Calcium dependence of phorbol 12,13-dibutyrate-induced force and myosin light chain phosphorylation in arterial smooth muscle

Phorbol dibutyrate (PDB) is an activator of protein kinase C and has been observed to cause a slow developing contraction in vascular smooth muscle. The mechanism of phorbol ester-induced contraction is unknown. We studied the Ca++-dependence of, and the degree of myosin light chain phosphorylation (MLC-P), during PDB-induced contractions in rabbit aortic rings. PDB elicited concentration-dependent contractions (3 X 10(-8) to 10(-6) M) in rabbit aortic rings incubated in normal (1.6 mM Ca++) physiologic salt solution (PSS). Addition of the Ca++-channel blocker nifedipine (0.1 microM) to PSS or removal or Ca++ from PSS significantly reduced the contractile responses to PDB. Depletion of Ca++ by repeated washes in O Ca++-PSS containing 10(-3) M ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid reduced, but did not eliminate, the responses to PDB. In PSS, PDB significantly increased the fraction of phosphorylated MLC/total MLC to 0.33 from a resting value of 0.20. Ca++ depletion reduced the resting fraction (MLC-P/MLC) to 0.14. PDB-stimulated contractions in Ca++-depleted tissues occurred in the absence of significant increases in MLC-P. Sodium nitroprusside partially relaxed PDB-induced contractions by approximately 50% whether elicited in the presence of 1.6 mM Ca++ or after Ca++ depletion. In both cases relaxation occurred in the absence of statistically significant decreases in MLC phosphorylation. Ca++-dependent MLC phosphorylation may account for a component of the PDB contractile response in rabbit aorta. Studies in the absence of Ca++ suggest that PDB may activate contraction without concomitant MLC-P.     

Phorbol dibutyrate contractions in rabbit aorta: calcium dependence and sensitivity to nitrovasodilators and 8-BR-cyclic GMP

Phorbol esters activate protein kinase C and induce contraction in vascular smooth muscle. The role of Ca and the sensitivity of this response to nitrovasodilators were evaluated in rabbit aortic rings. Phorbol dibutyrate (PDB) (0.01-10 microM) elicited a concentration-dependent contraction of rabbit aortic rings. Responses to PDB in a salt solution (SS) containing 2.54 mM CaCl2 were not significantly different from those in SS containing 0 Ca and 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Contractions to PDB (1 microM) in zero Ca SS were not reduced by depletion of the norepinephrine-sensitive pool of intracellular Ca. The Ca entry blockers verapamil (100 microM) and nifedipine (100 microM) did not affect PDB (1 microM) responses. Nitroprusside (0.1-10 microM) and nitroglycerin (0.1-10 microM) inhibited PDB contractions in both normal SS and Ca-free SS. 8-Br-cyclic GMP (100 microM) also inhibited PDB responses. Effects of PDB on 45Ca fluxes were evaluated in separate experiments. PDB (1 and 10 microM) elicited contraction, but no change in 45Ca uptake or efflux. In contrast KCl (80 mM) and norepinephrine (10 microM) elicited an increase in both influx and efflux, reflecting a rise in cytosolic Ca. The data suggest that PDB-induced contractions in rabbit aorta are independent of extracellular Ca and are not associated with a rise in cytosolic Ca as detected by calcium flux studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.     

Calcium-independent phosphorylation of smooth muscle myosin light chain by okadaic acid isolated from black sponge (Halichondria okadai)

Previously, we have shown that okadaic acid (OA), isolated from black sponge (Halichondria okadai) causes contraction even in the absence of Ca++ in the saponin-permealized taenia isolated from guinea pig cecum. In the present study, mechanism of action of OA was examined using native actomyosin extracted from chicken gizzard smooth muscle. In the absence of Ca++, OA (0.1-1 microM) induced superprecipitation and increased the Mg++-adenosine triphosphatase activity. The OA-induced superprecipitation was enhanced by Ca++ at a concentration (greater than 0.1 microM) which did not activate the calmodulin-dependent myosin light chain (MLC) kinase. The effect of OA was not affected by the calmodulin inhibitor, trifluoperazine, at a concentration (100 microM) needed to inhibit the Ca++-induced response, but was inhibited markedly by the nonselective kinase inhibitors, amiloride (1 mM) and K-252a (5 microM). The OA-induced superprecipitation in the absence of Ca++ was accompanied by phosphorylation of the 20 K dalton MLC, which also was enhanced by low concentration of Ca++ (greater than 0.1 microM). OA did not change the phosphatase activity which dephosphorylates the phosphorylated MLC. An activator of Ca++- and phospholipid-dependent protein kinase, 12-O-tetradecanoylphorbol 13-acetate (1 microM), did not modulate superprecipitation or phosphorylation of MLC in the presence and absence of OA. Furthermore, inhibitors of Ca++ and phospholipid-dependent protein kinase, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (400 microM) and polymyxin B (100 micrograms/ml), affected neither superprecipitation nor phosphorylation of MLC induced by OA. With a reconstituted system containing purified myosin and MLC kinase, OA induced only slight phosphorylation of MLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phorbol 12,13-dibutyrate on the vascular tone and on norepinephrine- and potassium-induced contractions of cat cerebral arteries

Phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), induced slow-developing sustained contractions in segments of cat middle cerebral arteries. PDB-induced responses were not affected by phentolamine (1 microM) and endothelium removal, and were reduced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (25 microM) and staurosporine (10 nM), PKC inhibitors. Forskolin (25 microM) produced a rapid and marked vasodilation in segments contracted with PDB. The 4 alpha-phorbol 12,13-didecanoate, an inactive compound, induced slight vasodilation. Preincubation with nifedipine diminished the responses elicited by PDB at all concentrations used. Ca-free medium containing 3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), but not 1 mM, markedly reduced the phorbol-induced responses at concentrations up to 10 nM. Nifedipine (0.1 microM) and forskolin (25 microM) produced a rapid and marked relaxation of PDB (10 nM)-evoked contractions in segments incubated in a Ca-free solution (1 mM EGTA), but PBD responses in 3 mM EGTA were not affected by nifedipine. PDB (10 and 100 nM) practically did not modify K-induced contractions, but reduced vasoconstrictions elicited by different norepinephrine concentrations; this effect was phorbol concentration and preincubation time-dependent. These results indicate that: 1) PDB induced PKC activation and contraction mainly produced by Ca entry (essentially at low PDB concentrations) through dihydropyridine-sensitive Ca channels; 2) the activated PKC has elevated sensitivity for Ca; 3) PKC may be involved in the alpha adrenoceptors desensitization, but did not play an important role in the norepinephrine-induced contraction in these arteries.     

Divergent pharmacological effects of three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine and calmidazolium, on isometric tension development and myosin light chain phosphorylation in intact bovine tracheal smooth muscle

To investigate the usefulness of calmodulin antagonists in intact cell systems, effects of three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine (CPZ) and calmidazolium on isometric tension development and myosin light chain (P-LC) phosphorylation in bovine tracheal smooth muscle strips were compared to inhibition of purified myosin light chain kinase activity. These antagonists inhibited the Ca++-calmodulin-induced activation of myosin light chain kinase in a concentration-dependent manner, with IC50 values of 1.0 (calmidazolium), 25 (W-7) and 65 microM (CPZ), respectively. Inhibitory effects of these antagonists were abolished with increasing concentrations of calmodulin. However, when these antagonists were used in intact smooth muscle strips, the gradation of potencies did not parallel the anticalmodulin activities. W-7 (100 microM) exhibited a similar extent of antagonism between the contractile responses to carbachol and KCl. The increase in P-LC phosphate content in response to 1-min stimulation with 10(-5) M carbachol was inhibited by W-7. CPZ exhibited an unexpectedly potent antagonism on carbachol-induced isometric tension development and P-LC phosphorylation. Atropine showed an antagonism similar to CPZ. CPZ and verapamil had similar antagonistic effects on KCI-induced contractions. Calmidazolium (50 microM) produced no significant inhibition on carbachol-induced isometric tension development and P-LC phosphorylation in intact smooth muscle strips. It may be concluded that 1) W-7 antagonizes the smooth muscle contraction through the inhibition of the initial increase in the P-LC phosphorylation; 2) CPZ produces effects other than calmodulin antagonism; and 3) calmidazolium is not effective in intact smooth muscle strips.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-coupled G proteins mediate contraction and Ca++ mobilization in isolated intestinal muscle cells.

Guanosine 5'-O-(gamma-thio)triphosphate (GTP[S]), NaF and cholecystokinin-octapeptide (CCK-8) were used to examine the participation of G proteins in agonist-induced contraction of smooth muscle cells isolated separately from circular and longitudinal muscle layer of guinea pig intestine. All three agents stimulated inositol 1,4,5-triphosphate (InsP3) production and protein kinase C activity to the same extent in permeabilized (GTP[S] and CCK-8) and nonpermeabilized (NaF and CCK-8) muscle cells. InsP3 production was 9 to 13 times higher in circular muscle cells consistent with preferential hydrolysis of phosphatidylinositol 4,5-biphosphate in this cell type. InsP3 production and protein kinase C activation in permeabilized muscle cells were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM). Maximal concentrations of GTP[S] (100 microM), CCK-8 (1 nM) and InsP3 (1 microM) elicited similar increases in [Ca++]i, net 45Ca++ efflux and contraction in permeabilized circular, but not longitudinal, muscle cells [( Ca++]i: 224 +/- 35 nM, 279 +/- 29 nM and 288 +/- 45 nM increase above basal level; 45Ca++ efflux: 35 +/- 2%, 34 +/- 3% and 37 +/- 3% decrease in cell Ca++ content; contraction: 26 +/- 2%, 24 +/- 2% and 25 +/- 2% decrease in cell length). The responses to GTP[S] and CCK-8 were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM) and heparin (10 micrograms/ml), whereas the response to InsP3 was abolished by heparin only. Maximal concentrations of NaF and CCK-8 elicited similar increases in [Ca++]i and contraction in nonpermeabilized circular and longitudinal muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris

Previously, we have reported that activation of muscarinic cholinergic receptors in the iris smooth muscle results in a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol and myo-inositol trisphosphate (IP3) and that the stimulated hydrolysis of this phospholipid correlates well with contraction. To determine whether or not there is a causal relationship between PIP2 breakdown and contraction, we have conducted correlative studies on the effects of carbachol (CCh) on PIP2 breakdown, measured as IP3 accumulation, myosin light chain (MLC) phosphorylation and contraction in the rabbit iris sphincter. We have also investigated the effects of time, temperature, atropine antagonism, Ca++ and C-kinase activators on the three measured responses. The data obtained can be summarized as follows: dose-response studies for IP3 accumulation, MLC phosphorylation and contraction revealed a close correlation between these responses; kinetic data on atropine antagonism showed that the three measured responses are competitively inhibited by the muscarinic antagonist; time course studies conducted at low temperature showed that the CCh-induced IP3 accumulation and MLC phosphorylation may precede contraction; time course studies on the effect of Ca++ on the three measured responses showed that IP3 release may account for the rapid phase of CCh-induced contraction and that extracellular Ca++ is essential for sustained MLC phosphorylation and the slow phase of contraction; the activity of phospholipase C, the enzyme involved in PIP2 hydrolysis, in membrane fragments from 32P-labeled sphincter muscle was found to be highly sensitive to Ca++, with half-maximal stimulation at about 1.1 microM Ca++; and phorbol 12,13-dibutyrate, but not phorbol 12-myristate 13-acetate, induced MLC phosphorylation and muscle contraction in a dose- and time-dependent manner. Phorbol 12,13-dibutyrate and ionomycin acted in a synergistic manner to elicit contraction. In conclusion, contractions by CCh in the iris sphincter may be explained on the basis of enhanced PIP2 turnover and its derived second messenger molecule(s); that there are consistent correlations, using different concentrations of CCh, atropine antagonism, time, temperature and Ca++, between the stimulated hydrolysis of PIP2, MLC phosphorylation and contraction. Finally, whereas the data presented favor the involvement of IP3 in the phasic component of the contractile response, the studies with phorbol 12,13-dibutyrate suggest that contractile regulation by 1,2-diacylglycerol, through activation of C-kinase, may be important during the tonic component of smooth muscle contraction.     

Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle

The Ca dependence of contraction and myosin phosphorylation was investigated in canine tracheal smooth muscle stimulated with carbachol, K or serotonin. Previous studies of tracheal muscle showed carbachol concentration-response curves for contraction and myosin phosphorylation were superposable. In contrast, there was a striking difference in the Ca++ sensitivities of tension and myosin phosphorylation when Ca++ concentration-response curves were constructed in the presence of 10(-7) M carbachol. Significant phosphorylation (greater than 0.3 moles phosphate/mole 20,000 dalton myosin light chain) was observed in the absence of active tension. In the present study, carbachol (10(-7) and 10(-6) M) and serotonin (10(-5) M) also induced significant myosin phosphorylation in low Ca++ solutions (0-0.025 mM CaCl2) without proportional increases in tension. K+ depolarization in Ca++-free physiological salt solution (60 mM KCl, 10(-6) M atropine) yielded phosphorylation not significantly different from basal levels. All stimulants induced active stress after readmission of Ca. The Ca++ dependence curve for myosin phosphorylation in muscles stimulated with carbachol was shifted up and to the left of the force curve. Atropine (10(-6) M) significantly reduced phosphorylation induced by carbachol in Ca++-free solutions, as did 3 X 10(-6) M nifedipine and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Phorbol 12-myristate, 13-acetate or phorbol 12,13-dibutyrate did not increase basal phosphorylation or phosphorylation in low Ca++ solutions, suggesting that protein kinase C did not phosphorylate myosin in this case. Myosin phosphorylation under these conditions is not sufficient to support contraction, and is reduced by treatments that decrease Ca++ entry.(ABSTRACT TRUNCATED AT 250 WORDS)     

An unusual Ca(2+) entry pathway activated by protein kinase C in dog splenic artery.

The characteristics of the Ca(2+) entry pathways that are activated by protein kinase C (PKC) in canine splenic artery were investigated. Phorbol 12, 13-dibutyrate (PDB) contracted tissues and increased Ca(2+) influx. PDB-induced contraction was reduced by preincubation of tissues in Ca(2+)-free Krebs' solution (1 mM EGTA) but was unaffected when Ca(2+)-free solution was applied after contraction was initiated with PDB. In contrast, (45)Ca influx and contraction induced by PDB were resistant to nifedipine, Cd(2+), Gd(3+), La(3+), or Ni(2+) whether added before or during exposure to PDB. Indeed, Cd(2+) reduced (45)Ca(2+) efflux and potentiated Ca(2+) influx, but not PDB-induced contraction. Norepinephrine (NE)-induced contractions were inhibited by preincubation in Ca(2+)-free Krebs' solution (1 mM EGTA). Nifedipine (10 microM) led to a small reduction in the NE-induced contraction but was without effect on (45)Ca(2+) influx. Pretreatment for 16 min with Cd(2+), Gd(3+), or La(3+) (each 1 mM) reduced or abolished NE-induced contraction and Ca(2+) influx. Application of these cations after exposure to NE did not affect (45)Ca(2+) influx but reduced tension. The Q(10) for the increase in (45)Ca(2+) influx was approximately 2 for high K(+) and NE, but 4 for PDB. The results suggest that stimulation of PKC in dog splenic artery activates a Ca(2+) entry pathway that is resistant to di- and trivalent cations. The inhibition of Ca(2+) influx by preincubating with cations during short-term exposure to NE may represent an action on Ca(2+) turnover that precedes activation of PKC.     

Phorbol-stimulated Ca(2+) mobilization and contraction in dispersed intestinal smooth muscle cells

This study examined the source of Ca(2+) mobilized by phorbol esters and its requirement for phorbol-induced contraction of smooth muscle cells isolated from the circular and longitudinal layers of guinea pig intestine. Phorbol-12-myristate-13-acetate caused rapid, sustained, concentration-dependent muscle contraction and increase in cystolic free [Ca(2+)](i) in muscle cells from both layers. Maximal contraction was similar to that elicited by receptor-linked agonists, whereas maximal [Ca(2+)](i) was 50% less. The increase in [Ca(2+)](i) was mediated by Ca(2+) release in circular, and Ca(2+) influx in longitudinal muscle cells; only the latter was abolished by methoxyverapamil and in Ca(2+)-free medium. [Ca(2+)](i) was essential for contraction in both cell types: contraction in longitudinal muscle cells was abolished by methoxyverapamil and in Ca(2+)-free medium; contraction in circular muscle cells was abolished only after depletion of Ca(2+) stores. Contraction was abolished by the protein kinase C (PKC) inhibitor calphostin C (1 microM), but was not affected by the myosin light chain kinase inhibitor KT5926 (1 microM), suggesting that activation of myosin light chain kinase was suppressed by phorbol-12-myristate-13-acetate or via PKC. Phorbol-induced contraction of permeabilized circular and longitudinal muscle cells was abolished by pretreatment with a common antibody to Ca(2+)-dependent PKC-alpha,beta,gamma, but was not affected by pretreatment with a specific PKC-epsilon antibody. This study demonstrates the ability of phorbol esters to mobilize Ca(2+) from different sources in different smooth muscle cell types and establishes the requirement of Ca(2+) for phorbol-induced contraction; the latter is exclusively mediated by Ca(2+)-dependent PKC isozymes.     

Plasma membrane calcium flux, protein kinase C activation and smooth muscle contraction

Isolated perfused rabbit ear arteries contract when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the calcium-activated, phospholipid-dependent protein kinase or C-kinase. Under conditions where the calcium concentration in the perfusate is 1.5 mM and the potassium concentration is 4.8 mM, there is a latent period of 70 +/- 19 min (mean +/- S.E.M., n = 10) between TPA addition and the onset of the contractile response. Once initiated, the contractile response is progressive and sustained. When perfusion conditions are altered in such a way as to modify calcium flux across the plasma membrane (i.e., raising the extracellular calcium concentration to 2.5 mM Ca++, raising the extracellular potassium concentration to 10 mM, and/or preincubating the tissues in media containing 100 nM Bay K 8644, a potent calcium channel agonist), the latency period between TPA addition and initiation of the contractile response is significantly reduced (2.5 mM Ca++, 37 +/- 7 min; 10 mM K+ and 2.5 mM Ca++, 11 +/- 3 min; 100 nM Bay K 8644 and 1.5 mM Ca++, 20 +/- 7 min; 100 nM Bay K 8644 and 2.5 mM Ca2+, 8.5 +/- 1.7 min; 10 mM K+ and 100 nM Bay K 8644, 11 +/- 5 min). Likewise, the combination of 2.5 mM calcium, 100 nM Bay K 8644, and 3.3 microM ouabain results in a contractile response 4.5 +/- 2.0 min after TPA addition (means +/- S.E.M., n = 4). Control tissues (absence of TPA addition) run simultaneously show no contractile responses to the various Ca++ flux regulators even after 90 min of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of endothelin-1 on guinea pig gallbladder smooth muscle in vitro.

The purpose of this study was to investigate the pharmacological activity of endothelin-1 (ET-1) on guinea pig gallbladder smooth muscle. Guinea pig gallbladder muscle strips were mounted in 10-ml siliconized organ baths containing Krebs' solution. After 1 hr of equilibration, ET-1 was added cumulatively. ET-1 induced slow-developing and long-duration contractile responses. The EC50 was approximately 10 nM. ET-1 was 5 times less potent than cholecystokinin (EC50, 2 nM), but 20 and 40 times more potent than carbachol (EC50, 200 nM) and histamine (EC50, 400 nM), respectively. The concentration-response curve to ET-1 was not affected by tetrodotoxin (0.1 microM) or by the muscarinic antagonist, atropine (10 microM). The neuronal N-type calcium channel blocker, omega-conotoxin (0.1 microM), had no significant effect on the ET-1 concentration-response curve. In contrast, the contractile effect to ET-1 was reduced markedly by removal of extracellular calcium or by the voltage-dependent calcium channel blockers nicardipine and diltiazem. Substitution of strontium (an inhibitor of intracellular calcium release) for Ca++ significantly reduced the response to ET-1. The cyclooxygenase inhibitor indomethacin had no significant effect on the contractile activity of ET-1. These finding suggest that ET-1 is a potent contractile stimulant of guinea pig gallbladder and that it acts directly on the smooth muscle. The activity depends on extracellular Ca++, suggesting involvement of Ca++ influx via the voltage-dependent Ca++ channel and on intracellular calcium.     

Potent stimulation of myofilament force and adenosine triphosphatase activity of canine cardiac muscle through a direct enhancement of troponin C Ca++ binding by MCI-154, a novel cardiotonic agent

In the present study we have analyzed a likely biochemical mechanism underlying the Ca++-sensitizing action of MCI-154 (6-[4-(4'-pyridyl)aminophenyl)-4,5-dihydro-3(2H)-pyridazinone hydrochloride), a novel cardiotonic agent, on the contractile protein system. MCI-154 (10(-7) to 10(-4) M) enhanced the tension development induced by -log molar-free Ca++ concentration (pCa) 5.8 in chemically skinned fiber from the canine right ventricular muscle in a concentration-dependent manner. At pCa 7.0, MCI-154 (10(-7) to 10(-4) M) markedly increased adenosine triphosphatase (ATPase) activities of canine myofibrils and reconstituted actomyosin. In myofibrils and reconstituted actomyosin, MCI-154 (10(-7) to 10(-4) M) caused a parallel shift of the pCa-ATPase activity relation curve to the left without affecting the maximum activity, suggesting an increase in Ca++ sensitivity. MCI-154 (10(-8) to 10(-4) M) had little effect on actin-activated, Mg++, Ca++ and (K+, EDTA)-ATPase activities of myosin. Ca++ binding to cardiac myofibrils or purified cardiac troponin was increased by 10(-4) M MCI-154. These results suggest that MCI-154 enhances Ca++ binding to cardiac troponin C to elevate the Ca++ sensitivity of myofilaments and thus may cause a positive inotropic action in cardiac muscle. MCI-154 may provide a valuable tool for studying the molecular mechanism by which Ca++ regulates the contractile system.     

Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A

Calyculin-A (CL-A), a novel marine toxin isolated from Discodermia calyx, caused contraction in the smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence (with 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) of external Ca++ at concentrations ranging from 1 X 10(-8) to 1 X 10(-6) M. In the presence of external Ca++, the contraction induced by CL-A was accompanied by an increase in the cytosolic free Ca++ concentration [( Ca++]cyt) as measured by the fluorescence indicator fura-2. Verapamil (3 X 10(-6) M) inhibited the increase in [Ca++]cyt, but not tension development caused by CL-A. In the absence of external Ca++, CL-A still caused contraction without changing [Ca++]cyt. Thus, from studies with intact smooth muscle it was demonstrated that, in the absence of external Ca++, CL-A can induce a contraction that was not accompanied by an increase in [Ca++]cyt. In permeabilized taenia, CL-A caused contraction in the absence of Ca++ (with 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid) at concentrations similar to those required to contract intact tissue. This contraction was inhibited by the nonselective kinase inhibitors such as amiloride (1 X 10(-3) M) and K-252a (2 X 10(-5) M). Low concentrations of Ca++ (approximately 1 X 10(-6) M) augmented the CL-A-induced contraction in the permeabilized taenia. In native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.     

Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-independent, inhibitory effects of cyclic adenosine 5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.     

Angiotensin II amplifies arterial contractile response to norepinephrine without increasing Ca++ influx: role of protein kinase C.

We investigated whether the enhanced contractile response to norepinephrine caused by a subthreshold concentration of angiotensin II was associated with an increased 45Ca++ influx or net uptake. Rabbit facial artery segments were mounted isometrically to measure the 45Ca++ influx and net uptake in response to norepinephrine. The contractile response to norepinephrine (3 microM) in the presence of angiotensin II (0.1 nM) was 149.5 +/- 7.4% of control. This response amplification was not associated with changes in norepinephrine-induced 45Ca++ influx or net uptake. Angiotensin II also potentiated the contractile response to caffeine obtained in a Ca(++)-free buffer containing ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (2 mM) to 148.0 +/- 4.8% of control. In both cases, the amplification was prevented by pretreatment with either staurosporine (10 nM) or calphostin C (100 nM), two inhibitors of protein kinase C. We conclude that angiotensin II potentiation of norepinephrine-induced vascular tone occurs in the absence of changes in stimulated Ca++ entry. This potentiation may be due to an increase in intracellular sensitivity to Ca++, possibly mediated by protein kinase C.     

Inhibition by amiloride of contractile elements in smooth muscle of guinea pig taenia cecum and chicken gizzard

The relaxant action of amiloride was investigated in the smooth muscles of guinea pig taenia ceci and chicken gizzard. Amiloride inhibited the contractions induced by high K+ (45.4 mM) and carbachol (10 microM) in the taenia with the concentrations needed to induce 50% inhibition (IC50) of approximately 41 microM. A prolonged incubation period (greater than 1 hr) was necessary to obtain the full inhibition of these contractions. The taenia gradually accumulated amiloride and the tissue/medium ratio exceeded 2.0 after a 120-min incubation period. Amiloride had no effect on the high K+-stimulated 45Ca++ uptake or the ATP content of the taenia. Amiloride inhibited the Ca++-induced contraction of the saponin-treated taenia with an IC50 of 186 microM. Amiloride (10-1000 microM) also inhibited superprecipitation and Mg++-adenosine triphosphatase activity of the gizzard native actomyosin as well as the phosphorylation of myosin light chain. The inhibition of the phosphorylation was antagonized competitively by ATP. Amiloride (1 mM) had no effect on the dephosphorylation of myosin light chain upon removal of Ca++ from reaction medium. Amiloride, at concentrations up to 1 mM, had not effect on calmodulin activity as monitored by the Ca++-calmodulin-activated erythrocyte membrane (Ca++ + Mg++)-adenosine triphosphatase and phosphodiesterase activities. In contrast to this, trifluoperazine inhibited the calmodulin activity at the concentration needed to inhibit the Ca++-induced contraction of the permeabilized taenia and the superprecipitation and the phosphorylation of myosin light chain of gizzard. We conclude that amiloride, unlike trifluoperazine, may inhibit directly the myosin light chain kinase activity to induce muscle relaxation.