Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle.

1. In smooth muscle, both Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx across the plasma membrane are responsible for the increase in the cytosolic Ca2+ level ([Ca2+]i). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca2+ pump, cyclopiazonic acid (CPA 10 microM), an inhibitor of the Ca(2+) -induced Ca2+ release, ryanodine, (10 microM), and an activator of the Ca(2+) -induced Ca2+ release, caffeine (20 mM), on [Ca2+]i and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2. CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3. In the presence of external Ca2+, noradrenaline (NA, 10 microM) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KCl (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca(2+) -free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca2+ in SR. High K+ was ineffective in the absence of external Ca2+. 4. In the presence of external Ca2+ and CPA, NA and high K+ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K+ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5. Dissociation of contraction from [Ca2+]i as measured with aequorin suggests that NA and high K+ increase Ca2+ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca2+ pump may regulate the Ca2+ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca2+, the SR Ca2+ pump may have little effect on regulation of Ca2+ level in the contractile compartment. Furthermore, the release of Ca2+ from SR seems to have little effect on the increase in the contractile Ca2+ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K+ in the presence of external Ca2+ in the ferret portal vein.     

Effects of a novel smooth muscle relaxant, KT-362, on contraction and cytosolic Ca2+ level in the rat aorta.

1. Inhibitory effects of a novel smooth muscle relaxant, KT-362 (5-[3-([2-(3,4-dimethoxyphenyl)-ethyl]amino)-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate), on contraction and the cytosolic Ca2+ level ([Ca2+]cyt) in isolated vascular smooth muscle of rat aorta were examined. 2. KT-362 inhibited the contractions induced by high K+ and noradrenaline. The inhibitory effect was antagonized by an increase in external Ca2+ concentration. A Ca2+ channel activator, Bay K 8644, did not change the effect of KT-362 on high K+-induced contraction. 3. [Ca2+]cyt, measured with fura-2-Ca2+ fluorescence, increased during the contractions induced by high K+ or noradrenaline. KT-362 decreased [Ca2+]cyt and muscle tension stimulated by high K+ or noradrenaline. By contrast, a Ca2+ channel blocker, verapamil, inhibited the noradrenaline-induced increase in [Ca2+]cyt with only partial inhibition of the noradrenaline-induced contraction and KT-362 inhibited the verapamil-insensitive portion of the contraction without changing [Ca2+]cyt. 4. In a Ca2(+)-free solution, noradrenaline and caffeine induced a transient contraction following a transient increase in [Ca2+]cyt. KT-362 inhibited the increments due to noradrenaline but not those induced by caffeine. 5. These results suggest that KT-362 inhibits vascular smooth muscle contraction by inhibiting Ca2+ channels, receptor-mediated Ca2+ mobilization, and receptor-mediated Ca2+ sensitization of contractile elements.     

Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle.

1. It has been shown that receptor agonists and activators of protein kinase C, phorbol esters, increase Ca2+ sensitivity of contractile elements in vascular smooth muscle. To discover if protein kinase C is involved in the agonist-mediated Ca2+ sensitization, we examined the effects of receptor agonists in the rat isolated aorta in which protein kinase C activity had been diminished by pretreatment with phorbol 12-myristate 13-acetate for 24 h. 2. In the aorta with protein kinase C activity, a high concentration (1 microM) of 12-deoxyphorbol 13-isobutyrate induced contraction and a low concentration (100 nM) potentiated high K(+)-induced contraction. In addition, prostaglandin F2 alpha induced greater contractions than high K+ at a given cytosolic Ca2+ level. The maximally effective concentrations of noradrenaline and endothelin-1 also induced greater contraction than high K+. In the aorta without protein kinase C activity, the contraction induced by 12-deoxyphorbol 13-isobutyrate and its potentiation of the high K(+)-induced contraction were abolished. However, prostaglandin F2 alpha, noradrenaline and endothelin-1 still induced a greater contraction than high K+. 3. In the aorta without protein kinase C activity, noradrenaline, endothelin-1 and prostaglandin F 2 alpha, but not 12-deoxyphorbol 13-isobutyrate, induced contractions in the presence of the Ca2+ channel blocker, verapamil, or in the absence of external Ca2+, by increasing Ca2+ sensitivity. 4. In the permeabilized preparations, inhibition of protein kinase C activity abolished the effect of potentiation of the Ca(2+)-induced contraction by 12-deoxyphorbol 13-isobutyrate although the potentiation of the contraction by prostaglandin F2 alpha did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Marked dissociation between intracellular Ca2+ level and contraction on exposure of rat aorta to lysophosphatidylcholine.

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) on exposure of the endothelium-denuded isolated rat aorta to palmitoyl-L-alpha-lysophosphatidylcholine. Lysophosphatidylcholine concentration-dependently induced a gradual increase in [Ca2+]i. Application of 10(-4) M lysophosphatidylcholine induced a large and sustained tonic increase in [Ca2+]i (the peak [Ca2+]i was 125.2 +/- 11.5% of the 80 mM K+-induced response) but only a small contraction (4.0 +/- 1.4% of the 80 mM K+ induced contraction). The sustained increase in [Ca2+]i was attenuated when extracellular Ca2+ was removed but it was unaffected by verapamil or 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7). Digitonin also produced a gradual increase in [Ca2+]i but with a pronounced contraction. Triton X-100 (0.1%) produced a marked elevation in [Ca2+]i with no detectable contraction. Triton X-100, however, caused a rapid leakage of fura PE-3. Treatment with 10(-4) M lysophosphatidylcholine for 1 or 2 h did not affect the contractile response induced by 80 mM K+ and this treatment did not release lactate dehydrogenase from the rat aorta. Treatment with lysophosphatidylcholine did not affect either the cyclic AMP level or the cyclic GMP level in endothelium-denuded aortic tissues. These results show that in the rat aorta lysophosphatidylcholine produces a large increase in [Ca2+]i (possibly in a non-contractile compartment) which does not induce contraction. Thus, the increase in [Ca2+]i induced by lysophosphatidylcholine (i) requires external Ca2+ but is not due to an increased Ca2+ influx through voltage-dependent L-type Ca2+ channels, (ii) is not primarily due to protein kinase C activation and (iii) is probably not due to a detergent action (like those of digitonin and triton X-100). The relative lack of a contractile response to lysophosphatidylcholine is not due to formation of cyclic AMP or cyclic GMP.     

Effects of endothelin on the mechanical activity and cytosolic calcium level of various types of smooth muscle.

1. Effects of porcine/human endothelin (endothelin-1), a novel vasoconstrictor peptide, on various smooth muscles were examined. 2. In rat aorta, endothelin (1 pM-30nM) induced contraction in a concentration-dependent manner. Removal of endothelium shifted the concentration-response curve to the left. When added during the sustained contraction induced by 0.1 microM noradrenaline, endothelin (1 nM) induced a relaxation that was inhibited by removing endothelium or by methylene blue. 3. In rat aorta without endothelium, endothelin (1-30 nM) increased cytosolic Ca2+ level [( Ca2+]cyt) followed by contraction. Endothelin induced less contraction than high K+ at a given [Ca2+] cyt when the concentration of endothelin was lower (1-3nm) and/or during the early phase of the contraction (less than 10 min). In contrast, endothelin induced a greater contraction than KCl after prolonged exposure to high concentrations (greater than 10 nM). 4. The increase in [Ca2+]cyt due to endothelin was strongly inhibited by 10 microM verapamil or 0.3 microM nicardipine although muscle contraction was only partially inhibited. 5.In Ca2+ -free solution, endothelin (30 nM) induced a transient increase in [Ca2+] cyt and a slow increase in muscle tension. After a prolonged incubation in Ca2+-free solution, endothelin (30 nM) still induced a slow increase in tension without changing [Ca2+]cyt. This contraction was inhibited by 1 microM sodium nitropusside or 10 microM forskolin. 6. In canine trachea and guinea-pig uterus, endothelin (30 nM) induced sustained contraction with an increase in [Ca2+]cyt. In the absence of external Ca2+, endothelin (30 nM) induced a sustained contraction in canine trachea without changing [Ca2+]cyt. In guinea-pig vas deferens, taenia caeci and ileal longitudinal muscle, endothelin induced small increases in [Ca2+]cyt and tension. 7. In permeabilized smooth muscles, endothelin (30 nM) did not change the muscle tone. 8. These results suggest that endothelin acts on the endothelium and increases the synthesis or release of endothelin-derived relaxing factor (EDRF). These results also suggest that endothelin acts directly on smooth muscle and increases [Ca2+]cyt by releasing Ca2+ from sites and increasing Ca2+ influx through the verapamil- and 1,4-dihydropyridine-sensitive pathway. Endothelin seems to decrease Ca2+ -sensitivity of contractile elements at lower concentrations and/or during the early phase of the contraction, whereas it increases Ca2+ -sensitivity at higher concentrations during the sustained phase of the contraction. Furthermore, endothelin induces a contraction that is not dependent on [Ca2+]cyt.     

Inhibitory effects of caffeine on contractions and calcium movement in vascular and intestinal smooth muscle.

1. The mechanism of the inhibitory effect of caffeine was investigated using vascular smooth muscle of rabbit aorta and intestinal smooth muscle of taenia isolated from guinea-pig caecum. 2. Caffeine, 0.5-10 mM, relaxed the sustained contraction induced by 65.4 mM KCl or 10(-6) M noradrenaline in aorta, and by 45.4 mM KCl or 10(-6) M carbachol in taenia. The inhibitory effect of caffeine on the high K+-induced contraction was antagonized by external Ca2+ but not by the Ca2 channel activators, Bay K 8644 (10(-7) M) or CGP 28,392 (10(-7) M). Forskolin (2 x 10(-7) M) potentiated the inhibitory effect of caffeine on the noradrenaline-induced contraction but not on the high K+- or carbachol-induced contraction. Caffeine induced a time- and concentration-dependent increase in the cyclic AMP content of aorta and forskolin caused a further augmentation. 3. 45Ca2+ uptake was increased by high K+ or noradrenaline in aorta and by high K+ or carbachol in taenia. The increments were inhibited by caffeine at concentrations needed to inhibit muscle contractions. 4. 45Ca2+ in the cellular releasable site in aorta was decreased either by noradrenaline or by caffeine. Simultaneous application of noradrenaline and caffeine did not induce an additive decrease. 5. In aorta treated with a Ca2+-free solution, caffeine induced only a small contraction. Noradrenaline induced a greater contraction which was inhibited by caffeine. After washout of caffeine and noradrenaline, the second application of noradrenaline induced a transient contraction suggesting that caffeine does not deplete the noradrenaline-sensitive store.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory effect of protein kinase C activator on contractility of rat vas deferens

The modulatory effect of the protein kinase C activator was examined on contraction of rat isolated vas deferens induced by constrictive agonists, noradrenaline (NA), ATP, BaCl2 and high K+. Phorbol 12,13-diacetate (PDA, 1 micromol/l) induced a transient extracellular Ca(2+)-dependent contraction while the inactive analogue, 4alpha-phorbol (1 micromol/l) had no effect. PDA significantly enhanced the peak amplitude of the contractile response to NA (0.1-10 micromol/l), ATP (100 micromol/l), Ba2+ (3 mmol/l) or high K+ (30 mmol/l). Staurosporine at 30 nmol/l reduced the enhancing effect of PDA on the agonist-induced contraction. NA (10 micromol/l) produced a phasic contraction followed by a sustained contraction, while ATP induced monophasic contraction. Pretreatment with nifedipine (10 nmol/l) had no effect on the phasic contraction induced by NA, but it significantly reduced ATP- or high K(+)-induced contraction. Staurosporine (30 nmol/l) alone attenuated the peak contractile response induced by NA or ATP but not by Ba2+. NA produced a transient contraction in Ca(2+)-free Krebs solution, and PDA (1 micromol/l) markedly enhanced this effect. These novel data indicate that activation of a protein kinase C-dependent mechanism not only affects contraction mediated by Ca2+ influx through voltage-sensitive Ca2+ channels, but also promotes intracellular Ca2+ release or intracellular Ca(2+)-mediated contractile mechanism in rat vas deferens.     

Ca2(+)-dependent and independent mechanisms of sustained contraction in vascular smooth muscle of rat aorta

Norepinephrine (NE), prostaglandin F2 alpha (PG), 12-deoxyphorbol 13-isobutyrate (DPB) and high K+ induced sustained increase in cytosolic Ca2+ [( Ca2+]i) and muscle tension in rat aorta. Verapamil, at the concentration which abolished the high K(+)-induced changes, also abolished the increase in [Ca2+]i due to NE, PG and DPB although contractions were only partially inhibited. Excess EGTA further decreased [Ca2+]i although a part of the contraction was not inhibited. These results indicate that the sustained contractions induced by NE, PG and DPB are due to increase in [Ca2+]i, increase in Ca2+ sensitivity of contractile elements, and a Ca2(+)-independent mechanism.     

Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery

Endothelin induced rapid increase followed by a decrease in cytosolic Ca2+ [( Ca2+]i) and a slow increase in muscle tension in the vascular smooth muscle strip of rat carotid artery. Thus, the endothelin-induced contraction was smaller, and it became gradually greater than high K-induced contraction at a given [Ca2+]i. In Ca2+-free solution, endothelin induced a transient increase in [Ca2+]i and a sustained contraction. These results suggest that endothelin-induced contraction is due to the increase in [Ca2+]i, the time-dependent change in Ca2+-sensitivity of contractile elements, and the mechanism which is independent of the increment in [Ca2+]i.     

Changes in the cytosolic Ca2+ concentration and Ca(2+)-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery

1. 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) Ca(2+)-sensitivity of the contractile apparatus in alpha-toxin permeabilized preparations. 2. 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. 3. 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. 4. The relationships between [Ca2+]i and tension suggested that the angiotensin-II-induced increase in the Ca(2+)-sensitivity of the contractile apparatus was maintained during the desensitization of smooth muscle contraction. 5. 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. 6. 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.     

Increased sensitivity of rat myometrium to the contractile effect of platelet activating factor before delivery.

1. The contractile effects of platelet activating factor (PAF) were compared in the myometrium isolated from non-pregnant and pregnant rats. 2. In the non-pregnant myometrium, PAF, at a concentration of 0.1 microM, did not change muscle tension and induced only a small transient contraction at 10 microM. 3. The contractile responses to PAF increased with the progress of gestation. In the late pregnant myometrium (21 day after gestation), PAF (0.1 nM-10 microM) induced large and relatively sustained contractions. The threshold concentration of PAF was decreased by approximately 10,000 times and the maximum contraction was increased 5 times by day 21 of gestation. 4. PAF (10 microM) increased the cytosolic Ca2+ concentration ([Ca2+]i) and muscle contraction to levels higher than those induced by high K+ in the pregnant rat myometrium (day 21). Verapamil (10 microM), a voltage-dependent Ca2+ channel blocker, decreased the stimulated [Ca2+]i and muscle tension to 49.6% and 22.7%, respectively, while the same concentration of verapamil completely inhibited the high K(+)-induced responses. 5. PAF (10 microM) induced a transient increase in [Ca2+]i with no contraction in the absence of external Ca2+ in the pregnant myometrium (day 21). 6. These results suggest that PAF induces contraction in rat myometrium by increasing Ca2+ influx. Although PAF released Ca2+ from stored sites, this Ca2+ does not seem to contribute to the PAF-induced contraction. Our major finding is that the sensitivity of the myometrium to PAF increased after gestation and that this may play a role in delivery.     

Inhibitory effects of procaine on contraction and calcium movement in vascular and intestinal smooth muscles.

1. The effects of procaine on muscle tension and 45Ca2+ movements were investigated in vascular smooth muscle of the rabbit aorta and intestinal smooth muscle of the taenia isolated from guinea-pig caecum. 2. Procaine (10 mM) induced a contraction in the taenia but had little effect on the resting tension in the aorta. 3. Procaine, 0.5-10 mM, relaxed the sustained contractions induced by 65.4 mM KCl and 10(-6) M noradrenaline in the aorta, and by 45.4 mM KCl, 10(-6) M carbachol and 10(-6) M histamine in the taenia. The inhibitory effect of procaine on the high K+-induced contractions was antagonized by external Ca2+ but not by the Ca2+ channel activators, Bay K 8644 and CGP 28,392. 4. 45Ca2+ uptake was increased by high K+ or noradrenaline in the aorta and by high K+ or carbachol in the taenia. The increments were inhibited by procaine at the concentrations needed to inhibit the muscle contractions. 5. In a Ca2+-free solution, noradrenaline and caffeine induced a transient contraction in the aorta, whereas a second application of each stimulant was almost ineffective. Addition of 1-10 mM procaine shortly before the first application of the stimulant inhibited the contraction. After washing the muscle with a Ca2+-free solution without procaine, the second application of the stimulant induced a greater contraction than that in control muscle without procaine pretreatment. 6. Noradrenaline and caffeine released 45Ca2+ from a cellular site in the aorta. Procaine inhibited the effects of these stimulants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different effects of verapamil on cytosolic Ca2+ and contraction in norepinephrine-stimulated vascular smooth muscle

Effects of verapamil on cytosolic Ca2+ levels ([Ca2+]cyt) and contraction in fura-2-loaded rat aorta were examined. Norepinephrine (NE) induced a greater contraction than KCl for a given increase in [Ca2+]cyt. Cumulative addition of verapamil decreased the NE-stimulated [Ca2+]cyt more strongly than contraction whereas verapamil decreased high K(+)-stimulated [Ca2+]cyt and contraction in parallel. In the presence of verapamil at a concentration needed to completely inhibit the high K(+)-induced increments, NE induced a transient increase, followed by a small sustained increase in [Ca2+]cyt which averaged 25% of that in the absence of verapamil. These changes were followed by a sustained contraction which averaged 60% of that in the absence of verapamil. In Ca2(+)-free solution, NE induced only a transient increase in [Ca2+]cyt whereas it induced a transient contraction, followed by a small sustained contraction. The second application of NE induced a small sustained contraction (10% of that in the presence of Ca2+) without increasing [Ca2+]cyt. These changes were not affected by verapamil. These results suggest that verapamil inhibits NE-induced increase in [Ca2+]cyt, but not the Ca2(+)-sensitization or Ca2(+)-independent contraction, and this may be the reason why the NE-induced contraction is less sensitive to verapamil than that induced by high K+.     

Inhibition of calcium channels by harmaline and other harmala alkaloids in vascular and intestinal smooth muscles.

Effects of harmaline and other harmala alkaloids on the contractions induced in the vascular smooth muscle of rabbit aorta and intestinal smooth muscle of taenia isolated from guinea-pig caecum were examined. In rabbit isolated aorta, harmaline inhibited the sustained contraction induced by 65.4 mM K+ with an IC50 (concentration needed for 50% inhibition) of 4.6 X 10(-5) M. This inhibitory effect on high K+-induced contraction was antagonized by raising the concentration of external Ca2+ but not by Bay K 8644, a Ca2+ channel facilitator. Harmaline also inhibited the sustained contraction induced by noradrenaline (10(-6) M) with an IC50 of 7.6 X 10(-5) M. The inhibitory effects on noradrenaline-induced contractions were not antagonized by raising the external Ca2+ concentrations or by Bay K 8644. In guinea-pig taenia, harmaline inhibited the 45.4 mM K+-induced contraction with an IC50 of 6.8 X 10(-5) M and the carbachol (10(-6) M)-induced contraction with an IC50 of 7.0 X 10(-5) M. The inhibitory effects on both high K+- and carbachol-induced contractions were antagonized by raising the external Ca2+ concentrations but not by Bay K 8644. Harmaline, at the concentrations needed to inhibit the muscle contraction, inhibited the increase in 45Ca2+ uptake induced by high K+, noradrenaline and carbachol in aorta and taenia. Harmaline did not change the cellular Na+ and ATP contents in resting and high K+ stimulated taenia. Other harmala alkaloids also inhibited the contractions in these smooth muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium regulation of smooth muscle contractility

Simultaneous measurements of cytoplasmic Ca2+ level [( Ca2+]i) and muscle contraction in smooth muscle indicated that [Ca2+]i gradually decreases during sustained contraction. This time-dependent dissociation has been explained by the latch bridge hypothesis, positive cooperativity between phosphorylated and non-phosphorylated crossbridges, involvement of cytoskeleton phosphorylation, or connection between myosin and actin filaments by caldesmon. Furthermore, it has been found that receptor agonists induce greater contraction than high K+ for a given increase in [Ca2+]i. This stimulus-dependent dissociation may be due to the receptor agonists-induced activation of protein kinase C which in turn decreases the inhibitory effect of calponin on the actin-myosin interaction, resulting in an apparent Ca2+ sensitization. Thus, the contractions induced by receptor agonists are due not only to the increase in [Ca2+]i but also to the increase in Ca2+ sensitivity of contractile elements. Ca2+ channel blockers inhibit the increase in [Ca2+]i but not the Ca2+ sensitization, and this may be the reason why these blockers are relatively weak inhibitors of the contraction induced by receptor agonists. By contrast, cyclic AMP and cyclic GMP decrease the Ca2+ sensitivity of contractile elements in addition to their effects to decrease [Ca2+]i.     

Mechanism of human urotensin II-induced contraction in rat aorta

Urotensin II induced sustained contraction with an EC(50) value of 2.29 +/- 0.12 nM in rat aorta. Urotensin II (100 nM) transiently increased cytosolic Ca(2+) level ([Ca(2+)](i)), followed by a small sustained phase superimposed with rhythmic oscillatory change. In the presence of verapamil and La(3+), the [Ca(2+)](i) oscillation was completely inhibited, although a small transient increase in [Ca(2+)](i) remained. The urotensin II-induced contraction was also partially inhibited by verapamil and La(3+). Combined application of verapamil, La(3+), and thapsigargin completely inhibited the increase in [Ca(2+)](i) with only partial inhibition of the contraction elicited by urotensin II. Urotensin II increased myosin light chain (MLC) phosphorylation to a level greater than that induced by 72.7 mM KCl (high K(+)). Pretreatment with Go6983 (PKC inhibitor), U0126 (MEK inhibitor), or SB203580 (p38MARK inhibitor) partially inhibited the urotensin II-induced contraction with no effects on the high K(+)-induced contractions. Wortmannin (MLC kinase inhibitor) only partially inhibited urotensin II-induced contraction, although it completely inhibited the high K(+)-induced contraction. These results suggest that urotensin II-induced contraction is mediated by the Ca(2+)/calmodulin/MLC kinase system and modulated by the Ca(2+) sensitization mechanisms to increase MLC phosphorylation. In addition, activations of PKC, p38MAPK, and ERK1/2 modulate the contractility mediated by urotensin II in rat aorta.     

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

1. 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. 2. 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. 3. 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. 4. 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 Ba(2+)-contractions was found to have no effect on [Ba2+]i, but it did enhance tension. 5. 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. 6. Pretreatment with calphostin-C, partially, but significantly inhibited the decrease in [Ca2+]i induced by AT-II. 7. 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.     

Essential role for class II phosphoinositide 3-kinase alpha-isoform in Ca2+-induced, Rho- and Rho kinase-dependent regulation of myosin phosphatase and contraction in isolated vascular smooth muscle cells

The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells, combined with the RNA interference-mediated gene-silencing technique, allowed us to determine the role of phosphoinositide 3-kinase (PI3K) class II alpha-isoform (PI3K-C2alpha) as a novel, Ca2+-dependent regulator of myosin light-chain phosphatase (MLCP) and contraction. The Ca2+-ionophore ionomycin induced a robust contractile response with an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The PI3K-C2alpha-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2alpha protein expression. The siRNA-mediated knockdown of PI3K-C2alpha, but not class I PI3K p110alpha, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. PI3K-C2alpha is uniquely less sensitive to the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) than the other PI3K members, including p110alpha. Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations showing that ionomycin and membrane depolarization induced Rho activation in vascular smooth muscle tissues in a Ca2+-dependent manner, ionomycin-induced contraction was dependent on Rho and Rho-kinase. Ionomycin induced phosphorylation of the MLCP-regulatory subunit myosin targeting protein 1(MYPT1) at Thr850 and the 20-kDa myosin light chain (MLC) in a Rho kinase-dependent manner. Knockdown of PI3K-C2alpha suppressed phosphorylation of both MYPT1 and MLC. The receptor agonist noradrenaline, which induced a rapid increase in the [Ca2+]i and Ca2+-dependent contraction, stimulated phosphorylation of MYPT1 and MLC, which was also dependent on Ca2+, PI3K-C2alpha, and Rho-kinase. These observations indicate that PI3K-C2alpha is necessary for Ca2+-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction.     

Calcium channel blocker-like action of 1,9-dideoxyforskolin in vascular smooth muscle.

The inhibitory effect of 1,9-dideoxyforskolin (DFK) on the contraction of rat aorta was compared with that of forskolin. DFK inhibited the contraction induced by high K+ more strongly than that induced by norepinephrine, whereas forskolin more strongly inhibited the norepinephrine-induced contraction. The inhibitory effect of DFK on high K(+)-induced contraction was antagonized by an increase in extracellular Ca2+ concentration. DFK inhibited the increase in cytosolic Ca2+ level and contraction in parallel whereas forskolin inhibited the contraction more strongly than the cytosolic Ca2+ level. These results suggest that DFK, but not forskolin, inhibits vascular smooth muscle contraction by a Ca2+ channel blocker-like action.     

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation.

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.