Kageyoshi Ono

Modulation of the delayed rectifier K+ current by isoprenaline in bull-frog atrial myocytes.

1. The effects of isoprenaline (ISO) on the calcium current (ICa) and delayed rectifier K+ current (IK) were examined using a tight‐seal whole‐cell voltage‐clamp technique in single cells from bull‐frog atrium to examine the ionic mechanism(s) of catecholamine‐induced action potential shape changes. 2. The effects of ISO on the action potential were dose‐dependent. Very low doses (5 x 10(‐9) M) prolonged the action potential. Higher doses (10(‐6) M) of ISO increased the plateau height, but shortened the action potential by accelerating the early repolarization phase. 3. ISO increased IK and ICa in a dose‐dependent fashion. Both of these effects were blocked by a beta‐receptor antagonist, propranolol (3 x 10(‐7) M). In contrast IK1, the inwardly rectifying K+ current, was not changed significantly by ISO. 4. The ISO‐induced increase in IK was observed in the presence of CdCl2 (3 x 10(‐4) M), indicating that this effect is not due to a Ca2(+)‐activated potassium current. 5. The reversal potential of IK in normal Ringer solution (‐83 +/‐ 2 mV) was not significantly changed by ISO. Thus, stimulation of the Na(+)‐K+ pump and a consequent hyperpolarizing shift in EK are not responsible for the increase in IK. 6. In the presence of ISO (10(‐6) M) the steady‐state activation curve (n infinity) for IK was consistently shifted to more negative values (by approximately 10 mV). The activation and deactivation kinetics of IK were also changed by ISO: activation was accelerated, deactivation was slowed. These ISO‐induced changes in IK result in an increase in IK at voltages corresponding to the plateau of the action potential. 7. ISO (10(‐6) M) increased ICa dramatically, approximately 6‐fold at 0 mV. At the same time, the time constant of ICa inactivation decreased significantly (34 +/‐ 4 ms control; 23 +/‐ 4 ms ISO). 8. These results confirm that low doses of sympathetic agonists acting via beta‐receptors increase ICa. Relatively high doses of beta‐receptor agonists increase both ICa and IK, but these two effects appear to be generated by different biophysical mechanisms. 9. These dose‐dependent changes in ICa and IK can explain the observed ISO‐induced changes in action potential shape. At doses of approximately 10(‐8) M ICa is increased, resulting in a more depolarized plateau and small lengthening of the action potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Negative Chronotropic Effect of Endothelin 1 Mediated Through ETA Receptors in Guinea Pig Atria

Endothelins exert potent excitatory cardiac effects by acting on specific receptors on myocytes. In this study, we have examined the signal transduction mechanism for the chronotropic effect of endothelins in guinea pig atria. A competition binding of [125I]endothelin 1 ([125I]ET-1) using the recently developed ETA receptor-selective antagonist BQ123 showed the presence of almost equal populations of ETA (44%) and ETB (56%) receptors in the guinea pig right atria. In a concentration-response study, endothelin 3 (ET-3), an agonist with higher affinity to ETB receptors than to ETA receptors, and sarafotoxin S6c (STXS6c), an ETB receptor-selective agonist, increased the rate of spontaneous beating at all concentrations tested (10 pmol/L to 100 nmol/L). In contrast, ET-1, a nonselective agonist, increased the heart rate at lower concentrations (10 pmol/L to 10 nmol/L) but decreased it at higher concentrations (30 to 100 nmol/L). When ET-1 (100 nmol/L) was applied in a single amount, heart rate was strongly increased; however, this increase was followed by a rapid decline in the response. ET-1 (100 nmol/L) but not ET-3 or STXS6c significantly reduced the heart rate when it was raised by isoproterenol (ISO, 300 nmol/L) either in the absence or presence of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Correspondingly, ET-1 significantly reduced the ISO-induced elevation of cAMP accumulation (19.1 +/- 1.7 pmol/mg protein [n = 8] and 12.6 +/- 1.2 pmol/mg protein [n = 7] in the absence and presence of ET-1, respectively; P < .01), which was also observed even in the presence of IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological analysis of the negative chronotropic effect of endothelin-1 in rabbit sinoatrial node cells

1 Electrophysiological effects of endothelin‐1 (ET‐1) were studied in rabbit sinoatrial node (SAN) using conventional microelectrode and whole‐cell voltage and current recordings. 2 In rabbit SAN, RT‐PCR detected ETA endothelin receptor mRNA. ET−1 (100 nm) increased the cycle length of action potentials (APs) from 305 ± 15 to 388 ± 25 ms; this effect was antagonised by the ETA receptor‐selective antagonist BQ−123 (1 μm). ET‐1 increased AP duration (APD50) by 22 %, depolarised the maximum diastolic potential (MDP) from −59 ± 1 to −53 ± 2 mV, shifted the take‐off potential by +5 mV and decreased the pacemaker potential (PMP) slope by 15 %. Under exactly the same experimental conditions, ET‐1 caused a positive chronotropic effect in guinea‐pig SAN with a decrease of 13 % in APD50, a shift of −4 mV in the take‐off potential and an increase of 8 % in the PMP slope. 3 Rabbit SAN exhibited two major cell types, distinguished both by their appearances and by their electrophysiological responses to ET‐1. Whereas the spontaneous pacing rate and the PMP slope were similarly decreased by ET‐1 (10 nm) in both cell types, ET‐1 depolarised MDP from −67 ± 1 to −62 ± 4 mV in spindle‐shaped cells but hyperpolarised it from −73 ± 1 to −81 ± 3 mV in rod‐shaped cells. ET‐1 decreased APD50 by 8 and 52 % and shifted the take‐off potential by +5 and −9 mV in spindle‐ and rod‐shaped cells, respectively. 4 ET‐1 decreased the high‐threshold calcium current (ICaL) by about 50 % in both cell types, without affecting its voltage dependence, and decreased the delayed rectifier K+ current (IK) with significant shifts (of +4.7 and +14.0 mV in spindle‐ and rod‐shaped cells, respectively) in its voltage dependence. It was exclusively in rod‐shaped cells that ET‐1 activated a sizeable amount of time‐independent inward‐rectifying current. 5 The hyperpolarisation‐activated current (If), observed exclusively in spindle‐shaped cells, was significantly increased by ET‐1 at membrane potentials between −74.7 and −84.7 mV whereas it was significantly decreased at more negative potentials. ET−1 significantly decreased the slope of the current‐voltage (I–V) relation of the If tail without changing its half‐maximum voltage. 6 The overall negative chronotropic influence of ET‐1 on the whole rabbit SAN is interpreted as resulting from the integration of its different actions on spindle‐ and rod‐shaped SAN cells through electrotonic interaction.

Desensitization of ETA endothelin receptor-mediated negative chronotropic response in right atria–species difference and intracellular mechanisms

1 Desensitization of ETA endothelin receptor (ETAR) was compared between the rat and guinea‐pig with regard to negative chronotropic response (NC) in the right atria (RA). 2 ET‐1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 μM) in both species, showing that ETAR and ETB endothelin receptor (ETBR) mediate NC and PC, respectively. 3 Repetitive applications of ET‐1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET‐1 produced virtually no response in the rat RA, whereas they produced BQ123‐sensitive NCs repetitively in guinea‐pig RA, exhibiting marked species difference in desensitization of ETAR‐mediated NC. 4 Pretreatment with staurosporine (100 nM) prevented desensitization of ETAR in the rat RA altogether. However, phorbol 12‐myristate 13‐acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ETAR. 5 Partial amino acid sequencing of ETARs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea‐pigs, rats, rabbits, bovines and humans. 6 In guinea pig RA, pretreatment with okadaic acid (1 μg ml−1) and PMA did not facilitate desensitization of ETAR whereas these agents successfully desensitized ETAR during combined stimulation of β‐adrenoceptor and ETAR by isoproterenol (300 nM) and ET‐1 (100 nM). 7 These results suggest that species differences in desensitization of ETAR are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ETAR appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by β‐adrenoceptor activation.

Organ culture of young rat vas deferens as an in vitro model for the study of denervation supersensitivity

To investigate whether organ culture is a suitable in vitro model for studying the mechanisms of denervation-induced supersensitivity, we cultured 1-week-old rat vas deferens for 3 days with a basic applied tension of 20 mg. Cultured muscles showed supersensitivity to norepinephrine and methacholine with concomitant elevation of the maximal response. To compare these changes with those caused by denervation, young rats were chemically denervated by injecting 6-hydroxydopamine, and consequent sensitivity changes were investigated. Denervated muscles showed non-specific supersensitivity to norepinephrine and methacholine but the maximal response did not increase. When these denervated muscles were organ-cultured, they showed no or only a slight increase in sensitivity to norepinephrine and methacholine, but the maximal response increased greatly. These observations led to the suggestion that the increase in sensitivity may be mediated through the same mechanisms as those for denervation supersensitivity. The elevation of the maximal response was suggested to be produced by the improvement of cell-to-cell conduction as well as some other unknown factor(s) probably specific to organ culture. Thus, it was concluded that organ-cultured 1-week-old rat vas deferens is a useful model to study the mechanisms of denervation supersensitivity.

Histamine H2 Receptor Antagonism by T-593: Studies on cAMP Generation in Hepa Cells Expressing Histamine H2 Receptor

Histamine H2 receptor antagonism by T-593 was investigated in Hepa cells expressing canine histamine H2 receptors. T-593 inhibited generation of cAMP in Hepa cells stimulated by 10–5 mol/l histamine with an IC50 value of 2.3 × 10–6 mol/l, (S)-(–)-T-593, one of the enantiomers comprising racemic T-593, inhibited cAMP generation with an IC50 value of 6.1 × 10–7 mol/l. On the other hand, the other enantiomer (R)-(+)-T-593 exhibited only a negligible effect. Incubation of the cell with (S)-(–)-T-593 for 60 min depressed the maximal response of the concentration-response curve of histamine with a nonparallel rightward shift. The slope of a Schild plot was 1.27. In contrast, (S)-(–)-T-593 caused a parallel rightward shift of the curve, with a Schild plot slope that did not significantly differ from unity, by treating the cells for 15 min. The H2 receptor-blocking action of (S)-(–)-T-593 remained almost unaffected after washing out the drug, whereas the effect of ranitidine was reversible after washing. These results suggest that T-593 possesses a time-dependent unsurmountable antagonistic action against histamine H2 receptor. T-593 may interact with the histamine H2 receptor molecule in a slowly associable and dissociable manner.