Developmental changes in action potential prolongation by K+-channel blockers in chick myocardium.

The effects of K(+)-channel blockers on the action potential duration of the myocardium were examined in isolated right ventricles from the 7 - 10-day-old, 11 - 13-day-old, and 14 - 20-day-old embryo and 1 - 7-day-old hatched chicks. E-4031 significantly prolonged action potential duration at all developmental stages examined; the prolongation was largest in the 11 - 13-day-old embryo and was accompanied by early after-depolarizations. Chromanol 293B showed smaller prolongation at all stages examined. Terfenadine prolonged action potential duration in the 11 - 13-day-old embryo, but not in other stages. Thus, the chick ventricular myocardium changes its repolarization properties during development.

Developmental Changes in Action Potential Prolongation by K+-Channel Blockers in Chick Myocardium.

The effects of K+-channel blockers on the action potential duration of the myocardium were examined in isolated right ventricles from the 7 - 10-day-old, 11 - 13-day-old, and 14 - 20-day-old embryo and 1 - 7-day-old hatched chicks. E-4031 significantly prolonged action potential duration at all developmental stages examined; the prolongation was largest in the 11 - 13-day-old embryo and was accompanied by early after-depolarizations. Chromanol 293B showed smaller prolongation at all stages examined. Terfenadine prolonged action potential duration in the 11 - 13-day-old embryo, but not in other stages. Thus, the chick ventricular myocardium changes its repolarization properties during development.

Chronic left atrial volume overload abbreviates the action potential duration of the canine pulmonary vein myocardium via activation of IK channel.

Electrophysiological properties of the pulmonary vein myocardium were assessed in a canine chronic atrioventricular block model resulting in left atrial volume overload. Five chronic atrioventricular block dogs and five sham-operated dogs were used. The heart was removed two months after a surgical procedure causing atrioventricular block, when atrial structural remodeling was established. Standard microelectrode penetrations were made with glass microelectrodes to obtain action potential signals of left atrium and pulmonary vein myocardia. The resting membrane potential in the pulmonary vein was more positive than that in the left atrium (-69 mV vs -74 mV) in both animal groups. The action potential duration at 50% repolarization of the pulmonary vein was shorter in the chronic atrioventricular block dogs than in the sham-operated dogs (38 ms vs 63 ms), whereas no significant difference was detected in the action potential duration of the left atrium between the two animal groups (67 ms vs 61 ms). The action potential duration of the pulmonary vein in the chronic atrioventricular block dogs was prolonged by charybdotoxin but not by iberiotoxin. Such prolongation was not observed in the normal pulmonary vein. These results suggest that long-term left atrial dilatation shortened the action potential duration of pulmonary vein myocardium, which may be associated with activation of the intermediate conductance Ca2+-activated K+ channel (IK channel).

Intracellular mechanisms and receptor types for endothelin-1-induced positive and negative inotropy in mouse ventricular myocardium.

We examined the intracellular mechanisms for endothelin-1-induced positive and negative inotropic components that coexist in the mouse ventricular myocardium using isolated ventricular tissue and myocytes from 4-week-old mice. In the presence of SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger, endothelin-1 produced positive inotropy. Endothelin-1, when applied to cardiomyocytes in the presence of SEA0400, did not change the peak amplitude of the Ca2+ transient but increased intracellular pH and Ca2+ sensitivity of contractile proteins. On the other hand, in the presence of dimethylamiloride (DMA), a specific inhibitor of the Na+-H+ exchanger, endothelin-1 produced negative inotropy. In cardiomyocytes, in the presence of DMA, endothelin-1 produced a decrease in peak amplitude of the Ca2+ transient. In the presence of both DMA and SEA0400, endothelin-1 produced neither positive nor negative inotropy. Positive inotropy was blocked by BQ-123 and negative inotropy by BQ-788. These results suggested that endothelin-1-induced positive inotropy is mediated by ET(A) receptors, activation of the Na+-H+ exchanger and an increase in intracellular pH and Ca2+ sensitivity and that the negative inotropy is mediated by ET(B) receptors, activation of the Na+-Ca2+ exchanger and decrease in Ca2+ transient amplitude.

Role of nitric oxide in Ca2+ sensitivity of the slowly activating delayed rectifier K+ current in cardiac myocytes.

Sarcolemmal Ca2+ entry is a vital step for contraction of cardiomyocytes, but Ca2+ overload is harmful and may trigger arrhythmias and/or apoptosis. To maintain the amount of Ca2+ entry within an appropriate range, cardiomyocytes have feedback systems that tightly regulate ion channel activities in response to the changes in intracellular Ca2+ concentration ([Ca2+]i), thereby regulating Ca2+ entry. In guinea pig ventricular myocytes, Ca2+ ionophore, A23187, induced suppression of the L-type Ca2+ currents (I(Ca,L)) and enhancement of the slowly activating delayed rectifier K(+) currents (I(Ks)). At a low stimulation rate, I(Ca,L) suppression and I(Ks) enhancement contributed to the A23187-induced APD shortening with a similar magnitude, whereas at a high stimulation rate, I(Ks) enhancement dominantly contributed to APD shortening. I(Ks) enhancement induced by A23187 was attributable to actions of nitric oxide (NO), because they were inhibited by an inhibitor of NO synthase (NOS) and by a NO scavenger. A23187-induced alterations of APD and I(Ks) were strongly suppressed by a NOS3 inhibitor, but barely affected by a NOS1 inhibitor, suggesting that NOS3 was responsible for NO release in this phenomenon. Inhibition of calmodulin (CaM), but not Akt, blocked the enhancement of I(Ks) by A23187. Thus, CaM-dependent NOS3 activation confers the selective Ca2+-sensitivity on I(Ks). Ca2+-induced I(Ks) enhancement and resultant APD shortening potentially act as a physiological regulatory mechanism of Ca2+ recycling, because they were observed at a physiological range of [Ca2+]i in cardiac myocytes and are induced by physiologically relevant Ca2+ loading, such as digitalis application and rise in extracellular Ca2+ concentration.

(R)-ACX, a pancreatic beta-cell selective sulfonylurea, does not aggravate myocardial ischemia-reperfusion damage.

The organ selectivity and the effect on myocardial ischemia-reperfusion injury of (R)-acetoxyhexamide ((R)-ACX), a novel sulfonylurea, were examined. (R)-ACX, as well as glibenclamide, concentration-dependently stimulated insulin release from INS-1 cell, a cell line derived from pancreatic beta-cells. The potency of (R)-ACX was about 1/10 of that of glibenclamide. In isolated guinea pig ventricular myocardial tissue, glibenclamide concentration-dependently inhibited the action potential shortening by NIP-121, an ATP-sensitive potassium channel opener, but (R)-ACX showed only slight inhibition. In isolated rat aortic rings contracted with norepinephrine, glibenclamide concentration-dependently inhibited the relaxation by NIP-121, while (R)-ACX showed only slight inhibition. In coronary-perfused guinea pig ventricular preparations, glibenclamide reduced the recovery of contractile force after ischemia-reperfusion, while (R)-ACX did not. In conclusion, (R)-ACX is a beta-cell selective sulfonylurea which, unlike glibenclamide, does not aggravate cardiac ischemia-reperfusion damage.

Endocardial endothelium-dependent positive inotropy by Ca2+ pump inhibitors: possible involvement of store-operated Ca2+ entry.

Positive inotropy by sarcoplasmic/endoplasmic reticulum Ca(2+) pump inhibitors was found and its mechanisms were analyzed pharmacologically. Thapsigargin and cyclopiazonic acid produced positive inotropy in isolated mouse left atria. The responses were inhibited by pretreatment of the endocardial surface with Triton X-100 or by indomethacin, which suggests that the inotropic responses were mediated by prostaglandin(s) released from the endocardial endothelium as well as acetylcholine-induced positive inotropy. The thapsigargin- and acetylcholine-induced positive inotropy was significantly inhibited by Gd(3+), La(3+) and lavendustin A, a tyrosine kinase inhibitor, but not by Ni(2+) and LOE908, a non-selective cation channel inhibitor. Gd(3+) and lavendustin A had no effect on the exogenously applied PGF(2)alpha-induced positive inotropy. In addition, acetylcholine did not induce any positive inotropy when applied after the application of thapsigargin. These results strongly suggest that thapsigargin- as well as acetylcholine-induced prostaglandin release from endocardial endothelium is mediated by store-operated Ca(2+) entry through Gd(3+)-sensitive channels and activation of tyrosine kinase.

Reduction by SEA0400 of myocardial ischemia-induced cytoplasmic and mitochondrial Ca2+ overload.

The cardioprotective effects of SEA0400, a novel Na(+)-Ca(2+) exchanger inhibitor, were examined in isolated guinea pig myocardial tissue and ventricular myocytes. In a coronary-perfused right ventricular tissue preparation, SEA0400 had no cardiosuppressive effect during normoxia and experimental ischemia, but enhanced the recovery of contractile force during reperfusion. SEA0400 had no effect on tissue ATP content during normoxia, but attenuated its decrease during ischemia. Treatment of ventricular myocytes with an ischemia mimetic solution (high K(+), glucose free, pH 6.0, gassed with N(2)) resulted in the depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca(2+) concentration, which had a similar time course. SEA0400 significantly delayed these changes. These results suggest that SEA0400 maintains mitochondrial function and tissue ATP content during ischemia through the inhibition of cytoplasmic and mitochondrial Ca(2+) overload.

Unique excitation-contraction characteristics of mouse myocardium as revealed by SEA0400, a specific inhibitor of Na+-Ca2+ exchanger.

The functional role of the sodium-calcium exchanger in mouse ventricular myocardium was evaluated with a newly developed specific inhibitor, SEA0400. Contractile force and action potential configuration were measured in isolated ventricular tissue preparations, and cell shortening and Ca2+ transients were measured in indo-1-loaded isolated ventricular cardiomyocytes. SEA0400 increased the contractile force, cell shortening and Ca2+ transient amplitude, and shortened the late plateau phase of the action potential. alpha-adrenergic stimulation by phenylephrine produced a sustained decrease in contractile force, cell shortening and Ca2+ transient amplitude, which were all inhibited by SEA0400. Increasing the contraction frequency resulted in a decrease in contractile force in the absence of drugs (negative staircase phenomenon). This frequency-dependent decrease was attenuated by SEA0400 and enhanced by phenylephrine. Phenylephrine increased the Ca2+ sensitivity of contractile proteins in isolated ventricular cardiomyocytes, while SEA0400 had no effect. These results provide the first pharmacological evidence in the mouse ventricular myocardium that inward current generated by Ca2+ extrusion through the sodium-calcium exchanger during the Ca2+ transient contributes to the action potential late plateau, that alpha-adrenoceptor-mediated negative inotropy is produced by enhanced Ca2+ extrusion through the sodium-calcium exchanger, and that the negative staircase phenomenon can be explained by increased Ca2+ extrusion through the sodium-calcium exchanger at higher contraction frequencies.

Cardioprotection without cardiosuppression by SEA0400, a novel inhibitor of Na+-Ca2+ exchanger, during ischemia and reperfusion in guinea-pig myocardium.

The effect of SEA0400, a novel Na+-Ca2+ exchanger inhibitor, on mechanical and electrophysiological parameters of coronary-perfused guinea-pig right ventricular tissue preparation was examined during no-flow ischemia and reperfusion. Contractile force and action potential duration were decreased during no-flow ischemia, while the resting tension was increased. Upon reperfusion, transient arrhythmias were observed and contractile force returned to less than 50% of preischemic values. SEA0400 (1 microM) had no effect on the decline in contractile force during the no-flow ischemia, but abolished the rise in resting tension. SEA0400 significantly improved the recovery of contractile force after reperfusion to about 80% of the preischemic value. SEA0400 had no effect on the action potential under normal conditions and during ischemia, but significantly improved the recovery of action potential duration after reperfusion. Enhancement of the recovery of contractile force during reperfusion by SEA0400 was also observed when the drug was applied only before and during the ischemic period and when the drug was applied only during reperfusion. The present results indicate that inhibition of Na+-Ca2+ exchanger either during ischemia or during reperfusion exerts cardioprotective effects and enhances the recovery of myocardial contractile function.

The mouse sino-atrial node expresses both the type 2 and type 3 Ca(2+) release channels/ryanodine receptors.

Ca(2+) released from intracellular Ca(2+) stores is shown to be involved in pacemaker activity in the sino-atrial (SA)-node. However, little is known about the molecular identity of the Ca(2+) release channel/ryanodine receptor (RYR) involved in pacemaker activity. We examined the mRNA distribution of three different RYR isoforms (RYR1, RYR2, and RYR3) in the mouse SA-node. RNase protection assay and in situ hybridization revealed that RYR2 mRNA expresses widely in the heart including the SA-node, while RYR3 mRNA expression is limited to the SA-node and to the right atrium. Thus, not only RYR2 but also RYR3 may participate in pacemaker activity.

Effect of SEA0400, a novel inhibitor of sodium-calcium exchanger, on myocardial ionic currents.

The effects of 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a newly synthesized Na(+)-Ca(2+) exchanger (NCX) inhibitor, on the NCX current and other membrane currents were examined in isolated guinea-pig ventricular myocytes and compared with those of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea (KB-R7943). SEA0400 concentration-dependently inhibited the NCX current with a 10 fold higher potency than that of KB-R7943; 1 microM SEA0400 and 10 microM KB-R7943 inhibited the NCX current by more than 80%. KB-R7943, at 10 microM, inhibited the sodium current, L-type calcium current, delayed rectifier potassium current and inwardly rectifying potassium current by more than 50%, but SEA0400 (1 microM) had no significant effect on these currents. These results indicate that SEA0400 is a potent and highly selective inhibitor of NCX, and would be a powerful tool for further studies on the role of NCX in the heart and the therapeutic potential of its inhibition.

2-Aminoethoxydiphenyl borate causes dissociation between membrane electrical and mechanical activity in guinea-pig urinary bladder smooth muscle.

Physiological functions of urinary bladder profoundly reflect smooth muscle mechanical activity. Urinary bladder smooth muscle itself produces myogenic rhythmic contraction, and this spontaneous mechanical event could be the fundamental determinant of urinary bladder functions. The spontaneous contraction of urinary bladder smooth muscle is thought to be triggered primarily by the action potential generated in this smooth muscle cell. Modulators of ion channels contributing to the configuration of action potential also affect urinary bladder smooth muscle mechanical activity as expected exactly from the effects on the electrical event. In the present study, we show that the frequency of action potential recorded in intact strip of guinea-pig urinary bladder smooth muscle is dramatically increased by 2-aminoethoxydiphenyl borate (2-APB; 30 microM) from 0.2 Hz to 1 Hz (approximately 500% increments). In contrast to an increasing effect expected from the membrane electrical alterations, mechanical activity (both contraction amplitude and frequency) of this smooth muscle is unexpectedly reduced by the same concentration of 2-APB to approximately 35% of the control. The present results firstly show an apparent dissociation of electrical-mechanical coupling in urinary bladder smooth muscle. The alteration of membrane electrical activity might not be the exclusive trigger mechanism responsible for the generation of spontaneous rhythmic contraction of this smooth muscle.

Pharmacological evidence that tetraethylammonium-sensitive, iberiotoxin-insensitive K+ channels function as a negative feedback element for sympathetic neurotransmission by suppressing omega-conotoxin-GVIA-insensitive Ca2+ channels in the relaxation of rabbit facial vein.

The facial vein isolated from various species relaxes in response to electrical field stimulation (EFS). EFS-elicited relaxation of the facial vein is mediated through the release of noradrenaline (NA) from sympathetic nerve endings and the subsequent activation of smooth muscle beta-adrenoceptors. The release of NA from sympathetic nerve endings in arterial tissues requires transmembrane Ca2+ influx, mediated predominantly by voltage-gated N-type Ca2+ channels. The present pharmaco-mechanical study was undertaken to determine whether the N-type channel is the exclusive pre-junctional Ca2+ channel mediating NA release from sympathetic nerve endings in the rabbit facial vein. Possible roles of K+ channels in the sympathetic neurotransmission were also examined, especially focusing on the contribution of voltage-dependent, Ca2+-activated K+ (BKCa) channels. An isolated ring preparation of the rabbit facial vein exhibited intrinsic myogenic tone which lasted for several hours when stretched. EFS produced frequency-dependent (0.25-2 Hz) relaxation in this preparation. EFS-elicited relaxation was abolished by tetrodotoxin (TTX, 1 microM), guanethidine (5 microM) or propranolol (1 microM), indicating that NA released from sympathetic nerve endings was mediating the relaxant response. NA-mediated neurogenic relaxation was almost eliminated by omega-conotoxin-GVIA (1 microM), an N-type Ca2+ channel blocker. On the other hand, tetraethylammonium (TEA, 2 mM) strongly potentiated EFS-elicited relaxation without affecting the relaxation induced by exogenously applied NA. This potentiation by TEA was not profoundly diminished by omega-conotoxin-GVIA (1 microM) alone or omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM, P-type channel blocker), but was almost abolished by omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM) plus omega-conotoxin-MVIIC (3 microM, N-, P- and Q-type channel blocker). The potentiating effect of TEA was not mimicked by iberiotoxin (100 nM) or charybdotoxin (3 microM), both of which block BKCa channels. These findings suggest that pre-junctional N-type Ca2+ channels play the predominant role in the sympathetic nerve transmission in the rabbit facial vein, as in peripheral arterial vascular beds. In addition, Ca2+ channels resistant to 1 microM omega-conotoxin-GVIA, most probably Q-type channels, appear to be present at the sympathetic nerve endings in the rabbit facial vein and contribute substantially to the regulation of NA release from the nerve endings. Prejunctional K+ channels, sensitive to TEA but pharmacologically distinct from iberiotoxin-sensitive BKCa channels, seem to be functionally coupled intimately with the omega-conotoxin-GVIA-resistant Ca2+ channels, and thus function as a negative feedback element in sympathetic neurotransmission in the rabbit facial vein.

Evidence for a significant role of a Gs-triggered mechanism unrelated to the activation of adenylyl cyclase in the cyclic AMP-independent relaxant response of guinea-pig tracheal smooth muscle.

Cyclic AMP is a key molecule in the regulation of airway smooth muscle tone. Increased cyclic AMP leads to relaxation of this smooth muscle and its inhibition results in the muscle contraction. A constitutive role for cyclic AMP in the contraction and relaxation of airway muscle is supported by the observations that direct activators of adenylyl cyclase, such as forskolin and membrane-permeable cyclic AMP analogues, relax this smooth muscle potently. This traditional view of the role for cyclic AMP is the basis for the idea that relaxation of airway smooth muscle mediated through adenylyl cyclase-linked, G(s)-coupled receptors, including the beta(2)-adrenoceptor, is achieved mainly by the elevation of cyclic AMP content [cyclic AMP-dependent mechanism(s)]. However, recent pharmacological and biochemical evidence raises a fundamental question concerning the role of cyclic AMP; can G(s)-coupled receptor-mediated relaxation of tracheal smooth muscle be attributed exclusively to cyclic AMP-dependent mechanism(s)? In the present study, we show that cholera toxin (CTX, 5 microg/ml), an activator of the heterotrimeric guanine-nucleotide-binding protein G(s), relaxes guinea-pig tracheal smooth muscle. CTX also elevates tissue cyclic AMP content by about 30-fold and this is practically abolished by an adenylyl cyclase inhibitor, SQ 22,536 (100 microM). However, unexpectedly, the relaxant response to CTX is not affected by SQ 22,536. These results firstly show that activation of G(s) is able to produce a relaxation in tracheal smooth muscle independently of the elevation of cyclic AMP. G(s)-triggered, cyclic AMP-unrelated cellular mechanism(s) seem(s) to play a substantial role in smooth muscle relaxation mediated through adenylyl cyclase-linked receptors. This mechanism may account in part for the cyclic AMP-independent relaxant response of tracheal smooth muscle.

Evidence for the possible involvement of Ca2+ entry blockade in the relaxation by class I antiarrhythmic drugs in the isolated pig coronary smooth muscle.

Effects of several Na+ channel blockers (i.e., class I antiarrhythmic agents), procainamide, quinidine, lidocaine, mexiletine, propafenone, were investigated in the isolated endothelium-denuded pig coronary artery focusing on the possible involvement of the blockade of Ca2+ channels and/or opening of K+ channels in the relaxant responses. All drugs except procainamide induced a concentration-dependent full relaxation of the coronary artery precontracted with high-KCl (30 mM, 80 mM). Inhibitions by procainamide of both high-KCl-induced contractions were less than 50% even at a concentration of 3 x 10(-2) M. Both high-KCl contractions were diminished by an L-type Ca2+ channel blocker, diltiazem, in a concentration-dependent manner. In contrast, cromakalim failed to inhibit 80 mM KCl-induced contraction. Tetrodotoxin (3 x 10(-5) M) did not affect the relaxant actions of the tested class I antiarrhythmic agents in high-KCl (80 mM)- or prostaglandin F2alpha-contracted muscle. The inhibitions by these class I antiarrhythmic agents of high-KCl-induced contraction were significantly attenuated when extracellular CaCl2 was increased from 2 mM to 20 mM. Furthermore, procainamide, quinidine, lidocaine, mexiletine as well as diltiazem decreased both cytoplasmic Ca2+ level ([Ca2+](cyt)) and muscle tension elevated by high-KCl in fura-2-loaded coronary preparations. These findings suggest that blockade of voltage-gated Ca2+ channels is involved in the relaxing action of these class I antiarrhythmic drugs in pig coronary artery. Blockade of Na+ channel and/or opening of K+ channels does not seem to play the principal role in the mechanism by which these antiarrhythmic drugs relax coronary artery.

Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP >> CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.

MaxiK channel mediates beta2-adrenoceptor-activated relaxation to isoprenaline through cAMP-dependent and -independent mechanisms in guinea-pig tracheal smooth muscle.

We examined the contribution of large-conductance, Ca(2+)-sensitive K+ (MaxiK) channel to beta2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between beta2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through beta2-type of adrenoceptor since the response was antagonized in a competitive fashion by a beta2-selective adrenoceptor antagonist butoxamine with a pA2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 microM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 microM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 microM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 microg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein Gs. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates beta2-adrenoceptor-activated relaxation; 2) both cAMP-dependent and -independent mechanisms underlie the functional coupling between beta2-adrenoceptor and MaxiK channel to induce muscle relaxation; and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between beta2-adrenoceptor and this ion channel.

MaxiK channel-triggered negative feedback system is preserved in the urinary bladder smooth muscle from streptozotocin-induced diabetic rats.

MaxiK channel, the large-conductance Ca2+-sensitive K+ channel, facilitates a negative feedback mechanism to oppose excitation and contraction in various types of smooth muscles including urinary bladder smooth muscle (UBSM). In this study, we investigated how the contribution of MaxiK channel to the regulation of basal UBSM mechanical activity is altered in streptozotocin-induced diabetic rats. Although the urinary bladder preparations from both control and diabetic rats were almost quiescent in their basal mechanical activities, they generated spontaneous rhythmic contractions in response to a MaxiK channel blocker, iberiotoxin (IbTx). The effect of IbTx on the mechanical activity was significantly greater in diabetic rat than in control animal. Similarly, the basal mechanical activity was increased with apamin, an inhibitor for some types of small conductance Ca2+-sensitive K+ channels, and this effect was more pronounced for diabetic rat. However, in both control and diabetic animals, IbTx action was stronger than that of apamin. Diabetes also enhanced the responses to BayK 8644, an L-type Ca2+ channel agonist. The extent of this enhancement in diabetic bladder vs. control was, however, almost the same as that attained with IbTx. Expression levels for MaxiK channel as well as apamin-sensitive K+ channels and L-type Ca2+ channel were not altered by diabetes, when determined as their corresponding mRNA levels. These results indicate that diabetes can potentially increase the basal UBSM mechanical activity. However, in diabetic UBSM, the main negative-feedback system triggered by MaxiK channel is still preserved enough to counteract the possible enhancement of this smooth muscle mechanical activity.

New aspects for the treatment of cardiac diseases based on the diversity of functional controls on cardiac muscles: diversity in the excitation-contraction mechanisms of the heart.

The waveform of the myocardial action potential (AP) triggering contraction differs among the species, developmental stage, and pathological state. The species difference in heart rate, which inversely correlates with body size, originates in the ion-channel mechanisms responsible for diastolic depolarization of the sinoatrial node. In some cases, such as the chronically AV-blocked dog and 11- to 13-day chick embryo, the repolarization reserve is decreased making the heart useful for drug evaluation. The degree of dependence of contraction on sarcoplasmic reticulum (SR) function increases during development. The large SR dependence and short AP of the adult mouse and rat support their rapid contraction under high heart rate. The function of the Na(+)/Ca(2+) exchanger is affected by AP waveform and ion concentrations; its major role is Ca(2+) extrusion, but under pathological conditions such as ischemia-reperfusion, it allows Ca(2+) influx and leads to myocardial injury, including loss of mitochondrial function. The role of mitochondria in ATP supply is less in the fetus where glycolysis plays a greater role. The pharmacological properties of the myocardium are affected by all of these factors and also by autonomic innervation and the hormonal status. Such comprehensive understanding is indispensable for the development of novel therapeutic strategies.