive antagonism against histamine the order of potency being tiotidine > oxmetidine > ranitidine > cimetidine. Oxmetidine was the only drug which at high concentrations (10^–6 M) decreased the maximum response of histamine probably because of non specific effects of the molecule already described in the literature. As it was expected, the H₁-receptor antagonist, mepyramine, exerted a non-competitive antagonism.     

Effects of H1-receptor antagonists on responding punished by histamine injection or electric shock presentation in squirrel monkeys

Squirrel monkeys were trained to press a key under a two-component schedule of food presentation. In the presence of either green or red stimulus lights, the 30th response produced a food pellet (fixed-ratio schedule). During the red stimulus lights (punishment component), the first response of each fixed ratio produced either an IV injection of histamine (100.0 g/kg/inj) or a brief electric shock (3.0 mA). Responding was selectively suppressed in either punishment component. Presession IM administration of chlorpheniramine (0.1 and 0.3 mg/kg), diphenhydramine (1.0 and 3.0 mg/kg), or pyrilamine (0.1 and 0.3 mg/kg) increased rates of responding punished by histamine but not those punished by electric shock. Presession administration of promethazine (0.1–3.0 mg/kg) or tripelennamine (0.1 and 0.3 mg/kg) also increased rates of responding punished by histamine in all subjects and response rates punished by electric shock in one of three subjects. Chlordiazepoxide (3.0–56.0 mg/kg) increased rates of responding punished by either histamine or electric shock. These results suggest that the punishing effects of histamine injection are mediated by H₁ receptors and that H₁-receptor antagonists increase rates of responding suppressed by punishment only under limited conditions including those in which histamine is the punishing stimulus.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

The Ca²⁺ paradox is an important phenomenon associated with Ca²⁺ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca²⁺ paradox.

EXPERIMENTAL APPROACH

Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope.

KEY RESULTS

The Ca²⁺ paradox was readily evoked by restoration of the extracellular Ca²⁺ following 10–20 min of nominally Ca²⁺-free superfusion. The Ca²⁺ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd³⁺, La³⁺) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca²⁺ content, assessed by caffeine application, gradually declined during Ca²⁺-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca²⁺ leak by tetracaine prevented Ca²⁺ paradox. The Na⁺/Ca²⁺ exchange (NCX) blocker KB-R7943 significantly inhibited Ca²⁺ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca²⁺ restoration. The SR Ca²⁺ content was better preserved during Ca²⁺ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence that (i) the Ca²⁺ paradox is primarily mediated by Ca²⁺ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca²⁺ depletion; and (ii) reverse mode NCX contributes little to the Ca²⁺ paradox, whereas inhibition of NCX during Ca²⁺ depletion improves SR Ca²⁺ loading, and is associated with reduced incidence of Ca²⁺ paradox in mouse ventricular myocytes.     

Ca2+ channel activating action of maitotoxin in cultured brainstem neurons

The actions of maitotoxin were studied using cultured brainstem cells and adrenal chromaffin cells. Maitotoxin induced a profound increase in the Ca2+ influx into cultured brainstem cells after a brief lag period. The maitotoxin-induced Ca2+ influx was suppressed by various voltage-dependent Ca2+ channel blockers such as Co2+, Mn2+, verapamil and diltiazem. Maitotoxin-catecholamine release in brainstem cells initiated to increase after a lag period of about 1 min and the increase continued even at 4 min after treatment, while in the adrenal chromaffin cells the release started after an about 1-min lag period to attain a maximum within first 2-min and gradually decrease thereafter. These results suggest that maitotoxin acts on Ca2+ channels to increase the Ca2+ influx, accompanied by enhancement of catecholamine release in the brainstem cells with a different temporal profile from that in the adrenal chromaffin cells.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells

Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex (DGC), causes skeletal muscular dystrophy and cardiomyopathy in BIO14.6 hamsters. Here, we studied the involvement of abnormal Ca2+ homeostasis in muscle degeneration and the protective effect of drugs against Ca2+ handling proteins in vivo as well as in vitro. First, we characterized the properties of cultured myotubes from muscles of normal and BIO14.6 hamsters (30-60 days old). While there were no apparent differences in the levels of expression of various Ca2+ handling proteins (L-type Ca2+ channel, ryanodine receptor, SR-Ca2+ ATPase, and Na+/Ca2+ exchanger), muscle-specific proteins (contractile actin and acetylcholine receptor), or DGC member proteins except SGs, BIO14.6 myotubes showed a high degree of susceptibility to mechanical stressors, such as cyclic stretching and hypo-osmotic stress as compared to normal myotubes, as evidenced by marked increases in creatine phosphokinase (CK) release and bleb formation. BIO14.6 myotubes showed abnormal Ca2+ homeostasis characterized by elevated cytosolic Ca2+ concentration, frequent Ca2+ oscillation, and increased 45Ca2+ uptake. These abnormal Ca2+ events and CK release were significantly prevented by Ca2+ handling drugs, tranilast, diltiazem, and FK506. The calpain inhibitor E64 prevented CK release, but not 45Ca2+ uptake. Some of these drugs (tranilast, diltiazem, and FK506) also exerted a significant protective effect for muscle degeneration in BIO14.6 hamsters and mdx mice in vivo. These observations suggest that elevated Ca2+ entry through sarcolemmal Ca2+ channels predominantly contributes to muscle degeneration and that the drugs tested here may have novel therapeutic potential against muscular dystrophy.     

Ca2+-calmodulin can activate and inactivate cardiac ryanodine receptors

Background and purpose:

Ca²⁺-calmodulin (Ca²⁺CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated.

Experimental approach:

We investigated the effects of low concentrations of Ca²⁺CaM (50–100 nmol·L⁻¹) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L⁻¹ CaM on sarcoplasmic reticulum (SR) Ca²⁺-release.

Key results:

Ca²⁺CaM activated or inhibited single RyR2, but activation was much more likely at low (50–100 nmol·L⁻¹) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L⁻¹ CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca²⁺-release events. Application of caffeine demonstrated that 50 nmol·L⁻¹ CaM reduced SR Ca²⁺ content.

Conclusions and implications:

We describe for the first time how Ca²⁺CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca²⁺-release and the use of caffeine suggests that this is a RyR2-mediated effect.     

Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation

The effects of 1-ethyl-2-benzimidazolinone (1-EBIO) and riluzole on human prostate cancer cells, LNCaP and PC-3, were evaluated using rubidium (86Rb(+)) efflux and proliferation assays. 1-EBIO and riluzole evoked concentration-dependent increases in 86Rb(+) efflux from LNCaP and PC-3 cells that were sensitive to inhibition by intermediate-conductance Ca(2+)-activated K(+) channel (IK(Ca)) blockers clotrimazole and charybdotoxin. Blockers of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, iberiotoxin, or small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel, apamin or scyllatoxin, had no effect. Concurrently, both 1-EBIO and riluzole evoked concentration-dependent increases in proliferation from human prostate cancer cell lines (LNCaP and PC-3 cells). Clotrimazole and charybdotoxin, but not iberiotoxin, apamin or scyllatoxin, inhibited 1-EBIO- and riluzole-evoked increases in proliferation from LNCaP and PC-3 cells. N-(3-(trifluoromethyl)phenyl)-N'-(2-hydroxy-5-chlorophenyl)urea (NS-1608) and 2-amino-5-(2-fluorophenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BK(Ca) channel openers had no effect on LNCaP and PC-3 proliferation. These results demonstrate that IK(Ca) channels play an important role in the regulation of human prostate cancer cell proliferation.     

Temporal responses of cutaneous blood flow and plasma catecholamine concentrations to histamine H1- or H2-receptor stimulation in man

Summary We have studied the effect of histamine and H₁- or H₂-receptor antagonists on cutaneous blood flow and catecholamine release in man.Histamine was infused alone or in combination with mepyramine, an H₁-antagonist or cimetidine, an H₂-antagonist for 2 h. Cutaneous blood flow was measured continously with a laser Doppler flowmeter, and noradrenaline and adrenaline concentrations were determined in blood samples drawn every 15 min.The infusion of histamine caused an immediate and sustained vasodilatation. The Concomitant infusion of mepyramine prevented the immediate vasodilatation, but had no effect on the sustained response. The Concomitant infusion of cimetidine was without effect on the immediate vasodilatation, but abolished the sustained response. Infusion of the antagonists alone had no effect on cutaneous blood flow.Histamine caused a rapid and sustained increase in plasma noradrenaline, while the increase during concomitant H₁-receptor blockade was delayed but achieved the level observed during the histamine infusion. The response to histamine during H₂-receptor blockade was small and transient. The rise in plasma adrenaline was not significant.These findings suggest that histamine causes an immediate cutaneous vasodilatation through H₁-receptors and a more sustained response through H₂-receptors. The vasodilatation is accompanied by an increase in plasma catecholamine concentrations. Despite the continuous infusion of histamine, blood flow decreased during the last hour of histamine infusion, while the plasma noradrenaline concentration was still elevated.     

In vivo effects of some histamine H1-receptor antagonists on monoamine metabolism in the mouse brain

Summary The in vivo effects of four Hr-antagonists, diphenhydramine, chlorpheniramine, mepyramine, and promethazine, on the metabolism of noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) were investigated in the whole mouse brain. Diphenhydramine and chlorpheniramine had no significant effect on levels of NA, 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), DA, and 5-HT, but they significantly decreased levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA). In particular chlorpheniramine markedly decreased 5-HIAA levels at doses as low as 1 mg/kg, i. p. Mepyramine significantly decreased 5-HIAA levels but not those of other substances. High doses of promethazine significantly decreased NA levels but markedly increased those of MHPG, DOPAC, HVA, 5-HT, and 5-HIAA. The DA reduction induced by -methyl-p-tyrosine (-MT) was significantly inhibited by diphenhydramine, chlorpheniramine, and promethazine, but the -MT-induced NA decrease was significantly enhanced by promethazine. The 5-HIAA accumulations induced by probenecid were significantly inhibited by chlorpheniramine and mepyramine. These results suggest: (1) Diphenhydramine and chlorpheniramine inhibit the turnover of both DA and 5-HT by blocking their neuronal uptake. (2) Promethazine and mepyramine inhibit DA and 5-HT turnover, respectively, as a result of the inhibition of the uptake mechanism. (3) Promethazine increases NA turnover by enhancing NA release. The discriminative effects of these drugs on the monoamine systems may be related to some differences in their CNS actions. Send offprint requests to K. Saeki at the above address     

Effects of presynaptic modulators on Ca2+-induced noradrenaline relase from cardiac sympathetic nerves

Summary In order to elucidate the mode of action of the Ca²⁺-antagonistic inhibitor nifedipine, its effect on Ca²⁺-mediated action potentials and transmembrane slow inward current in papillary muscles of guinea pigs and cats was studied.Nifedipine (0.5 mg/l1.4×10^–6M) depressed upstroke velocity and overshoot of the Ca²⁺-mediated action potential and reduced the transmembrane slow inward current by about 50%, but the kinetics of inactivation and recovery from inactivation were not affected. The decrease of upstroke velocity was accompanied by a proportional diminution of isometric contractile force. This indicates that nifedipine exerts its Ca²⁺-antagonistic effect on excitation-contraction coupling in mammalian ventricular myocardium by inhibition of the transmembrane Ca²⁺ inward current. The inhibitory action of nifedipine on contractile tension development could be neutralized by an augmentation of the extracellular Ca²⁺ concentration from 2 mM to 4 mM or by -receptor stimulation (isoproterenol) that promotes the transmembrane Ca²⁺-rich medium or under the influence of isoproterenol the upstroke velocity of the Ca²⁺-mediated action potentials rose even above the initial values which were measured prior to the nifedipine administration.     

Involvement of Ca(2+)-induced Ca2+ releasing system in interleukin-1beta-associated adenosine release

Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.     

Activation of K+ channel by 1-EBIO rescues the head and neck squamous cell carcinoma cells from Ca2+ ionophore-induced cell death

Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular Ca²⁺ ([Ca²⁺]_i)-dependent signaling affects the fate of cancer cells. Here we investigate the role of Ca²⁺-activated K⁺ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of different cell lines; SNU-1076, OSC-19 and HN5. Treatment with 1 µM ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of Ca²⁺-activated Cl^- channels (Ano-1) and Ca²⁺-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward K⁺ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Consistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the Ca²⁺ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.     

K+ channel openers,cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery

Summary The effects of K⁺ channel openers, cromakalim and an acetoxyl derivative of KRN 2391 (Ki 4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca²⁺]_i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP₃) production induced by the thromboxane A₂ analogue, U46619, in canine coronary arteries. Upon single dose applications of U46619 at 300 nmol/l, phasic and tonic increases in [Ca²⁺]_i and force were seen, which were almost abolished by cromakalim (10 mol/l) and Ki4032 (100 mol/l).In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction. Cromakalim (0.01–10 mol/l) and Ki4032 (0.1–100 mol/l) concentration-dependently inhibited the increases in [Ca²⁺]_i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K⁺ channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/l KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K⁺ channel openers inhibits the production of IP₃ and Ca ²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.Correspondence to T. Yanagisawa at the above address     

Effects of centrally administered H2 antagonists in the behavioral despair test

Injection of the histamine-2 (H₂) receptor antagonist cimetidine into the lateral ventricles of mice produced a dose-related reduction in swimming in the behavioral despair test. This response can be attenuated by intracerebroventricular (ICV) injection of the histamine-1 (H₁) receptor antagonist chlorpheniramine, or the H₂ receptor agonist impromidine, given simultaneously with cimetidine. At doses which blocked cimetidine, neither chlorpheniramine nor impromidine alone had effects on swimming. A similar decrease in swimming behavior was also seen after ICV injections of the non-imidazole H₂ antagonist, BMY 25,368. This effect of BMY 25,368 was also attenuated by chlorpheniramine and impromidine. These results suggest that H₁ and H₂ receptors in the brain may mediate opposing behavioral effects.     

Mechanisms of Na+ and Ca2+ influx into respiratory neurons during hypoxia

Changes in intracellular Na+ and Ca2+ in inspiratory neurons of neonatal mice were examined by using ion-selective fluorescent indicator dyes SBFI and fura-2, respectively. Both [Na+]i and [Ca2+]i signals showed rhythmic elevations, correlating with the inspiratory motor output. Brief (2-3 min) hypoxia, induced initial potentiation of rhythmic transients followed by their depression. During hypoxia, the basal [Na+]i and [Ca2+]i levels slowly increased, reflecting development of an inward current (Im). By antagonizing specific mechanisms of Na+ and Ca2+ transport we found that increases in [Na+]i, [Ca2+]i and Im due to hypoxia are suppressed by CNQX, nifedipine, riluzole and flufenamic acid, indicating contribution of AMPA/kainate receptors, persistent Na+ channels, L-type Ca2+ channels and Ca2+-sensitive non-selective cationic channels, respectively. The blockers decreased also the amplitude of the inspiratory bursts. Modification of mitochondrial properties with FCCP and cyclosporine A decreased [Ca2+]i elevations due to hypoxia by about 25%. After depletion of internal Ca2+ stores with thapsigargin, the blockade of NMDA receptors, Na+/K+ pump, Na+/H+ and Na+/Ca2+ exchange, the hypoxic response was not changed. We conclude that slow [Na+]i and [Ca2+]i increases in inspiratory neurons during hypoxia are caused by Na+ and Ca2+ entry due to combined activation of persistent Na+ and L-type Ca2+ channels and AMPA/kainate receptors.