Koki Shigenobu

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.

Inhibition of myocardial L- and T-type Ca2+ currents by efonidipine: possible mechanism for its chronotropic effect

Effects of efonidipine, a dihydropyridine phosphonate Ca2+ channel antagonist, on the guinea-pig heart were compared with those of nifedipine. In the sino-atrial node, 1 microM efonidipine produced increase in cycle length accompanied by prolongation of the phase 4 depolarization which was not prominent with 0.1 microM nifedipine. In ventricular myocytes, both efonidipine and nifedipine produced inhibition of the L-type Ca2+ current, nifedipine being tenfold more potent than efonidipine. Efonidipine also inhibited the T-type Ca2+ current at higher concentrations but nifedipine did not. Both Ca2+ channel antagonists had no or only a weak effect on K+ currents. In addition, 40 microM Ni2+, which selectively inhibited the T-type Ca2+ current, had no effect on myocardial Ca2+ transients and contractile force. In conclusion, efonidipine was shown to have inhibitory effects on both L- and T-type Ca2+ currents, which may contribute to its high negative chronotropic potency.

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 (ICa,L) and enhancement of the slowly activating delayed rectifier K+ currents (IKs). At a low stimulation rate, ICa,L suppression and IKs enhancement contributed to the A23187-induced APD shortening with a similar magnitude, whereas at a high stimulation rate, IKs enhancement dominantly contributed to APD shortening. IKs 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 IKs 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 IKs by A23187. Thus, CaM-dependent NOS3 activation confers the selective Ca2+-sensitivity on IKs. Ca2+-induced IKs 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.

Effects of Ca2+ channel antagonists on sinus node: Prolongation of late phase 4 depolarization by efonidipine

Effects of various Ca2+ channel antagonists on the action potential configuration of rabbit sino-atrial node tissue were examined with standard microelectrode techniques. All Ca2+ channel antagonists decreased the maximum rate of phase 0 depolarization (Vmax) and increased the cycle length. The potency order to increase the cycle length was nisoldipine = verapamil > nifedipine = clentiazem > efonidipine > diltiazem. The potency order to decrease Vmax and to shift the threshold potential to a positive direction was the same as that to increase the cycle length, indicating that the major mechanism of negative chronotropism was inhibition of the L-type Ca2+ current. All Ca2+ channel antagonists except efonidipine shifted the maximum diastolic potential to the positive direction, decreased the action potential amplitude and prolonged the action potential duration. The effects of nifedipine were slightly weaker than those of other drugs when compared at equally bradycardiac concentrations. These differences may reflect differences in drug effects on currents other than the L-type Ca2+ current. A characteristic feature of efonidipine was selective suppression of the later phase of pacemaker depolarization with no effect on action potential amplitude and duration. Similar suppression of the later phase was observed with 50 microM Ni2+, which is reported to inhibit the T-type, but not L-type, Ca2+ current. Thus, efonidipine appears to suppress selectively the later phase of pacemaker depolarization through inhibition of both L- and T-type Ca2+ currents, which may be the underlying mechanism for its reported potent negative chronotropic but weak inotropic activity.

Endothelium-dependent vasodilator effects of platelet activating factor on rat resistance vessels.

1 To elucidate the mechanisms of the powerful and long‐lasting hypotension produced by platelet activating factor (PAF), its effects on perfusion pressure in the perfused mesenteric arterial bed of the rat were examined. 2 Infusion of PAF (10−11 to 3 × 10−10m; EC50 = 4.0 × 10−11m; 95%CL = 1.6 × 10−11–9.4 × 10−11m) and acetylcholine (ACh) (10−10 to 10−6m; EC50 = 3.0 ± 0.1 × 10−9m) produced marked concentration‐dependent vasodilatations which were significantly inhibited by treatment with detergents (0.1% Triton X‐100 for 30s or 0.3% CHAPS for 90s). 3 Pretreatment with CV‐6209, a PAF antagonist, inhibited PAF‐ but not ACh‐induced vasodilatation. 4 Treatment with indomethacin (106m) had no effect on PAF‐ or ACh‐induced vasodilatation. 5 These results demonstrate that extremely low concentrations of PAF produce vasodilatation of resistance vessels through the release of endothelium‐derived relaxing factor (EDRF). This may account for the strong hypotension produced by PAF in vivo.

Intrasarcomere [Ca2+] gradients and their spatio-temporal relation to Ca2+ sparks in rat cardiomyocytes

1 Line‐scan analyses of spontaneous Ca2+ sparks, non‐propagating local rises in Ca2+ concentration, and the early phase of Ca2+ transients in cardiomyocytes were performed with a rapid‐scanning laser confocal microscope (Nikon RCM8000) and fluo‐3. 2 On electrical stimulation, points at which rise in Ca2+ began earliest were observed at regular spacings of 1.82 ± 0.26 μm (mean ± s.d.) along the longitudinal axis of the cell. The points were heavily stained with di‐2‐ANEPEQ, which stains the T‐tubules, indicating that they were at the Z‐line. 3 The points where spontaneous Ca2+ sparks originated coincided with the points which showed faster Ca2+ elevation, i.e. the Z‐line. 4 In some cases where a Ca2+ spark had occurred within about 30 ms before the evoked Ca2+ transient, fast elevation of Ca2+ was not observed at the corresponding Z‐line, indicating the presence of a refractory period in Ca2+ release from the SR. 5 The present results provide visual evidence for Ca2+ release from the junctional sarcoplasmic reticulum in cardiomyocytes. The presence of a refractory period in Ca2+ release after Ca2+ sparks provided new evidence that the normal Ca2+ transient may be the summation of Ca2+ sparks.

Sustained negative inotropism mediated by α‐adrenoceptors in adult mouse myocardial developmental conversion from positive response in the neonate

1 Inotropic responses to α‐adrenoceptor stimulation and the effects of antagonists were examined in isolated ventricular preparations from neonatal and adult mice. 2 Phenylephrine, in the presence of propranolol, produced positive inotropic responses in neonates up to 1 week after birth, while it produced negative inotropic responses in mice older than 3 weeks. 3 Both positive and negative responses to phenylephrine in neonates and adults, respectively, were antagonized by prazosin, WB4101 (2‐([2,6‐dimethoxyphenoxyethyl]aminomethyl)‐1,4‐benzodioxane) and 5‐methylurapidil, but not by atropine, yohimbine or chlorethylclonidine. 4 Noradrenaline (NA) produced positive inotropic responses both in the neonate and adult; the responses were observed in a lower concentration‐range in the neonate than in the adult. WB4101 produced a significant leftward shift of the concentration‐response curve for noradrenaline in adult preparations while only a slight rightward shift was observed in the neonate. 5 Our results demonstrate the presence of α‐adrenoceptor‐mediated inotropic responses in the mouse ventricular myocardia. The response to phenylephrine changes from a positive to a negative effect during postnatal development. The responses are mediated by α1‐adrenoceptors, and modulate the overall inotropic response to NA in the adult.

Expression and function of multidrug resistance P-glycoprotein in a cultured natural killer cell-rich population revealed by MRK16 monoclonal antibody and AHC-52

Natural killer (NK) cells have been reported recently to be the highest in expressing multidrug resistance (MDR) P-glycoprotein among normal mature lymphoid cells. Using a cultured NK cell-rich population, we have examined the expression and function of P-glycoprotein, in particular its role in NK cell-mediated cytotoxicity, by employing two MDR-reversing agents (nicardipine and AHC-52, a nicardipine analog almost devoid of calcium channel blocking activity) and monoclonal antibody against P-glycoprotein (MRK-16). The expression of P-glycoprotein was detected by flow cytometry and polymerase chain reaction of reverse transcribed mRNA. P-glycoprotein was functional in terms of rhodamine dye excretion and its susceptibility to the MDR-reversing agents. Since the concentration of nicardipine required for 50% inhibition (IC50) of rhodamine dye excretion (2 microM) was close to that of AHC-52 (5 microM), it was suggested that their inhibitory effects were not due to calcium channel blocking activity, and that ACH-52 is a selective inhibitor for P-glycoprotein. The IC50 of nicardipine for NK cell-mediated cytotoxicity (33 microM) was also close to that of AHC-52 (26 microM), indicating that P-glycoprotein is involved in NK cell-mediated cytotoxicity. In support of this, MRK16 inhibited NK cell-mediated cytotoxicity in a concentration-dependent manner. Both binding of target cells to NK cells and post-binding events were affected by AHC-52, suggesting that P-glycoprotein is involved in several steps in NK cell-mediated cytotoxicity.

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.

Involvement of maxi-KCa channel activation in atrial natriuretic peptide-induced vasorelaxation

Large conductance, voltage- and Ca2+-sensitive K+ (maxi-KCa) channels play an important role in the regulation of vascular smooth muscle excitability and contractility. The activity of maxi-KCa channels is modified by a variety of intracellular messengers including cGMP, as well as by voltage and Ca2+. In the present study, we investigated the functional relevance of maxi-KCa channels in atrial natriuretic peptide (ANP)-mediated vasorelaxation in the isolated rat mesenteric artery. ANP produced concentration-dependent relaxation in the de-endothelialized rat mesenteric artery . Iberiotoxin, a specific blocker of maxi-KCa channels, greatly attenuated the ANP-induced vasorelaxation. Similarly, a large portion of the vascular relaxation induced by 8-Bromo-cGMP, a membrane permeable analogue of cGMP, was inhibited by iberiotoxin. These results indicate that activation of maxi-KCa channels contributes substantially to the vascular relaxation produced by ANP in the rat mesenteric artery. Intracellular cGMP, increased by ANP, and the subsequent activation of cGMP-dependent protein kinase (PKG) may play a central role in the activation of maxi-KCa channels in the ANP-produced vascular relaxation.