Hitonobu Tomoike

Low‐voltage‐activated calcium current in rat aorta smooth muscle cells in primary culture.

1. Electrical and pharmacological properties of the low‐voltage‐activated Ca2+ current (ICa, LVA) in rat aorta smooth muscle cells (SMC) in primary culture were examined, particularly in comparison with the high‐voltage‐activated Ca2+ current (ICa, HVA). Both types of Ca2+ currents were recorded in external solution containing 20 mM‐Ca2+, using the whole‐cell voltage‐clamp technique. 2. ICa, LVA was evoked by step depolarizations to potentials more positive than ‐60 mV from a holding potential of ‐100 mV, and reached a peak in the current‐voltage (I‐V) relationship around ‐30 mV. ICa, HVA was activated at ‐20 mV, and reached a peak at +20 mV. 3. The intracellular dialysis of 5 mM‐F‐ irreversibly suppressed ICa, HVA, with time, while it has little effect on the ICa, LVA. The ICa, LVA could be separated from the ICa, HVA by either selecting the holding and test potential levels or by perfusing intracellularly with F‐. 4. The ratio of peak amplitude of Ba2+, Sr2+ and Ca2+ currents in the respective I‐V relationship was 1.6:1.2:1.0 for high‐voltage‐activated Ca2+ channels and was 1.0:1.4:1.0 for low‐voltage‐activated ones. 5. The inactivation phase of ICa, HVA was fitted by a sum of double‐exponential functions, the time constants of which were larger when the current was carried by Ba2+ than by Ca2+. The inactivation time course of ICa, LVA was fitted by a single‐exponential function, and the time constant was practically the same when the current was carried by Ba2+ or by Ca2+. Activation and inactivation processes of ICa, LVA were potential‐dependent. 6. The steady‐state inactivation curve of ICa, LVA was fitted by the Boltzmann equation, having a mid‐potential of ‐80 mV and a slope factor of 5.0. The recovery time course from steady‐state inactivation was fitted by a sum of two exponential functions. The time constants of the faster phase were 230 and 380 ms, and those of slower phase were 2.8 and 1.8 s at the repolarization potentials of ‐120 and ‐100 mV, respectively. 7. The amplitude of ICa, LVA depended on the external Ca2+ concentration ([Ca2+]o), approaching saturation at 95 mM [Ca2+]o. 8. Various polyvalent cations blocked both types of Ca2+ current reversibly in the order (IC50 in M): La3+ (8 x 10(‐8)) greater than Cd2+ (6 x 10(‐6)) greater than Ni2+ (1 x 10(‐5)) greater than Zn2+ (2 x 10(‐5)) for ICa, HVA, and La3+ (6 x 10(‐7)) greater than Zn2+ (3 x 10(‐5)) greater than Cd2+ (4 x 10(‐4)) greater than Ni2+ (6 x 10(‐4)) for ICa, LVA.(ABSTRACT TRUNCATED AT 400 WORDS)

Coronary artery spasm induced in miniature swine: Angiographic evidence and relation to coronary atherosclerosis

In a swine model of coronary artery spasm, the pathogenetic role of coronary atherosclerosis was examined. Following endothelial balloon denudation of the left circumflex coronary artery (LCX), male miniature swine were fed a laboratory chow diet containing 2% cholesterol. Although there was no difference in the extent of coronary vasoconstrictive response to histamine, serotonin, and ergonovine between the left anterior descending coronary artery (LAD) and the LCX before the denudation, a constrictive response was significantly augmented along the denuded portion of the LCX 1 and 3 months after the denudation. Augmented vasoconstrictive responses to phenylephrine were never evidenced. Histamine was the most potent vasoactive agent, and coronary artery spasm was provoked repeatedly by intracoronary or intravenous administration of histamine in the presence or absence of cimetidine. The spasm was provoked only in the denuded portion of the LCX, the same area which was angiographically normal before the occurrence of the spasm. Histologically, atherosclerotic changes were predominant along the denuded portion of the LCX. Topologic correlation was suggested between the site of the spasm and the site of coronary atherosclerosis. It is concluded that in this swine model of coronary artery spasm, atherosclerotic changes may be an important causative factor, in terms of an activation of multiple receptor-operated calcium channels in the coronary artery.

Role of ATP-sensitive K+ channel on ECG ST segment elevation during a bout of myocardial ischemia. A study on epicardial mapping in dogs.

BackgroundATP-sensitive K+ channels are activated when the myocardium becomes ischemic. However, the role of the ATP-sensitive K+ current in the emergence of ECG ST changes during ischemia remained unclarified. Methods and ResultsThe left anterior descending coronary artery (LAD) was cannulated and perfused with arterial blood from the carotid artery through a bypass tube in 8 anesthetized, open-chest dogs. An array of 60 unipolar electrodes mounted on a sock was used to record epicardial electrograms of the whole heart. Pinacidil (10 μg kg-1 min-1), an ATP-sensitive K+ channel opener, was infused into the bypass tube for 2 minutes, and the electrograms were recorded before and after the infusion. The elevation of the ST segment and the increase of QRST area were observed spatially over the LAD-perfused region. At the electrode showing the largest ST segment elevation, the activation recovery interval, an index of action potential duration, was shortened from 202±9 to 111±18 milliseconds (P<.001). These electrographic changes were similar to those noted in 2-minute coronary occlusion (n=8). The extent of ST segment elevation during coronary occlusion was attenuated after the intravenous pretreatment with glibenclamide (0.3 mg/kg), a blocker of the KATP channel (n=5) ConclusionsThe findings of this study suggest that the activation of ATP-sensitive K+ channels during a bout of acute myocardial ischemia plays an important role in the emergence of ECG ST elevation.

Attenuation of endothelium-related relaxation and enhanced responsiveness of vascular smooth muscle to histamine in spastic coronary arterial segments from miniature pigs.

Mechanism of coronary spasm was examined regarding endothelium-related relaxation and contraction produced by smooth muscle cells of spastic vessels isolated from Göttingen miniature pigs. In these pigs, coronary artery spasm was documented angiographically in vivo three months after endothelial denudation, and spastic and control segments of the coronary artery were suspended in organ chambers at their optimal length for generating tension. Applications of KC1 (118 mM), acetylcholine(10-9 to 10-4 M), and PGF2α (10-8 to 3 ± 10-5 M) produced similar tension, at the respective doses, in both the spastic and control coronary arteries. During increasing concentrations of histamine (l0-8 to 3 ± 10-4 M; n= 14) and serotonin (10-9 to 10-5 M; n= 13), the maximum tension of the spastic vessel was 136 ± 6 and 97 ± 4%, respectively, of the tension produced by 118 mM KC1. That is significantly larger than seen in the control vessels: 98 ± 4 and 74 ± 4%, respectively. The ED50 to histamine and serotonin was also significantly less in the spastic vessels. After mechanical removal of the endothelium, the tension generated during the cumulative administration of histamine (n = 8) but not serotonin (n = 8) was larger in the spastic than the control vessels, thereby suggesting the presence of augmented responses of the smooth muscle to histamine in the spastic vessels. The increase in maximum tension after mechanical denudation was greater in the control than the spastic vessels in cases of histamine and serotonin. Endothelium-dependent relaxations due to serotonin (n = 5) and A23187 (n = 5) were tested under conditions of precontraction by PGF2α (10-5 M). The maximum relaxation induced by serotonin was 31 ± 4 and 67 ± 8% (p<0.01) in the spastic and the control vessel, respectively. A23187 relaxed completely the spastic vessel, to a similar extent as seen in the control vessels; however, the ED50 of relaxation evoked by A23187 was 2.7 ± 0.6 ± 10-8 and 6.3 ± 1.4 ± 10-9 M (p<0.01) in the spastic and the control vessels, respectively. Thus, both increased responsiveness of smooth muscle to histamine and impairment of endothelium-dependent relaxation to serotonin and to histamine may play an important role in the hypercontraction observed in coronary artery spasm in these miniature pigs.

Endothelium determines flow-dependent dilation of the epicardial coronary artery in dogs

The mechanisms of epicardial coronary artery dilation after reactive hyperemia were studied in instrumented conscious dogs. A pair of ultrasonic crystals, an electromagnetic flow probe and a cuff occluder were placed on the left circumflex coronary artery in 12 mongrel dogs under sterile conditions. Reactive hyperemia after 20 s of coronary occlusion dilated the epicardial coronary artery by 120 +/- 14 micron (3.8 +/- 0.6%, p less than 0.01) from 3.167 +/- 0.345 mm. This reactive dilation was abolished by flow-limiting coronary stenosis. However, vasodilation after nitroglycerin was 168 +/- 26 micron (5.1 +/- 0.5%) and 162 +/- 27 micron (4.9 +/- 0.6%), respectively, before and after flow limitation. After removal of the endothelium by a balloon catheter, dilation of the epicardial coronary artery after reactive hyperemia was markedly attenuated to 7 +/- 4 micron (p less than 0.01 versus before denudation), despite the presence of a similar degree of reactive hyperemia. The extent of coronary dilation after nitroglycerin was unchanged before and after de-endothelialization. Thus, the endothelium contributed to reactive dilation but not to the nitroglycerin-induced dilation. The negative feedback control of coronary diameter to changes in flow velocity may relate to the regulation of coronary artery tone.

Serum Levels of Soluble Form of Fas Molecule in Patients with Congestive Heart Failure

Compared with soluble Fas molecule (sFas, an inhibitor of Fas-mediated apoptosis) in normal volunteers, the serum level of sFas significantly increased by 41% in New York Heart Association (NYHA) class III (p <0.05) and by 97% in NYHA class IV patients with congestive heart failure (p <0.001). Furthermore, sFas showed correlations with soluble forms of TNF receptor-p55 (RI) and -p75 (RII) (r = 0.68 and r = 0.56) which inhibit activities of TNF alpha.

Duration of ischemia is vital for collateral development: repeated brief coronary artery occlusions in conscious dogs.

The effects of two types of repeated transient coronary artery occlusions on collateral development were examined in chronically instrumented, conscious dogs. A 2-minute coronary occlusion at 32-minute intervals (group 1, n=ll) or a 15-second occlusion at 4-minute intervals (group 2, n=7) were repeated day and night without interruption. In both groups, the total duration of coronary occlusions each day was the same (90 minutes). Before and after repetitive occlusions of either group, effects of transient 2-minute coronary occlusion on regional segment shortening in the ischemic area were examined to assess the functional state of the collateral vessels. In group 1, systolic segment shortening in the area rendered ischemic was reduced to -97.8±17.7percent; of the preocclusive control value during 2 minutes of coronary occlusion. After 125-478 repetitive occlusions (3-11 days), the degree of hypokinesia during the 2-minute occlusion was significantly improved to -0.6±4.6percent; of the preocclusive value (p<0.001 vs. before the repetition). In group 2, it remained unchanged even after 3,500-5,450 repetitive occlusions (11-16 days): -111.8±8.2percent; before and -111.4±13.8percent; after the repetition of 15-second occlusions (NS). The ratio of peripheral coronary arterial pressure to aortic pressure during transient coronary occlusion, measured by selective catheterization, was significantly higher in group 1 than in group 2 (64.4±5.3percent; vs. 20.7±1.3percent;, p<0.001). These findings suggest that myocardial ischemia of 2 minutes but not 15 seconds is vital to provide effective stimuli for angiogenesis.

Role of coronary artery spasm in progression of organic coronary stenosis and acute myocardial infarction in a swine model. Importance of mode of onset and duration of coronary artery spasm.

BackgroundCoronary spasm may play an important role in progression of organic coronary stenosis and myocardial infarction, but the mechanisms responsible for these complications are not known. This study aimed to examine whether the mode of onset and the duration of coronary spasm influenced progression of organic coronary stenosis and acute myocardial infarction in a swine model of coronary spasm. Methods and ResultsGottingen miniature pigs were subjected to cholesterol feeding, balloon-induced coronary arterial denudation, and x-ray irradiation. Five months later, coronary spasm was induced by intracoronary injection of serotonin. In 10 pigs, coronary spasm was provoked abruptly and maintained for 25 minutes by five repeated intracoronary injections of serotonin (10 μg/kg) every 5 minutes (group A, abrupt onset and short duration). In group B, coronary spasm was provoked gradually by intracoronary injections of serotonin at graded doses of 0.1, 0.3, and 0.6 μg/kg every 5 minutes and was then maintained for 25 minutes in four pigs (group BR, gradual onset and short duration) and for 120 minutes in six pigs (group B2, gradual onset and long duration) by repeated intracoronary injections of serotonin (10 μg/kg) every 5 minutes. Intramural hemorrhage was noted histologically at the spastic site more frequently in group A with abrupt onset (nine of 10 pigs) than in group B with gradual onset (two of 10 pigs) (p<0.01). Progression of organic coronary stenosis due to intramural hemorrhage was noted in seven pigs (six pigs in group A and one pig in group B), including three cases of total coronary occlusion. Evidence for the evolution of acute myocardial infarction (serial ECG findings, left ventriculograms, and histological findings) was noted in one pig (7%) of group A or B1 with short duration and in five of six pigs (83%) in group B2 with long duration (p<0.01 versus group A and Bi). ConclusionThese results indicate that 1) intramural hemorrhage was frequently induced by coronary spasm of abrupt but not of gradual onset, 2) intramural hemorrhage resulted in acute progression of coronary stenosis and sometimes resulted in persistent total coronary occlusion leading to acute myocardial infarction, and 3) prolonged coronary spasm resulted in acute myocardial infarction without progression of organic coronary stenosis.

Serotonin-induced coronary spasm in a swine model. A minor role of defective endothelium-derived relaxing factor.

BackgroundCoronary spasm may be caused by endothelial dysfunction, vascular smooth muscle hyperreactivity, or both. We aimed to determine the relative role of endothelial dysfunction and vascular smooth muscle hyperreactivity in the pathogenesis of coronary artery spasm in the swine model in vivo. Methods and ResultsIn G6ttingen miniature pigs given a high cholesterol diet, a segment of the left coronary artery was denuded and irradiated with x-ray (total, 30 Gy). Three months after endothelial denudation and irradiation, vasomotor responses of the denuded and control sites to agonists were assessed by quantitative arteriography. Serotonin (10 μg/kg) provoked coronary spasm at the denuded site (diameter reduction, 79±6%) associated with ST elevation but not at the nondenuded control site (21±6%). Intracoronary infusion of N

Intramural hemorrhage and endothelial changes in atherosclerotic coronary artery after repetitive episodes of spasm in x-ray-irradiated hypercholesterolemic pigs.

-nitro-L-arginine methyl ester (LNNA, an inhibitor of endothelium- derived nitric oxide) of 1 and 3 mg/kg potentiated constriction evoked with serotonin (1, 3, 10 μg/kg) at the control site but did not alter it at the denuded site. However, serotonin-induced constriction after LNNA was still less at the control site (31±3%) than at the denuded site (80±5%). Endotheliumdependent vasodilation with substance P (0.1, 1, 10 ng/kg), which was inhibited by LNNA, was less (P<.01) at the denuded site than at the control site, whereas vasodilation with the nitrovasodilator SIN-i (0.1, 1, 10 ng/kg) was comparable between the two sites. Histological study revealed regenerated endothelial cells and intimal thickening at the denuded site ConclusionsThe results suggest that the denuded segment of the coronary artery with regenerated endothelium was associated with defective endothelium-dependent vasodilation mediated by nitric oxide and vascular smooth muscle hyperreactivity to serotonin. However, provocation of coronary spasm with serotonin resulted primarily from vascular smooth muscle hyperreactivity but not by defective nitric oxide production in this swine model.