Yoshio Tanaka

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+‐Ca2+ 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; 1u2003μM SEA0400 and 10u2003μM KB‐R7943 inhibited the NCX current by more than 80%. KB‐R7943, at 10u2003μM, inhibited the sodium current, L‐type calcium current, delayed rectifier potassium current and inwardly rectifying potassium current by more than 50%, but SEA0400 (1u2003μM) 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.

Aging, ion channel expression, and vascular function

Cardiovascular disease remains the leading cause of death in the United States, and aging is one of the main risk factors for its development. Coronary arteries nurture the heart, but as age progresses, they suffer changes that make them stiffer, thicker, and with higher spontaneous contractile activity. Even in the absence of pathological atherosclerotic lesions, these changes make the coronary arteries at risk for vasospasm and the individual at risk for myocardial ischemia and heart failure. Thus, knowledge of the molecular mechanisms involved in the vascular physiology, disease, and aging of the coronary circulation is required to develop strategies to preserve the quality of life of an increasingly aging population. One of the key factors that regulate coronary arterial tone is the activity of K+ channels in the vascular smooth muscle cells (SMCs). In particular, voltage-dependent and Ca(2+)-activated K+ (BKCa) channels, which are abundant in the coronary SMCs, are targets of vasoconstrictors and vasorelaxants, and play a key role in determining arterial tone and diameter. Aging induces a reduction in the density of the alpha-subunit of BKCa channels in coronary smooth muscle, lowers baseline endothelial release of the relaxant nitric oxide (NO), and increases the response to endothelial constrictor factors and K+. Thus, aging induces the remodeling of important proteins involved in the excitability and contractility of the coronary circulation. Altogether, these changes increase the risk of coronary artery vasospasm, myocardial ischemia, and infarct in the elderly.

Evidence for the primary role for 4-aminopyridine-sensitive Kv channels in β3-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles

Gastrointestinal smooth muscles exhibit relaxation in response to the stimulation of β-adrenoceptors with catecholamines. Subtypes of β-adrenoceptors which mediate catecholamine-elicited relaxations in gastrointestinal smooth muscles are predominantly atypical β-adrenoceptors including β3-adrenoceptors. Gastrointestinal smooth muscle relaxations mediated via β3-adrenoceptors can occur independently of intracellular cyclic adenosine monophosphate (AMP) elevation. One of the mechanisms responsible for cyclic AMP-independent smooth muscle relaxation following activation of Gs protein-coupled receptors could be activation of voltage-gated K+ channels. In the present study, possible contribution of two types of K+ (large-conductance, Ca2+-sensitive and voltage-gated K+, BKCa; voltage-gated, Kv) channels to β3-adrenoceptor-mediated, cyclic AMP-independent relaxations was compared in gastric fundus and duodenum smooth muscles isolated from the guinea-pig. In these gastrointestinal smooth muscles, three catecholamines ((–)-isoprenaline, (–)-noradrenaline and (–)-adrenaline) and two β3-adrenoceptor agonists ((R*, R*)-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (±)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy] -1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((±)-CGP12177A)) elicited a concentration-dependent relaxation in the presence of β1- and β2-adrenoceptor antagonists. The relaxations were unaffected by an adenylyl cyclase inhibitor, SQ-22,536 (100xa0μM), which indicates their characteristic of cyclic AMP-independency. On the other hand, the SQ-22,536-resistant, β3-adrenoceptor-mediated relaxant components were potently attenuated when the tone was raised using high-KCl (80xa0mM) or in the presence of a Kv channel blocker, 4-aminopyridine (4-AP, 1–3xa0mM). Iberiotoxin (100xa0nM), a selective blocker of BKCa channels which significantly contribute to cyclic AMP-independent vascular smooth muscle relaxations induced through activation of Gs protein-coupled receptors, did not apparently show any inhibitory effects on SQ-22,536-resistant, β3-adrenoceptor-mediated relaxations in these gastrointestinal smooth muscles. The present results indicate that 4-AP-sensitive Kv channels play a primary role in β3-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles. In these smooth muscles, BKCa channels seem to apparently contribute insignificantly to cyclic AMP-independent relaxations following stimulation of β3-type of adrenoceptors.

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, Gs-coupled receptors, including the β2-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 Gs-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, 5xa0μg/ml), an activator of the heterotrimeric guanine-nucleotide-binding protein Gs, 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 (100xa0μM). However, unexpectedly, the relaxant response to CTX is not affected by SQ 22,536. These results firstly show that activation of Gs is able to produce a relaxation in tracheal smooth muscle independently of the elevation of cyclic AMP. Gs-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.

Possible Involvement of Prostaglandins F2α and D2 in Acetylcholine-Induced Positive Inotropy in Isolated Mouse Left Atria

The inotropic action of prostaglandins PGF_2α, PGD₂ and PGE₂ on isolated mouse left atria was characterized and compared with the positive inotropic action of acetylcholine, which has previously been shown to be mediated by prostaglandins released from the endocardial endothelium. PGF_2α, PGD₂ and PGE₂ produced positive inotropic responses; the time course of the change in contractile force induced by PGF_2α and PGD₂ was about the same as that by acetylcholine, while that by PGE₂ was slower. Fluprostenol and sulprostone, FP and EP receptor agonists, respectively, had positive inotropic effects while BW-245C, a DP receptor agonist, had no effect. AH-6809, a DP receptor antagonist, had no inhibitory effect on the positive inotropic response to PGD₂. Dimethylamiloride, an inhibitor of Na⁺/H⁺ exchange, inhibited the positive inotropic response to PGF_2α, PGD₂ and acetylcholine, but not PGE₂. Fluorometric pH measurement with carboxy-SNARF-1-loaded atrial myocytes revealed no change in intracellular pH on application of PGF_2α. PGF_2α and PGD₂ significantly prolonged the duration of the atrial action potential while PGE₂ had no significant effect. These findings suggest that prostaglandins induce positive inotropic response in mouse atria through FP and EP receptor stimulation and that the former mechanism mediates in part the positive inotropic response to acetylcholine.

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

Abstract. 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; 30xa0µM) from 0.2xa0Hz to 1xa0Hz (~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 ~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.

Function of β1-adrenoceptors and mRNA expression of β1- and β2-adrenoceptors in guinea-pig esophagus

beta-Adrenoceptor subtypes mediating relaxation were examined by using pharmacological and molecular analyses in guinea-pig esophageal muscularis mucosae. (-)-Isoprenaline-induced relaxations were antagonized by (+/-)-propranolol (pA2 = 8.47+/-0.07), a selective beta1-adrenoceptor antagonist, (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP20712A; pA(2)=9.43+/-0.09), and a selective beta(2)-adrenoceptor antagonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI-118,5511; pA2 = 7.11+/-0.04), indicating that beta(1)-adrenoceptors but not beta2- or beta3-adrenoceptors were essentially involved in beta-adrenoceptor-mediated relaxations. However, the expression of messenger RNA (mRNA) for beta1- and beta2-adrenoceptors, but not for beta3-adrenoceptors, was detected by reverse transcription-polymerase chain reaction (RT-PCR). These results clearly suggest that not all beta-adrenoceptor mRNA expressed strictly reflect functional receptors in guinea-pig esophageal muscularis mucosae.

Pharmacological characterization of α1-adrenoceptor in mouse iliac artery

Abstract Subtypes of α 1 -adrenoceptor-mediated contraction to noradrenaline in the mouse iliac artery were determined (pharmaco-mechanically). Prazosin, 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) and 5-methylurapidil shifted the concentration–response curve for noradrenaline to the right, giving the pA 2 values of 9.30, 9.55 and 8.71, respectively. 8-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]-ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) shifted the concentration–response curve for noradrenaline to the right and the pA 2 value was 6.62. These results indicate that the contractile response to noradrenaline in the mouse iliac artery is predominantly mediated by the α 1A -adrenoceptor subtype.