Kazuhide Nishimaru

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

Decreased Expression of Voltage- and Ca2+-Activated K+ Channels in Coronary Smooth Muscle During Aging

Abstract— Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca2+-activated K+ channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming &agr; subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52±9 channels/pF (3 months old) to 18±5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by ≈50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed ≈80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and ≈45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively (≈70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction

The voltage-dependent and Ca2+-activated K+ channel (MaxiK, BK) and the cellular proto-oncogene pp60c-Src (c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its α subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by ≈16 mV. hSlo inhibition by c-Src is due to channel direct phosphorylation because: (i) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by ≈10 mV, and (ii) immunoprecipitated hSlo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSlo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.

Functional and molecular evidence of MaxiK channel β1 subunit decrease with coronary artery ageing in the rat

Large‐conductance, voltage‐ and Ca2+‐activated K+ channels (MaxiK, BK) are key regulators of vascular tone. Vascular MaxiK are formed by the pore‐forming α subunit and the modulatory β1 subunit, which imprints unique kinetics, Ca2+/voltage sensitivities and pharmacology to the channel. As age progresses, α subunit functional expression and protein levels diminish in coronary myocytes. However, whether ageing modifies β1 subunit expression or the mechanism of α subunit reduction is unknown. Thus, we examined functional and pharmacological characteristics of MaxiK, as well as α and β1 transcript levels in coronary myocytes from young and old F344 rats. The mechanism of age‐dependent α subunit protein reduction involves its transcript downregulation. A corresponding loss of β1 transcripts was also detected in old myocytes, suggesting a proportional age‐dependent decrease of β1 to α subunit protein. Indeed, MaxiK channel properties, defined by coassembly of β1 and α subunits, were equivalent in young versus old, for example in terms of (i) activation kinetics, (ii) sensitivity to Ca2+ levels > 1 μm (iii) dehydrosoyasaponin‐I‐induced activation, and (iv) iberiotoxin blockade. Consistent with less MaxiK expression/function in older myocytes, the ability of iberiotoxin to contract coronary rings was reduced ∼50% with ageing confirming our previous findings. 5‐Hydroxytryptamine (5‐HT) contractile efficacy was reduced by iberiotoxin pretreatment in young > old coronary arteries (explained by larger iberiotoxin‐induced contraction and decreased dynamic range for 5‐HT contraction in young versus old) with no apparent differences in nitroglycerine‐induced relaxation. We propose that the age‐related MaxiK reduction involves a parallel decrease of α and β1 functional expression via a transcript downregulatory mechanism; a major impact on basal and possibly stimulated coronary contraction may contribute to altered coronary flow regulation and coronary morbidity in the elderly.

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.

Positive and negative inotropic effects of muscarinic receptor stimulation in mouse left atria

In isolated mouse left atria, acetylcholine (ACh) produced a biphasic inotropic response; a transient decrease in developed tension was followed by an increase. Both negative and positive responses were concentration dependent and were inhibited by atropine. The negative and positive inotropic responses were also observed with a nonselective muscarinic stimulant, oxotremorine-M, but not with an M1-receptor selective stimulant, McN-A343. Pirenzepine, an M1-receptor antagonist, inhibited both negative and positive inotropic responses at high concentrations. Gallamine, an M2-receptor antagonist, inhibited the negative response. Hexahydro-siladifenidol hydrochloride, p-fluoro analog (p-F-HHSiD), an M3-receptor antagonist, inhibited the positive response with no effect on the negative phase. In pertussis toxin (PTX) treated preparations, negative inotropic response to ACh was not observed. These results suggest that the negative and positive inotropic responses to acetylcholine in mouse atria are mediated by M2 and M3 receptors, respectively. The negative phase, but not the positive phase, was mediated by a PTX-sensitive G protein.

Role of sarcoplasmic reticulum in myocardial contraction of neonatal and adult mice

Changes in action potential parameters by and inotropic responses to nicardipine, verapamil, ryanodine and cyclopiazonic acid were examined in isolated ventricular myocardial preparations from neonatal and adult mice. The action potential of both neonatal and adult mice had a unique configuration with little evidence of a plateau at depolarized membrane potential; the action potential duration was significantly larger in neonatal preparations. Nicardipine had no effect on action potential parameters in the adult while it significantly shortened the action potential duration at 50% repolarization in the neonate. Ryanodine significantly shortened the action potential duration at 80% repolarization at both ages: the shortening was significantly larger in the adult when compared with the neonate. The contraction of ventricular preparations from adult mice were relatively resistant to nicardipine and verapamil. Nicardipine or verapamil, even at 10(-5) M, only decreased the contractile force to 70% of control values; the decrease was much less than that reported in other experimental species such as chick, guinea pig or rabbit. In the neonate, 10(-5) M nicardipine or verapamil decreased the contractile force to 30% of control values. Ryanodine had a potent negative inotropic effect both in the neonate and adult; the effect was significantly larger in the adult. Cyclopiazonic acid produced a decrease in contractile force and prolongation of the time required for relaxation; both effects were significantly larger in the adult. These results suggest that the contraction of the adult mouse myocardium is highly dependent on SR function and less dependent on transsarcolemmal Ca2+ influx when compared with the myocardium of the neonatal mouse and that of other species.

Acetylcholine-induced positive inotropy mediated by prostaglandin released from endocardial endothelium in mouse left atrium

Abstract. The possible involvement of the endocardial endothelium in the positive inotropic response of the mouse left atrium to acetylcholine was examined pharmacologically. In mouse left atria, acetylcholine produced a biphasic inotropic response: a transient decrease in contractile force followed by a late increase. The positive response was not affected by the presence of phentolamine and propranolol, but was almost abolished by pretreatment of the preparation with 1% Triton X-100, which denudes the endocardium of its endothelium. Nordihydroguaiaretic acid, NG-nitro-L-arginine, BQ-123 and BQ-788 had no effect on the inotropic responses to acetylcholine, but indomethacin completely abolished the positive response. Prostaglandins and their analogues had a positive inotropic effect with a potency order PGF2α>PGD2>PGE2>U46619, whereas beraprost had no effect. Neither Triton X-100 pretreatment nor the presence of indomethacin affected the positive inotropic effect of PGF2α. Acetylcholine and PGF2α prolonged the action potential duration similarly. These results suggest that the acetylcholine-induced positive inotropic response in mouse left atria is mediated by prostaglandin released from the endocardial endothelium.

Function and clustered expression of MaxiK channels in cerebral myocytes remain intact with aging

The incidence of stroke increases significantly in the aging population where stroke related deaths boost at >75 years and survivors are often permanently disabled. Aging is known to decrease cerebral blood flow likely due to an increase in arterial tone. Although MaxiK channels are key regulators of cerebral arterial tone their pattern of expression and function in cerebral blood vessels during aging is unknown. Using specific antibodies against the alpha-subunit of MaxiK channels and current recordings, we now demonstrate that in aging cerebral myocytes, MaxiK channels remain healthy. Furthermore, we show for the first time that in the vasculature, MaxiK channels are expressed in clusters. Clusters have an estimated radius of approximately 200 nm in young rats (3-5 month old Fisher 344 rats) which remains normal in old (25-30 month rats) cerebral myocytes. Consistent with a healthy MaxiK channel expression in old cerebral arteries, MaxiK current density, kinetics and Ca(2+) sensitivity were practically identical in young and old myocytes. Sensitivity to nanomolar concentrations of dehydrosoyasaponin-I that activates channels formed by alpha and beta subunits is also the same in young and old myocytes. These results demonstrate that MaxiK channels maintain normal expression during cerebral aging which is in sharp contrast to our previous finding of loss of expression in aging coronary arteries. It seems therefore, that cerebral myocytes have developed a protective anti-aging mechanism leading to the continued expression of MaxiK channels.

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.