Hiromichi Yamamoto

Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture

In the presence of endothelin, there was a rapid increase in the 45Ca++ efflux from primary cultured rat vascular smooth muscle cells, both in physiological salt solution and in calcium free medium containing 2 mM EGTA. The 45Ca++ influx was not affected. The endothelin-induced, transient increase in cytosolic calcium concentration is probably mainly due to release of calcium from the intracellular store in vascular smooth muscle cells.

Mechanisms of caffeine-induced contraction and relaxation of rat aortic smooth muscle.

1. Using front‐surface fluorimetry and Fura‐2, we determined the effects of caffeine on cytosolic calcium concentration ([Ca2+]i) and on tension of strips of the rat thoracic aorta. We also determined the effects of caffeine on 45Ca2+ influx into the strips. The objective was to elucidate the mechanism of contraction and relaxation in vascular smooth muscle, as induced by caffeine. 2. In normal physiological salt solution (PSS), caffeine induced a transient tension development, while it induced a biphasic change in [Ca2+]i. The initial transient peak in [Ca2+]i which coincided with tension development was followed by a sustained increase. Thus, changes in tension did not follow changes in [Ca2+]i. In Ca(2+)‐free PSS, both the caffeine‐induced contraction and the increase in [Ca2+]i were transient. It was suggested that in both the presence and absence of extracellular Ca2+, the transient increase in [Ca2+]i was due to the release of Ca2+ from the intracellular store. Although the sustained increase in [Ca2+]i depended on extracellular Ca2+, it was not affected by diltiazem, a Ca2+ antagonist. 3. Caffeine inhibited the increase in [Ca2+]i and tension development during 118 mM‐K+ depolarization, in a concentration‐dependent manner. 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. Pretreatment of the strips with ryanodine did not alter the inhibitory effects of caffeine. 4. Caffeine inhibited the increased [Ca2+]i and developed tension during stimulation by 10(‐5) M‐noradrenaline, in a concentration‐dependent manner. 5. Dibutyryl cAMP (10(‐4) M) inhibited both high K(+)‐induced and noradrenaline‐induced tension development. Inhibition of an increase in [Ca2+]i in relation to the inhibition of tension during noradrenaline stimulation was much greater than that in 118 mM‐K+ depolarization. 6. Although caffeine per se had no effect on 45Ca2+ influx in the strips in normal PSS, caffeine did inhibit the increase in 45Ca2+ influx stimulated by 118 mM‐K+ or by 10(‐5) M‐noradrenaline, to a similar extent and with similar IC50 values. 7. The characteristic features of the effects of caffeine on vascular smooth muscle, i.e. the transient nature of contraction and the relaxation of precontracted strips could be explained as follows: caffeine is able to reduce [Ca2+]i after releasing Ca2+ from intracellular stores; however, this may play a minor role. Independent of the [Ca2+]i reduction, the second messenger, cAMP, might directly influence the [Ca2+]i‐tension relationship, and if so, would play a major role.

Endothelin induces the Ca2+-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.

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

SummaryBy 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 μmol/l) markedly (98%) decreased the MgATP-dependent 45Ca2+ content in the non-mitochondrial Ca2+ stores in the presence of 1 μmol/l free Ca2+. Heparin (1–100 μg/ml) dose-dependently inhibited this Ca2+ release by IP3. In Ca2+-free solution, heparin (100 μg/ml) inhibited the increases in 45Ca2+ efflux rate evoked by 10 μmol/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 Ca2+ content in skinned cells. These results suggest that heparin (1–100 μg/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.

Myosin phosphorylation and Ca2+ sensitization in porcine coronary arterial smooth muscle stimulated with endothelin-1

We examined the mechanism of endothelin-1-induced contraction in the porcine coronary artery by assessing the changes in myosin light chain phosphorylation and cytosolic Ca2+ concentration ([Ca2+]i) determined by fura-2 and front-surface fluorometry. Endothelin-1 induced a rapid phosphorylation of myosin both in the presence and in the absence of extracellular Ca2+. During the later phase of contraction (5-30 min), the tension and [Ca2+]i were maintained, while the phosphorylated myosin decreased in the presence of extracellular Ca2+. At a similar extent of [Ca2+]i elevation, endothelin-1 induced a greater tension development than did 118 mM K(+)-depolarization and this was not attributed to a greater degree of phosphorylation of myosin. In the Ca(2+)-depleted strips, endothelin-1 induced a slow and transient contraction without an increase in either [Ca2+]i or myosin phosphorylation. Therefore, in addition to the activation of mechanisms of [Ca2+]i-myosin phosphorylation, endothelin-1 may activate additional calcium regulatory mechanisms which may thus increase the Ca2+ sensitivity of contractile elements while also enhance and prolong the contractions in the porcine coronary artery.

Changes in the cytosolic Ca2+ concentration and Ca2+-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery

To investigate the underlying mechanism for the angiotensin II‐induced desensitization of the contractile response during the prolonged stimulation of the vascular smooth muscle, we determined the effects of angiotensin‐II on (1) cytosolic Ca2+ concentration ([Ca2+]i) and tension using fura‐2‐loaded medial strips of the rabbit femoral artery, (2) 45Ca2+ influx in ring preparations, and (3) Ca2+‐sensitivity of the contractile apparatus in α‐toxin permeabilized preparations. In the presence of extracellular Ca2+, high concentrations of angiotensin‐II elicited biphasic increases in [Ca2+]i and tension, which consisted of initial transient and subsequent lower and sustained phases. The 45Ca2+ influx initially increased after the application of 10−6 M angiotensin‐II, and thereafter gradually decreased. At 20 min after the application, there was a discrepancy between the level of [Ca2+]i and the extent of 45Ca2+ influx. The relationships between [Ca2+]i and tension suggested that the angiotensin‐II‐induced increase in the Ca2+‐sensitivity of the contractile apparatus was maintained during the desensitization of smooth muscle contraction. When 10−6 M angiotensin‐II was applied during the sustained phase of contraction induced by 118 mm K+‐depolarization, at 10 min after the application, the [Ca2+]i levels were significantly lower and the tension levels were significantly higher than those prior to the application of angiotensin‐II. In conclusion, the decrease in [Ca2+]i, which is partially due to the inhibition of the Ca2+ influx, is mainly responsible for the desensitization evoked by high concentrations of angiotensin‐II, and angiotensin‐II seems to activate additional mechanisms which inhibit Ca2+ signaling during prolonged stimulation.

Extracellular Ca(2+)-dependent potentiation by cocaine of serotonin- and norepinephrine-induced contractions in rat vascular smooth muscle.

Using front-surface fluorometry, we determined the effects of cocaine on force and cytosolic Ca2+ concentration ([Ca2+]i) in the rat aorta. We also examined the effects of cocaine on 45Ca2+ influx. Cocaine (10(-7) to 10(-4) M) alone did not alter the resting level of [Ca2+]i and force. Cocaine (< 10(-4) M), in a concentration-dependent manner, potentiated the 10(-6) M serotonin (5-HT)-induced or 10(-8) M norepinephrine (NE)-induced sustained increase in [Ca2+]i and force in the presence of extracellular Ca2+, whereas it had no potentiating effects in Ca(2+)-free solution. Similar potentiating effects of cocaine were observed in pharmacologically denervated strips. Cocaine (10(-5) M) produced a leftward shift of concentration-response curves for both 5-HT- and NE-induced increases in [Ca2+]i and force with no effect on the maximal response or the relations between [Ca2+]i and force. Cocaine (10(-5) M also accelerated the 45Ca2+ influx during activation by 10(-6) M 5-HT or by 10(-8) M NE. Cocaine (> 10(-3) M) inhibited 5-HT-, NE-, and high-K+ depolarization-induced contractions accompanied by decreases in [Ca2+]i in normal physiological salt solution and 5-HT- or NE-induced transient increase in [Ca2+]i and force in Ca(2+)-free physiological salt solution. Thus, low concentrations of cocaine potentiate NE- or 5-HT-induced contraction by augmenting the increase in [Ca2+]i. These potentiating effects may derive from either an increase in the affinity of the receptors to agonists or an increase in the Ca2+ influx. On the other hand, high concentrations of cocaine (> 10(-3) M) have a relaxant effect on vascular smooth muscle, as a result of a decrease in [Ca2+]i.

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.

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.

The mechanism of the decrease in cytosolic Ca2+ concentrations induced by angiotensin II in the high K(+)-depolarized rabbit femoral artery.

Using front‐surface fluorometry of fura‐2‐loaded strips, and measuring the transmembrane 45Ca2+ fluxes of ring preparations of the rabbit femoral artery, the mechanism underlying a sustained decrease in the cytosolic Ca2+ concentration ([Ca2+]i) induced by angiotensin II (AT‐II) was investigated. The application of AT‐II during steady‐state 118 mM K+‐induced contractions caused a sustained decrease in [Ca2+]i following a rapid and transient increase in [Ca2+]i, while the tension was transiently enhanced. When the intracellular Ca2+ stores were depleted by thapsigargin, the initial rapid and transient increase in [Ca2+]i was abolished, however, neither the sustained decrease in [Ca2+]i nor the enhancement of tension were affected. Depolarization with 118 mM K+ physiological salt solution containing 1.25 mM Ba2+ induced a sustained increase in both the cytosolic Ba2+ concentration ([Ba2+]i) level and tension. However, the application of 10−6 M AT‐II during sustained Ba2+‐contractions was found to have no effect on [Ba2+]i, but it did enhance tension. After thapsigargin treatment, AT‐II neither decreased nor increased the enhanced Ca2+ efflux rate induced by 118 mM K+‐depolarization, whereas AT‐II did increase the enhanced 45Ca2+ influx and the 45Ca2+ net uptake induced by 118 mM K+‐depolarization. Pretreatment with calphostin‐C, partially, but significantly inhibited the decrease in [Ca2+]i induced by AT‐II. These findings therefore suggest that AT‐II stimulates Ca2+ sequestration into the thapsigargin‐insensitive Ca2+ stores, and thus induces a decrease in [Ca2+]i in the high external K+‐stimulated rabbit femoral artery.