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Biomedical subjects

H Kanaide

Publications and source records attributed to H Kanaide.

At least 127 records · Page 7Linked to original sources

Endothelin induces the Ca(2+)-transient in endothelial cells in situ.

Using front-surface fluorometry of fura-2 and valvular strips of the pig aorta, we recorded changes in the cytosolic Ca2+ concentration, [Ca2+]i, of endothelial cells in situ, quantitatively, and investigated the effects of endothelin-1 and -3 on these endothelial cells. Both endothelin-1 and -3 elevated [Ca2+]i of a peak (the first phase) and sustained type. This first phase is considered to be due to a release of Ca2+ from intracellular storage sites. The sustained phase depended on extracellular Ca2+ and is considered to be due to an influx of Ca2+ through the plasma membrane. At equimolar concentrations, the peak elevations of [Ca2+]i induced by endothelin-1 were much higher than those induced by endothelin-3. We suggest that, in endothelial cells in situ, endothelin-1 mobilizes stored Ca2+ and may activate Ca(2+)-sensitive pathways, including the release of prostacyclin and endothelium-derived relaxing factors, more potently than does endothelin-3.

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Dextran sulfate inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned and cultured smooth muscle cells.

Dextran sulfate inhibited the inositol 1,4,5-trisphosphate (IP3)-induced decrease in 45Ca2+ content, in a dose-dependent manner, in saponin-skinned and primary cultured smooth muscle cells from the rat aorta. The maximum inhibition was observed at 3-10 mg/ml, and the IC50 was about 173 micrograms/ml. Dextran sulfate also inhibited the IP3-induced increase in 45Ca2+ efflux rate, but did not affect the caffeine-induced Ca2+ release. Dextran sulfate inhibited the specific binding of [4,5-32P]IP3 to the skinned cells, thereby indicating that it may have an effect on the IP3 receptor. Dextran (without the sulfates) had no inhibitory effect on either the IP3-induced Ca2+ release or on the specific binding of [4,5-32P]IP3. Thus, sulfate groups on the molecule of dextran sulfate may play an important role in the inhibition of the IP3-induced Ca2+ release from intracellular stores.

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Temporal changes in the calcium-force relation during histamine-induced contractions of strips of the coronary artery of the pig.

1. We examined temporal changes in the relationship between cytosolic calcium concentrations ([Ca2+]i) and developed tension during histamine-induced contractions of strips of the coronary artery of the pig, by making use of simultaneous measurements of fura-2 fluorescence and force. 2. The relationship between [Ca2+]i and developed tension observed with cumulative applications of extracellular Ca2+ ([Ca2+]o), ranging from 0 mM to 10 mM, during 118 mM K(+)-depolarization was similar to that observed in chemically skinned strips of the porcine coronary artery, as noted by other investigators. [Ca2+]i at 0 mM [Ca2+]o, at 50% of maximum, and at maximum tension development were 76 nM, 424 nM, and 3050 nM, respectively. 3. Cumulative applications of histamine induced dose-dependent increases in [Ca2+]i and tension and the extent of tension for a given change in [Ca2+]i increased, i.e. greater effectiveness of [Ca2+]i-tension relationship, than seen with K(+)-depolarization. 4. When histamine 10(-5) M was applied, [Ca2+]i abruptly rose and reached the first peak within several seconds. After a slight dip at 30 s, [Ca2+]i reached a second peak at 3 min, and then gradually declined. On the other hand, tension developed rapidly reached a maximum at 4 min, then gradually declined. The relation between [Ca2+]i and tension in the early, rising phase of contraction was similar to that obtained during depolarization. At the time of maximum tension development, the relation was greater than that observed during depolarization, which persisted in the phase of declining tension. 5. To examine the role of protein kinase C in the increased effectiveness of [Ca2+]i-tension relation of histamine-induced contractions, we used 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7), as an inhibitor. Exposure of strips to H-7, i0-' M, reduced the effectiveness of [Ca2+]i-tension relation toward the level observed during depolarization, while it had no effect on the [Ca2 ]i-tension relation in the early phase of contraction either in the presence or absence of extracellular Ca2 + 6. In intact smooth muscle of the coronary artery of the pig, the relationship between [Ca2+]i and developed tension varies during contraction. Histamine, a receptor-mediated stimulus, induced a greater tension for a given change in [Ca2+]i during the steady state of contraction than did depolarization. The greater effectiveness of contraction, particularly in the later phase, seems to be mediated by activation of an H-7-sensitive mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Effects of glibenclamide on cytosolic calcium concentrations and on contraction of the rabbit aorta.

1. Using fluorometry of fura-2 and rabbit aortic strips, we studied the effects of glibenclamide (GLB), a sulphonylurea anti-diabetic drug and an inhibitor of opening of K+ channels, on cytosolic calcium concentrations ([Ca2+]i) and on force development. 2. Both high K(+)-depolarization and noradrenaline (NA) increased [Ca2+]i and force, in a concentration-dependent manner, in the presence of extracellular Ca2+ (1.25 mM). However, force development in relation to [Ca2+]i ([Ca2+]i-force relationship) observed with NA was much greater than that observed with K(+)-depolarization. 3. Pretreatment with GLB (10(-6)-10(-4) M) for 10 min partially inhibited, in a concentration-dependent manner, both [Ca2+]i elevation and the force development induced by 118 mM K(+)-depolarization or NA 10(-5) M in the presence of extracellular Ca2+. The [Ca2+]i-force relationship induced by both 118 mM K+ physiological salt solutions and by NA 10(-5) M in the GLB-treated strips overlapped that obtained in the non-treated strips, thereby suggesting that GLB has no effect on the Ca2(+)-sensitivity of the intracellular contractile apparatus. Only high concentrations (10(-4) M) of GLB decreased [Ca2+]i and the force, when applied after the force induced by 118 mM K+ PSS or NA 10(-5) M reached the maximum level. 4. In the absence of extracellular Ca2+, NA induced a transient increase in [Ca2+]i and in the force and these increases were inhibited when the vascular strips were pretreated with GLB for 10 min. The [Ca2+]i-force relationship obtained in the GLB-treated strips overlapped that in the non-treated ones. 5. An ATP-sensitive K+ channel opener, cromakalim (10-5M) reduced the increased [Ca2 + ]i and force induced by 25mm K+-depolarization and NA 10-SM. Subsequent application of GLB concentrationdependently reversed this relaxant effect of cromakalim on the NA-induced contraction (IC50 = 2x 10 7 M). Complete reversal of the effect was observed with 10IsM GLB. 6. We suggest that GLB inhibits both high K+-depolarization- and NA-induced contraction of the rabbit aorta, by decreasing [Ca2+]i and with no effect on the [Ca2+]i-force relationship. However, when NA-induced contractions were inhibited by a K+-channel opener, GLB reversed this inhibitory effect by inhibiting K+-channel opening and increasing [Ca2 +].

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Cytosolic calcium transients differ between porcine coronary arterial and aortic smooth muscle cells in primary culture.

Using quin 2 microfluorometry of porcine vascular smooth muscle cells in primary culture at 25 degrees C, we investigated differences in cytosolic calcium transients between epicardial coronary artery and aorta. Both in coronary arterial and aortic smooth muscle cells, histamine induced transient and dose-dependent elevations of cytosolic calcium concentrations, with a similar time course and EC50 (coronary artery, 1.4 x 10(-7) M; aorta, 1.8 x 10(7) M). However, a transient and dose-dependent elevation of cytosolic calcium concentrations was induced by norepinephrine in aortic smooth muscle cells (EC50 = 2.5 x 10(-7) M) but not in coronary arterial smooth muscle cells. Isoproterenol, which produced no change in cytosolic calcium concentrations in aortic vascular smooth muscle cells, significantly and dose dependently decreased concentrations of calcium in coronary arterial smooth muscle cells (EC50 = 1.5 x 10(7) M). Dibutyryl cAMP decreased the concentration of cytosolic calcium both in the coronary arterial and aortic vascular smooth muscle cells with a similar time course and EC50 (coronary artery, 9.8 x 10(-6) M; aorta, 1.1 x 10(-5) M). Intracellular concentration of cAMP was increased in response to isoproterenol, as determined with radioimmunoassay of the coronary arterial smooth muscle cells but not in the aortic cells. Thus, the characteristics of receptors on the sarcolemma may play a key role in the regulation of responsiveness of vascular smooth muscle cells to various vasoactive substances. Aortic smooth muscle cells are alpha-receptor dominant, and activation results in a transient elevation of cytosolic calcium concentrations. The epicardial coronary arterial smooth muscle cells are beta-receptor dominant, and activation results in an increase in cAMP and a reduction of cytosolic calcium concentrations. These results may account for the poor contraction, or relaxation, of epicardial coronary artery induced by sympathetic stimulation and exogenously applied catecholamines.

Aminoquinolines↗

Characteristics of the histamine-sensitive calcium store in vascular smooth muscle. Comparison with norepinephrine- or caffeine-sensitive stores.

Using the microfluorometry of an intracellularly trapped calcium indicator dye, quin2, characteristics of intracellular Ca2+ store sites sensitive to histamine, norepinephrine, or caffeine were investigated using rat vascular smooth muscle cells in primary culture at 25 degrees C. With similar time courses, both histamine- and the norepinephrine-sensitive Ca2+ store sites were readily depleted in Ca2(+)-free medium and almost completely replenished by loading the cells with 1.0 mM Ca2+ solution for 3 min, while the caffeine-sensitive Ca2+ store site was little affected. In the absence of extracellular Ca2+, transient elevations of cytosolic Ca2+ repeatedly appeared in response to repetitive applications of histamine, norepinephrine, or caffeine, with progressive reductions in peak levels. Histamine released Ca2+ from the norepinephrine-sensitive store site and norepinephrine released Ca2+ from the histamine-sensitive one. On the other hand, caffeine had little effect on the histamine- and/or the norepinephrine-sensitive Ca2+ store site in Ca2(+)-free medium, and vice versa. We propose that the location and mechanisms of release of Ca2+ of the histamine-sensitive Ca2+ store site are identical with events at the norepinephrine-sensitive site, and differ from the caffeine-sensitive one, in vascular smooth muscle cells in primary culture.

Aminoquinolines↗

Heparin specifically inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned rat aortic smooth muscle cells in primary culture.

By measuring the 45Ca2+ movement in saponin-skinned primary cultured rat aortic smooth muscle cells, we examined the specificity of the inhibitory effect of heparin on the IP3-induced Ca2+ release. IP3 (100 mumol/l) markedly (98%) decreased the MgATP-dependent 45Ca2+ content in the non-mitochondrial Ca2+ stores in the presence of 1 mumol/l free Ca2+. Heparin (1-100 micrograms/ml) dose-dependently inhibited this Ca2+ release by IP3. In Ca2(+)-free solution, heparin (100 micrograms/ml) inhibited the increases in 45Ca2+ efflux rate evoked by 10 mumol/l IP3. De-N-sulfated heparin did not inhibit the IP3-induced Ca2+ release. Hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C and 2,6-disulfated D-glucosamine had no inhibitory effects on the IP3-induced Ca2+ release. High concentrations (over 1 mg/ml) of heparin inhibited the 45Ca2+ influx and decreased the 45Ca2+ content in skinned cells. These results suggest that heparin (1-100 micrograms/ml) specifically inhibits the IP3-induced increase in Ca2+ permeability of Ca2+ stores and that three sulfate groups at different locations on the molecule of heparin, two at the D-glucosamine and one at the iduronic acid, may be important for this action, in skinned vascular smooth muscle cells, in culture.

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Release of intracellularly stored Ca2+ by inositol 1,4,5-trisphosphate--an overview.

1. Inositol 1,4,5-trisphosphate (I(1,4,5)P3) releases Ca2+ from ATP-dependent Ca2+ stores in permeabilized cells and in microsomal fractions. 2. Various factors affect the amount of Ca2+ released by I(1,4,5)P3. 3. The molecular mechanism involved in the I(1,4,5)P3-induced Ca2+ release is now being investigated and I(1,4,5)P3-specific receptors and/or specific release channels are being given special attention. 4. While the I(1,4,5)P3-sensitive Ca2+ stores are presumed to locate at the endoplasmic reticulum, the relation between the I(1,4,5)P3- and the agonist-sensitive Ca2+ stores remains to be elucidated.

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Effects of diltiazem on calcium concentrations in the cytosol and on force of contractions in porcine coronary arterial strips.

1. Using front-surface fluorometry with fura-2-loaded porcine coronary arterial strips, we simultaneously measured effects of a Ca2+ antagonist, diltiazem, on cytosolic Ca2+ concentrations [( Ca2+]i) and on tension development. 2. In the presence of extracellular Ca2+ (1.25 mM), histamine concentration-dependently induced abrupt (the first component) and then sustained (the second component) elevations of [Ca2+]i. In the absence of extracellular Ca2+, histamine induced transient elevations of [Ca2+]i, and the time course was similar to that of the first component observed in the presence of extracellular Ca2+. Histamine caused a greater contraction for a given change in [Ca2+]i than did potassium, at [Ca2+]i over 300 nM. 3. Diltiazem, 10(-8)M to 10(-5)M, concentration-dependently inhibited the second component of [Ca2+]i elevation and tension development induced by histamine (10(-5) M). Only at higher concentrations (over 10(-5) M) did diltiazem inhibit the first component of increases in [Ca2+]i and tension development induced by histamine, both in the presence and absence of extracellular Ca2+. 4. Diltiazem (10(-6) M) inhibited increases in [Ca2+]i and tension development induced by cumulative applications of extracellular Ca2+ during K(+)-depolarization. The curve of [Ca2+]i against tension of these Ca2(+)-induced contractions obtained in diltiazem-treated strips overlapped with that obtained in untreated strips. This suggests that diltiazem has no direct effects on contractile elements. 5. In contrast, the histamine-induced Ca2(+)-tension curve (second component) was shifted in parallel to the left by diltiazem. 6. We conclude that diltiazem, at therapeutic concentrations, specifically inhibits extracellular Ca2+- dependent increases in [Ca2 +]i, with no effects on the release of Ca2 + from intracellular store sites or on Ca2 +-sensitivity of the contractile elements involved in the contractions induced by elevations of [Ca2 +]i.

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Front-surface fluorometry with fura-2 and effects of nitroglycerin on cytosolic calcium concentrations and on tension in the coronary artery of the pig.

1. By use of front-surface fluorometry and fura-2-loaded strips of the coronary artery of the pig, the effects of nitroglycerin (NG) on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension development were measured simultaneously. 2. Both high K+ depolarization and histamine increased [Ca2+]i and tension in a concentration-dependent manner. However, the tension development in relation to the [Ca2+]i increase ([Ca2+]i-tension relation) observed with histamine was much greater than that observed with K+ depolarization. 3. NG reduced in a concentration-dependent manner both [Ca2+]i and tension, irrespective of whether the vascular strips were in a resting state or during exposure to high K+ or to histamine stimulation. However, the extent of reduction in tension (relaxation) was greater than that expected from the reduction in [Ca2+]i based on the [Ca2+]i-tension relationship observed with K(+)-depolarization. 4. In the absence of extracellular Ca2+, NG depleted stored Ca2+ and also inhibited Ca2+ release from histamine-sensitive stores, but had no effect on the caffeine-sensitive stores. NG inhibited the caffeine-induced tension development with no change in [Ca2+]i. 5. We suggest that NG relaxes the coronary artery of the pig by reducing [Ca2+]i and also by directly controlling contractile elements through second messengers not related to changes in [Ca2+]i.

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Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture.

Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type ICa) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type ICa, without affecting T-type ICa. L-type ICa was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type ICa is halved by 0.1 microM nicardipine, 3.0 microM diltiazem, 0.6 microM verapamil, and 0.1 microM flunarizine, whereas T-type ICa is halved by the same drugs at 0.6, 30, 30, and 0.1 microM, respectively. Diltiazem and verapamil accelerated the decay of L-type ICa and cumulatively blocked L-type ICa during repetitive step depolarizations elicited every 30 seconds ("use-dependent block"). Diltiazem and verapamil neither changed the decay of T-type ICa nor showed a use-dependent block of T-type ICa. Nicardipine and flunarizine blocked both L- and T-type ICa from the first depolarization step after drug treatment ("tonic block") and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively) were smaller than those for the resting state of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 microM) of nicardipine initially potentiated T-type ICa and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.

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Effects of okadaic acid on cytosolic calcium concentration and contraction of porcine coronary artery.

We simultaneously measured cytosolic Ca concentrations ([Ca]i) and tension in fura-2-loaded porcine coronary artery and evaluated the effects of okadaic acid (OA), C44H66O13, a potent phosphatase inhibitor, on [Ca]i and tension. OA over 10(-6) M induced dose-dependent contractions. There were no differences in the time course and the extent of developed tension between the presence and absence of extracellular Ca. Only in the presence of extracellular Ca did OA induce increase in [Ca]i. During contractions induced by cumulative applications of extracellular Ca in high K(+)-depolarizing solution, OA 10(-6) M decreased the maximum tension development during [Ca]i increase, with no effects on the Kd value and Hill coefficient. OA seems to inhibit Ca-dependent contractions by reducing Ca-effectiveness but without affecting Ca-sensitivity. OA-induced contraction are independent of extracellular Ca and [Ca]i.

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Does allopurinol prevent myocardial injury as a result of hypoxia-re-oxygenation in rats?

We made use of the xanthine oxidase inhibitor allopurinol and examined changes related to myocardial injury of the rat heart during hypoxia-re-oxygenation. The rat heart was perfused using the Langendorff method. With low-oxygen perfusion for 60 min in a solution saturated with mixed gases of 95% N2 + 5%O2, contractile tension did not develop and tension development was not restored upon re-oxygenation. During hypoxia, the resting tension increased (4.1 g) in the absence of allopurinol. In the allopurinol-administered group (100 microM), contractile tension did not develop during hypoxia; however, the development of tension was restored (18%) upon re-oxygenation. The elevation of resting tension was less (3.2 g) during hypoxia. All events related to the myocardial injury (inhibition of Na+, K(+)-ATPase activities, generation of malondialdehyde, extracellular leakage of creatine kinase) after low-oxygen perfusion for 60 min and re-oxygenating perfusion for 30 min were mild in the allopurinol treated group, compared with findings in the non-administered group. Tissue ATP at 10 min after low-oxygen perfusion was of a significantly high value in the allopurinol treated group (13.2 mumols/g dry weight), compared with findings in the group not given the drug (8.4 mumol/g dry weight). Sixty minutes after low-oxygen perfusion, tissue ATP in the allopurinol group also remained high, compared with the group not given the drug. Although the intensity of the epicardial NADH fluorescence indicated that the extent of inhibition of aerobic energy production during 10 min of low-oxygen perfusion was the same for both groups, lactate was produced in large quantities in the allopurinol treated group, hence energy generation advanced with glycolysis. These observations suggest that allopurinol prevents myocardial injury as a result of hypoxia-re-oxygenation. In the low-oxygen perfusion period, generation of energy is maintained and improved with glycolysis and there is a reduction in the generation of free radicals and an inhibition in lipid peroxidation.

Allopurinol↗

Endothelin-induced Ca-independent contraction of the porcine coronary artery.

Front surface fluorometry and porcine coronary arterial strips loaded with fura-2 were used to investigate the effect of endothelin on cytosolic Ca concentrations, [Ca]i, and on contractile force, the objective being to elucidate the mechanism of action. Both in the presence and absence of extracellular Ca, endothelin induced rapid and dose-dependent increases in [Ca]i and in contraction. When caffeine-sensitive and histamine-sensitive intracellular Ca stores were depleted, in Ca-free medium, a transient contraction but no increase in [Ca]i followed the subsequent application of endothelin. This Ca-independent component was largely inhibited by the relative protein kinase C inhibitor, H-7, but not inhibited by W-7, calmodulin antagonist. This component is probably linked to activation of protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in primary cultures.

We made use of quin2 microfluorometry to determine the effects of endothelin (ET) on cytosolic free Ca2+ concentrations [Ca2+]i) in rat aortic smooth muscle cells in primary culture. In Ca2+-containing medium, ET induced a rapid and sustained elevation of [Ca2+]i. In the latter component, in particular, the elevation of [Ca2+]i was inhibited by diltiazem. In Ca2+-free medium, ET induced a rapid and transient [Ca2+]i elevation, which was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca2+ store was practically depleted by repeated treatment with caffeine in Ca2+-free media, ET did not elevate [Ca2+]i. Thus, it was suggested that ET induces [Ca2+]i elevation not only by extracellular Ca2+-dependent, mechanisms but also by releasing Ca2+ from the intracellular store, and that the ET-sensitive Ca2+ store may overlap with the caffeine-sensitive one, in cultured vascular smooth muscle cells.

Aminoquinolines↗

Voltage-dependent anion-selective channels in cultured smooth muscle cells of the rat aorta.

1. Properties of the voltage-dependent anion-selective channel in cultured smooth muscle cells of the rat aorta were studied using the patch-clamp technique. 2. The channel had a single channel conductance of 346 +/- 4 pS (n = 43, mean +/- SEM) with symmetrical 142 mM-Cl- solution in inside-out patch configurations. 3. The channel was activated spontaneously at a potential range -20 approximately +20 mV and inactivated more rapidly with increases to more positive or negative potentials. 4. The channel was selective for anions and the permeability ratio for monovalent anion was Br-:Cl-:HCOO-:CH3COO-:propionate-:aspartate- = 1.1:1:0.7:0.4: less than 0.02: less than 0.02. 5. The openings of the channels were observed more frequently in inside-out membrane patches than in cell-attached ones, and were independent of intracellular free Ca concentrations. 6. The density of this channel was estimated to be 1.3/micron2. 7. Physiological roles of the channel were discussed.

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