Teruyuki Yanagisawa

PHARMACOLOGICAL PROFILE OF A NEW CORONARY VASODILATOR DRUG, 2‐NICOTINAMIDOETHYL NITRATE (SG‐75)

1. In anaesthetized, open‐chest dogs, 2‐nicotinamidoethyl nitrate (SG‐75) administered intravenously (0.3–1 mg/kg) or intraduodenally (3 mg/kg) produced decreases in systemic blood pressure, coronary resistance, heart rate and an increase in coronary sinus outflow, but virtually no change in myocardial oxygen consumption and atrioventricular conduction. The effects of SG‐75 administered intraduodenally emerged within a few minutes after dosing and lasted over about 1 h.

Cytoplasmic calcium and the relaxation of canine coronary arterial smooth muscle produced by cromakalim, pinacidil and nicorandil

1 In order to investigate the vasodilator mechanisms of the K+ channel openers, cromakalim, pinacidil and nicorandil, we measured changes in cytoplasmic Ca2+ concentration ([Ca2+]i) simultaneously with force by a microfluorimetric method using fura‐2, a calcium indicator, in canine coronary arterial smooth muscle cells. 2 The three K+ channel openers all produced a concentration‐dependent reduction of [Ca2+]j in 5 and 30mM KCl physiological salt solution (PSS) but failed to affect [Ca2+]i in 45 and 90 mM KC1‐PSS. 3 Cromakalim only partly inhibited (‐45%) the 30 mM KCl‐induced contractures, whereas pinacidil and nicorandil nearly abolished contractions produced by 45 mM, 90 mM and 30 mM KC1‐PSS. 4 Tetrabutylammonium (TBA), a nonselective K+ channel blocker, or glibenclamide, a supposed adenosine 5′‐triphosphate (ATP)‐sensitive K+ channel blocker, abolished the reduction of [Ca2+]i caused by the three K+ channel openers and the relaxant effect of cromakalim, whereas they only slightly attenuated the relaxant effects of pinacidil and nicorandil. 5 The increase in [Ca2+]i produced by 45 or 90 mM KCl‐PSS in the presence of pinacidil or nicorandil was abolished by 10−5m verapamil, indicating that the increase in [Ca2+]i was caused by the influx of extracellular Ca2+ and that pinacidil and nicorandil did not affect the voltage‐dependent Ca2+ channel directly. 6 The [Ca2+]i‐force relationship in the presence of cromakalim was not distinguishable from that of control. 7 The [Ca2+]i‐force curve was shifted to the right by pinacidil and nicorandil. 8 These results show that cromakalim is a more specific K+ channel opener than pinacidil and nicorandil, and that vasodilatation produced by cromakalim in this study is predominantly a result of a reduction of [Ca2+]i due to the closure of voltage‐dependent Ca2+ channels by hyperpolarization. In contrast, additional mechanisms are involved in the vasodilator actions of pinacidil and nicorandil. One of these is related to a reduction in the sensitivity of contractile proteins to Ca2+. The latter mechanism of nicorandil is akin to that of nitroglycerin. K+ channels opened by these K+ channel openers may be ATP‐sensitive ones which are blocked by glibenclamide.

Nitroglycerin relaxes canine coronary arterial smooth muscle without reducing intracellular Ca2+ concentrations measured with fura‐2

1 Changes in cytoplasmic Ca2+ concentration ([Ca2+]i) were measured simultaneously with force by a microfluorometric method using a calcium indicator, fura‐2, in canine coronary arterial smooth muscle cells. 2 Depolarization by high (30–90 mm) KCl‐physiological salt solution (PSS) produced concentration‐dependent increases in force and [Ca2+]i. 3 The KCl‐induced increase in [Ca2+]i was abolished by Ca2+‐free conditions and almost abolished by verapamil 10−5m, suggesting that it was entirely due to the increased Ca2+ influx through voltage‐dependent Ca2+ channels. 4 The [Ca2+]i‐force relationship in the presence of verapamil was not distinguishable from that of control. 5 Nitroglycerin produced a concentration‐dependent, reversible contraction of the coronary artery that had been contracted by high KC1‐PSS, without reduction of the increased [Ca2+]i. 6 The KCl‐induced increase in [Ca2+]i was not affected by nitroglycerin and in the presence of nitroglycerin it was abolished by 10−5m verapamil suggesting that it was caused by the influx of extracellular Ca2+. 7 The [Ca2+]i‐force curve was shifted to the right by nitroglycerin. 8 It is likely that nitroglycerin relaxes the coronary arterial smooth muscle by reducing the amount of myosin light chain phosphorylation even in the presence of raised [Ca2+]i produced by increased Ca2+ influx following depolarization.

Coronary Vasodilator and Cardiac Effects of Optical Isomers of Verapamil in the Dog

Summary Coronary vasodilator and cardiac effects of (-) and (+) verapamil were investigated in two kinds of canine heart preparations. When administered intravenously to anesthetized open-chest dogs, (-) verapamil was only 1.5–2 times as potent as (+) verapamil in increasing coronary sinus outflow and in decreasing mean arterial pressure and coronary resistance. However, (-) verapamil was about 5 times as potent in producing a negative chronotropic effect, about 10 times as potent in producing a negative dromotropic effect, and about 15 times as potent in decreasing myocardial oxygen consumption as (+) verapamil. When injected into the anterior septal artery in the isolated, blood-perfused papillary muscle preparation of the dog, (-) verapamil was about 15 times as potent as (+) verapamil in producing a negative inotropic effect. However, in increasing blood flow through the anterior septal artery (-) verapamil was only about 2.5 times as potent as (+) verapamil. These results indicate that in equieffective doses in producing coronary vasodilatation (+) verapamil is far less cardiodepressant than the (-) isomer.

MECHANISMS UNDERLYING THE CARDIOVASCULAR ACTION OF A NEW DIHYDROPYRIDINE VASODILATOR, YC‐93

1.The mechanisms underlying the cardiovascular action of YC‐93, a new dihydropyridine vasodilator with cyclic AMP phosphodiesterase inhibitory activity, was investigated by comparing its effects wth those of papaverine in various isolated, blood‐perfused heart preparations of the dog.

BRL 38227 (levcromakalim)-induced hyperpolarization reduces the sensitivity to Ca2+ of contractile elements in canine coronary artery

Potassium (K+) channel openers decrease intracellular free Ca2+ concentrations ([Ca2+]i by hyperpolarizing the membrane and deactivating the Ca(2+)-channels. To examine whether the hyperpolarization produced by K(+)-channel openers has other effects on the mechanical activity of vascular smooth muscle, we investigated the effects of levcromakalim (BRL 38227) on membrane potential, [Ca2+]i, as measured with fura-2, and force of contraction induced by 30 mmol/l KCl-physiological salt solution (PSS), in canine coronary arteries. BRL 38227 hyperpolarized the membrane and reduced increases in [Ca2+]i and in contractile force induced by 30 mmol/l KCl-PSS. The [Ca2+]i-contractile force curve, determined in the presence of BRL 38227, was located to the right of the control curve determined by decreasing extracellular Ca2+ concentrations ([Ca2+]o) in 30 mmol/l KCl-PSS. The [Ca2+]i-contractile force curve, determined by decreasing extracellular K+ concentrations ([K+]o), was also located to the right of that determined by decreasing [Ca2+]o in 30 mmol/l KCl-PSS. The effect of BRL 38227, a reduction in the Ca(2+)-sensitivity of contractile elements, was antagonized by the ATP-sensitive K(+)-channel blocker, glibenclamide (10(-6) or 10(-5) mol/l). These results suggest that the membrane hyperpolarization induced by BRL 38227, or the repolarization caused by reducing [K+]o, decreases the Ca(2+)-sensitivity of contractile elements of vascular smooth muscle.

Effect of 2-nicotinamidethyl nitrate (SG-75) on the membrane potential of left atrial muscle fibres of the dog

Summary1.2-Nicotinamidethyl nitrate (SG-75) (10−6–10−4 mol/l) and methacholine (MeCh) (10−7–10−5 mol/l) hyperpolarized the resting membrane potential, and produced increases in amplitude and maximum rate of rise and a decrease in duration of the action potential of left atrial muscle fibres of the dog.2.Changes in membrane potentials produced by SG-75 were not modified by atropine at a concentration (3×10−7 mol/l) which greatly antagonized those induced by MeCh.3.The decrease in resting membrane potential for a 10-fold increase in extracellular K+ concentration ([K+]0) was greater in the presence than in the absence of SG-75 or MeCh.4.SG-75- and MeCh-induced changes in membrane potentials were not affected by the replacement of NaCl by Na isethionate in perfusion fluid.5.SG-75- and MeCh-induced changes in membrane potentials were not modified by ouabain (10−6 mol/l).6.The “slow response” of left atrial muscle fibres was obtained by increasing the [K+]0 to 27 mM in the presence of isoprenaline (10−6 mol/l). SG-75 and MeCh primarily produced a decrease in duration of the “slow response”. At higher concentrations SG-75 and MeCh hyperpolarized the resting membrane potential and decreased the amplitude and maximum rate of rise of the “slow response”. Unlike SG-75 and MeCh, verapamil primarily decreased the amplitude and maximum rate of rise of the “slow response” without changes in duration of the “slow response” and resting membrane potential.7.The above results suggest that the mechanism of action of SG-75 on left atrial muscle fibres may be an increase in potassium conductance of the membrane without mediation through muscarinic receptors.

IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for Arf6 that interacts with PSD-95 at postsynaptic density of excitatory synapses

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-d-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAa receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6.

CHARACTERIZATION OF ADRENOCEPTORS MEDIATING POSITIVE INOTROPIC RESPONSES IN THE VENTRICULAR MYOCARDIUM OF THE DOG

1 The pharmacological characteristics of adrenoceptors mediating the positive inotropic action in the dog heart were assessed by the use of blood‐perfused papillary muscles and isolated strips of ventricular myocardium. 2 On the blood‐perfused papillary muscle driven at 2 Hz and in sinus node preparations, phenylephrine induced positive inotropic and chronotropic responses in the same dose range and was much less potent than isoprenaline. The dose‐response curve for the chronotropic action of phenylephrine was parallel to that of isoprenaline, whilst the dose‐response curve for the inotropic action of phenylephrine was less steep than that of isoprenaline. 3 The infusion of pindolol, a β‐adrenoceptor blocking agent, at a rate of 1 μg/min, shifted the isoprenaline dose‐response curves to the right, and to the same extent, in both papillary muscle and sinus node preparations. In contrast to isoprenaline, the antagonism of phenylephrine by pindolol was noncompetitive. Phentolamine did not affect the positive inotropic and chronotropic actions of phenylephrine. 4 On isolated ventricular strips α‐adrenoceptor blockade by 10−6m phentolamine did not affect dose‐response curves to phenylephrine or dopamine. Pindolol shifted the dopamine dose‐response curves to the right in a competitive manner and those of phenylephrine in a noncompetitive manner. 5 On ventricular strips from reserpine‐pretreated dogs phenylephrine and tyramine dose‐response curves were shifted markedly to the right and downwards. Desipramine (10−5m) which enhanced the action of noradrenaline considerably reduced the myocardial responses of phenylephrine. 6 Papaverine (10−5m) decreased the threshold concentration of phenylephrine required to stimulate the myocardium and shifted phenylephrine dose‐response curves to the left. 7 Raising the temperature from 32°C to 37°C shifted phenylephrine dose‐response curves to the right; when the temperature was raised from 37°C to 42°C the affinity of the drug was not changed. 8 Other α‐adrenoceptor stimulants, methoxamine and clonidine, decreased the active tension of ventricular strips. The responses to noradrenaline and adrenaline (in the presence of pindolol; 3 × 10−8m) were not affected by phentolamine (10−6m). 9 The results indicate that adrenoceptors mediating positive inotropic responses in the dog ventricle are of the /J‐type and that post‐synaptic α‐adrenoceptors are not involved. Phenylephrine acts mainly by releasing noradrenaline from adrenergic nerve endings and partly by a weak direct action on β‐adrenoceptors.

Modified Cardiovascular L-type Channels in Mice Lacking the Voltage-dependent Ca2+ Channel β3 Subunit

The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. Of the four β subunits reported to date, the β3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the β3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in β3-null mice. Western blot analysis indicated a significant reduction in expression of the α1 subunit in the plasma membrane of β3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in β3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, β3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the β3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the β3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.