A major role for the rho-associated coiled coil forming protein kinase in G-protein-mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea

1 G protein-mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho-associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y-27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-induced contraction in alpha-toxin- or beta-escin-permeabilized rabbit trachea. 2 Although pre-treatment with Y-27632 dose-dependently inhibited GTPgammaS (10 microM)-induced Ca2+ sensitization of alpha-toxin-permeabilized trachea, a Y-27632-insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPgammaS response in the presence of Y-27632 (100 microM). 3 GF 109203X (5 microM) abolished 1 microM 4beta-phorbol 12, 13-dibutyrate (PDBu)-induced, but only partially inhibited the GTPgammaS-induced Ca2+ sensitization. A combination of Y-27632 (100 microM) and GF 109203X (5 microM) totally abolished the GTPgammaS response. 4 GTPgammaS caused only a small contraction in the absence of Ca2+. Wortmannin (30 microM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+-induced contraction. ATP-triggered contraction of the strip which had been treated with calyculin A (1 microM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 microM), but not by Y-27632 (100 microM), in the presence of GTPgammaS and Ca2+. 5 GTPgammaS, but not PDBu, contracted the beta-escin-permeabilized trachea in the absence of Ca2+, but the presence of Ca2+-independent MLCK. 6 We conclude that ROCK plays a primary role in G-protein-mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.     

Effect of a peptide inhibitor of protein kinase C on G-protein-mediated increase in myofilament Ca(2+)-sensitivity in rabbit arterial skinned muscle.

1. To investigate the role of protein kinase C in the increase mediated by guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins) in the sensitivity of the contractile proteins to Ca2+ in vascular smooth muscle, the effect of a novel peptide inhibitor of protein kinase C (PKC19-36) on Ca(2+)-induced contraction and myosin light chain (MLC) phosphorylation was studied in the presence and absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in beta-escin-skinned smooth muscle strips of rabbit mesenteric artery. For comparison, the effects were also observed of PKC19-36 on the action of phorbol 12,13-dibutylate (PDBu, an activator of PKC) on the two Ca(2+)-induced responses. 2. In beta-escin-skinned strips treated with ionomycin, Ca2+ (0.1-3 microM) concentration-dependently produced contraction in parallel with an increase in MLC-phosphorylation. GTP gamma S (10 microM) and PDBu (0.1 microM) each shifted both the Ca(2+)-force and Ca(2+)-MLC-phosphorylation relationships to the left without a significant change in either maximum response. The relationship between force and MLC-phosphorylation was not modified by either GTP gamma S or PDBu, indicating that the sensitivity of MLC-phosphorylation to Ca2+ is enhanced by both GTP gamma S and PDBu. 3. PKC19-36 itself modified neither the contraction nor MLC-phosphorylation induced by Ca2+ but it did block the PDBu-induced enhancement of these two Ca(2+)-induced responses. By contrast, PKC19-36 did not modify the GTP gamma S-induced enhancement of the two Ca(2+)-induced responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF-2 cells.

1. The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor- and thapsigargin-mediated increases in [Ca2+]i in DDT1 MF-2 smooth muscle cells. 2. After pretreatment of cells with GF109203X (5 microM, 45 min), the histamine (100 microM)-induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 +/- 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 +/- 26%) in the presence of the PKC inhibitor. 3. The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration-dependent manner (IC50: 3.1 +/- 1.1 microM). 4. Histamine-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X. 5. The PKC activating phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), strongly reduced histamine-induced Ins(1,4,5)P3 formation (58 +/- 16%). This effect was reversed by GF109203X (5 microM). Furthermore, PMA diminished histamine evoked Ca2+ release (50 +/- 6%) and blocked Ca2+ entry completely. 6. The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2(+)-ATPase with thapsigargin (1 microM), was strongly reduced (57 +/- 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long-term pretreatment of cells with PMA (1 microM, 48 h) did not abolish this effect of GF109203X (48 +/- 3% inhibition). 7. In permeabilized DDT, MF-2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 microM) was markedly reduced (42 +/- 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function. 8. Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 +/- 11%), by histamine (24 +/- 6%) and also by thapsigargin (121 +/- 10%). The GF109203X- and the thapsigargin-induced uptake of 45Ca2+ were not additive. 9. These data suggest that GF109203X reduces the filling-state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store-dependent (capacitive) Ca2+ entry.     

Mechanism underlying H2O2-induced inhibition of acetylcholine-induced contraction in rabbit tracheal smooth muscle

The mechanism underlying the inhibition by H2O2 of acetylcholine-induced contraction was investigated in epithelium-denuded strips of rabbit trachea. Acetylcholine (10 microM) generated a phasic, followed by a tonic increase in both the intracellular Ca2+ concentration ([Ca2+]i) and force. Although the acetylcholine-induced tonic contraction was around 9 times the high K+ (80 mM)-induced one, the two stimulants induced similar [Ca2+]i increases (around 0.2 microM), indicating that acetylcholine generates tonic contraction via increases in both [Ca2+]i and myofilament Ca2+-sensitivity. H2O2 (30 microM) (a) enhanced the acetylcholine-induced tonic (not phasic) increase in [Ca2+]i but attenuated both phases of the acetylcholine-induced contraction and (b) enhanced the high K+-induced increase in [Ca2+]i but did not modify the high K+-induced contraction. In beta-escin-skinned strips, application of acetylcholine in the presence of GTP enhanced the contraction induced by 0.3 microM Ca2+ so that its amplitude became similar to that induced by 1 microM Ca2+. H2O2 (30 microM) attenuated the contraction induced by 0.3 microM Ca2+ (alone or in the presence of acetylcholine) but not those induced by higher concentrations of Ca2+ alone (0.5 microM and 1 microM). These results indicate that H2O2 acts directly on contractile proteins in rabbit tracheal smooth muscle to inhibit the contraction induced by low concentrations of Ca2+ (<0.5 microM). An action of H2O2 that increases [Ca2+]i (and thereby masks this reactive-oxygen-induced inhibition of myofilament Ca2+-sensitivity) is apparent in the presence of high K+ but not of acetylcholine. Thus, in rabbit tracheal smooth muscle H2O2 downregulates myofilament Ca2+-sensitivity more potently during acetylcholine-induced contraction than during high-K+-induced contraction, leading to an effective inhibition of the former contraction.     

Studies of the effect of glyceryl trinitrate and cyclic GMP on calcium turnover in bovine mesenteric artery

It was recently observed that the relaxation induced by glyceryl trinitrate (GTN) showed a biphasic concentration-response curve; a high-sensitivity component represented by concentrations less than 1 nM and a low-sensitivity component represented by concentrations greater than 1 nM. The effect of two glyceryl trinitrate concentrations (0.1 nM and 1 microM) were tested on the uptake of 45Ca2+ to tissue pieces of bovine mesenteric arteries (BMA) as well as on the uptake of 45Ca2+ to a microsomal preparation of BMA. The effect of GTN and 8-Br-cGMP was also studied on the IP3-induced release of Ca2+ from the microsomal preparation preloaded with 45Ca2+. The influence of IP3 and GTN on the activity of Ca2(+)-ATPase in the microsomal preparation was tested as well. The phenylephrine-stimulated uptake of Ca2+ to tissue pieces of BMA was significantly reduced by the high GTN-concentration (1 microM) but not by the lower concentration. The uptake of Ca2+ to the microsomal preparation was significantly stimulated by the two GTN-concentrations tested, as well as by 8-Br-cGMP (0.1 mM). The calcium release induced by IP3 (1 microM) from the microsomal preparation was inhibited by both the low and the high GTN-concentration and by 8-Br-cGMP (0.1 mM). The Ca2(+)-ATPase activity was stimulated by both GTN-concentrations tested while it was inhibited by IP3. It is concluded that GTN is able to induce a reduction of the free intracellular Ca2+ by several mechanisms, which are of importance for the relaxation represented by the high-affinity component. The low-affinity component in addition reduces the inflow of Ca2+ over the plasma membrane.     

Endothelin-1 acts via protein kinase C to block KATP channels in rabbit coronary and pulmonary arterial smooth muscle cells

We investigated the effects of the vasoconstrictor endothelin-1 (ET-1) on the whole-cell ATP-sensitive K+ (KATP) currents of smooth muscle cells that were isolated enzymatically from rabbit coronary artery (CASMCs) and pulmonary artery (PASMCs). The size of the KATP current did not differ significantly between CASMCs and PASMCs. ET-1 reduced the KATP current in a concentration-dependent manner, and this inhibition was greater in PASMCs than in CASMCs (half-inhibition values of 12.20 nM and 1.98 nM in CASMCs and PASMCs, respectively). However, the level of inhibition induced by other vasoconstrictors (angiotensin II, norepinephrine, and serotonin) were not significantly different between CASMCs and PASMCs. Pretreatment with the protein kinase C (PKC) inhibitors staurosporine (100 nM) and GF 109203X (1 microM) prevented ET-1-induced inhibition of the KATP current in both arterial smooth muscle cell preparations. The PKC activators phorbol-12,13-dibutyrate (PDBu) and 1-olelyl-2-acetyl-sn-glycerol (OAG) reduced the KATP current in dose-dependent manner. Although the numbers of ET receptors were not significantly different between the 2 arterial smooth muscle cell preparations, the effects of PDBu and OAG were greater on PASMCs. ET-1-induced inhibition of the KATP current was unaffected by the PKA inhibitor Rp-cAMPs (100 microM) and PKA inhibitory peptide (5 microM).     

Protein kinases participate in the contraction in response to levobupivacaine in the rat aorta

Levobupivacaine is a long-acting amide local anesthetic that intrinsically produces vasoconstriction both in vivo and in vitro. Levobupivacaine increases intracellular calcium concentrations ([Ca(2+)](i)) in vascular smooth muscle cells. The goals of this in vitro study were to investigate whether levobupivacaine-induced contraction is associated with increased Ca(2+) sensitivity and to identify the protein kinases involved in mediating contraction in response to levobupivacaine in isolated rat aortic smooth muscle. The effect of levobupivacaine and potassium chloride (KCl) on the [Ca(2+)](i) and tension was measured simultaneously with acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative levobupivacaine concentration-response curves were generated in the presence or absence of the following antagonists: GF 109203X; Y-27632; genistein; SP600125; PD 98059; and SB 203580. Levobupivacaine-induced protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) phosphorylation and Rho-kinase (ROCK-2) membrane translocation were detected in rat aortic vascular smooth muscle cells using Western blotting. The slope of the [Ca(2+)](i)-tension curve for levobupivacaine was higher than that for KCl. Y-27632, GF 109203X, and SP600125 attenuated levobupivacaine-induced contraction in a concentration-dependent manner. Genistein, PD 98059, and SB 203580 attenuated levobupivacaine-induced contraction. Pretreatment with GF 109203X and Y-27632 inhibited levobupivacaine-induced PKC phosphorylation and Rho-kinase (ROCK-2) membrane translocation, respectively. Pretreatment with SP600125 or PD 98059 attenuated the levobupivacaine-induced phosphorylation of JNK and ERK, respectively. These results indicate that levobupivacaine-induced contraction involving an increase in myofilament Ca(2+) sensitivity involves the primary activation of Rho-kinase-, PKC-, and JNK-mediated pathways of rat aortic smooth muscle.     

Effect of KC399, a newly synthesized K+ channel opener, on acetylcholine-induced electrical and mechanical activities in rabbit tracheal smooth muscle.

1. Effects of KC399, an opener of ATP-sensitive K+ channels were investigated on membrane potential, isometric force and intracellular Ca2+ ([Ca2+]i) mobilization induced by acetylcholine (ACh) in smooth muscle from the rabbit trachea. 2. In these smooth muscle cells, ACh (0.1 and 1 microM) depolarized the membrane in a concentration-dependent manner, KC399 (1-100 nM) hyperpolarized the membrane whether in the presence or absence of ACh. When the concentration of ACh was increased, the absolute values of the membrane potential induced by the maximum concentration of KC399 were less negative. 3. ACh (0.1 to 10 microM) concentration-dependently produced a phasic, followed by a tonic increase in both [Ca2+]i and force. KC399 (above 3 nM) lowered the resting [Ca2+]i and attenuated the ACh-induced phasic and tonic increases in [Ca2+]i and force, in a concentration-dependent manner. The magnitude of the inhibition was greater for the ACh-induced tonic responses than for the phasic ones. Nicardipine (0.3 microM), a blocker of the L-type Ca2+ channel, attenuated the ACh-induced tonic, but not phasic, increases in [Ca2+]i and force. KC399 further attenuated the ACh-induced tonic responses in the presence of nicardipine. 4. In beta-escin-skinned strips, Ca2+ (0.3-10 microM) produced a contraction in a concentration-dependent manner. KC399 (0.1 microM) had no effect on the Ca(2+)-force relationship in the presence or absence of ATP with GTP. However, at a very high concentration (1 microM), this agent slightly shifted the relationship to the right and attenuated the maximum Ca(2+)-induced contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by protease-activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation

1 The present study examined effects of agonist enzymes and receptor-activating peptides for protease-activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. 2 Thrombin at 0.03-0.1 microM and the PAR-1-activating peptide SFLLR-NH2 at 3-100 microM or TFLLR-NH2 at 10-50 microM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH2 at 10-100 microM elicited only small contraction. Trypsin at 0.08 microM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH2 at 1000 microM exhibited no effect. 3 The contractile responses of the duodenal strips to TFLLR-NH2 and to SLIGRL-NH2 were partially attenuated by the L-type calcium channel blocker nifedipine (1 microM), the protein kinase C inhibitor GF109203X (1 microM) and the tyrosine kinase inhibitor genistein (15 microM), but were resistant to indomethacin (3 microM) and tetrodotoxin (1-10 microM). 4 The relaxation of the preparations exerted by TFLLR-NH2 was unaffected by indomethacin (3 microM), propranolol (5 microM), NG-nitro-L-arginine methyl ester (100 microM) and tetrodotoxin (1-10 microM). This relaxation was resistant to either GF109203X (1 microM) or genistein (15 microM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 microM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 microM), completely abolished the PAR-1-mediated duodenal relaxation, and significantly enhanced the PAR-1-mediated contraction. 6 These findings demonstrate that PAR-1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR-2 plays a minor excitatory role in the muscle, and that PAR-4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR-1 and PAR-2 activation are mediated, in part, by activation of L-type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR-1 activation occurs via activation of apamin-sensitive, but charybdotoxin-insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.     

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) in vascular smooth muscle

The inhibitory effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca2+ level ([Ca2+]i) were examined using isolated rabbit aorta loaded with a fluorescent Ca2+ indicator, fura-2. TMB-8 (100 microM) decreased the high K(+)-induced increase in muscle tension, and [Ca2+]i and 45Ca2+ influx to their respective resting levels. TMB-8 (100 microM) almost completely inhibited the increase in [Ca2+]i and 45Ca2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 microM) inhibited the remaining portion of the contraction without additional decrease in [Ca2+]i. The inhibitory effect of TMB-8 on high K(+)-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca2+ concentrations or by the Ca2+ channel activators, CGP 28,392 and by Bay K8644. In Ca(2+)-free solution, norepinephrine-induced transient increases in [Ca2+]i and muscle tension and 100 microM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca2+]i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 microM) did not inhibit the Ca(2+)-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

Potentiation of beta-adrenoceptor function in bovine tracheal smooth muscle by inhibition of protein kinase C

To examine the role of contractile agonist-induced activation of protein kinase C (PKC) in functional antagonism of airway smooth muscle contraction by beta-adrenoceptor agonists, we examined the effects of the specific PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide) on isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by various concentrations of methacholine and histamine. In the absence of GF 109203X, the potency of isoprenaline (pD(2)) was gradually reduced at increasing methacholine- and histamine-induced smooth muscle tones, but the maximal relaxation (E(max)) was decreased only at higher concentrations of methacholine. In the presence of GF 109203X, pD(2) values were significantly increased for both methacholine- and histamine-induced contractions. Moreover, isoprenaline E(max) values in the presence of high concentrations of methacholine were also increased. Although both methacholine- and histamine-induced contractions were slightly reduced by GF 109203X, the changes in isoprenaline pD(2) could only partially be explained by reduced contractile tone. In contrast to isoprenaline, forskolin-induced relaxations were not affected by GF 109203X. The results indicate that PKC activation contributes to the reduced beta-adrenergic responsiveness induced by methacholine and histamine, which may involve uncoupling of the beta-adrenoceptor from the effector system. Since many mediators and neurotransmitters in allergic airway inflammation can activate PKC, this cross talk may be important in the reduced bronchodilator response of patients with severe asthma.     

Modulation of calmodulin function and of Ca2+-induced smooth muscle contraction by the calmodulin antagonist, HT-74

The relationship between the functions of calmodulin (CaM) and Ca2+-induced smooth muscle contraction was investigated using a newly synthesized CaM antagonist, 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4- -phenylpiperidinyl)propyl]benzenesulfonamide (HT-74). We noted a selectivity of HT-74 for CaM, compared to other calcium-binding proteins and target enzymes of CaM. As HT-74 had no significant effect on the intensity of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence in the presence of the Ca2+-CaM complex, the HT-74-binding sites may differ from those of naphthalenesulfonamides and phenothiazines which decrease ANS fluorescence. The Ca2+ binding to CaM was inhibited significantly by 1.0 microM HT-74, in sharp contrast to phenothiazines and naphthalenesulfonamides which increase the extent of the Ca2+ binding to CaM. Increasing CaM concentrations reversed the HT-74-induced inhibition of CaM-dependent enzymes such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase, with Ki values of 0.5 microM and 0.4 microM, respectively. In the presence of 0.3 microM HT-74, potassium-depolarized rabbit aortic strips pre-contracted with 0.3 mM CaCl2 relaxed, and this relaxation was completely reversed by the addition of an excess amount of CaCl2 (10 mM). This compound shifted the dose-response curve for CaCl2 to the right, in a competitive manner. However, HT-74 inhibited the phenylephrine-induced contraction elicited in Ca2+-free solution and the calcium ionophore A23187-induced contraction in the presence of calcium ion. Therefore, this agent affects intracellular actions of Ca2+ rather than membrane receptors or the influx of Ca2+. HT-74 is a CaM antagonist which binds to CaM in a manner different from that heretofore reported. It inhibits Ca2+ binding to CaM and produces a competitive inhibition of Ca2+-induced contractions of depolarized vascular smooth muscle.     

Effects of glutathione S-transferase inhibitors on nitroglycerin action in pig isolated coronary arteries

1. The present study was designed to clarify the role of glutathione S-transferase (GST) in the vasorelaxation response and development of tolerance to nitroglycerin (GTN) using GST inhibitors. 2. In pig isolated coronary arteries, GST activity was significantly changed to 77 and 82, or 69% of the control level (100%) following treatment with bromosulphophthalein (BSP; 10-3 and 10-4 mol/L) or ethacrynic acid (ETA; 10-4 mol/L), both GST inhibitors, respectively, but not following treatment with 10-3 and 10-4 mol/L GTN (GST activity 97 and 98% of control, respectively). 3. In KCl-contracted coronary artery strips pre-incubated with 10-5 and 10-4 mol/L GTN, 10-4 and 10-3 mol/L BSP or 10-4 mol/L ETA, concentration-dependent relaxations produced by GTN were significantly decreased compared with control. 4. 8-Bromo cGMP (8-Br-cGMP), a membrane-permeable cGMP analogue, produced concentration-dependent relaxations in GTN-pretreated arterial strips that were identical to control responses. However, there was weak but significant decrease in concentration-dependent relaxations in response to 8-Br-cGMP in BSP- and ETA-pretreated arteries. 5. The cGMP content in coronary arteries was significantly increased with GTN, GTN + BSP or GTN + ETA to similar high levels compared with control. 6. The results of the present study show that BSP and ETA decrease GTN- and 8-Br-cGMP-induced vasorelaxation, but have no effect on the GTN-induced increase in cGMP content in coronary arteries, suggesting a possibility that the GST inhibitors may have depressant actions on GTN- and 8-Br-cGMP-induced vasorelaxation through direct inhibition of the vasorelaxation of vascular smooth muscle themselves, in addition to having inhibitory effects GST activity.     

Inhibitory mechanism of monensin on high K+-induced contraction in guniea-pig urinary bladder

In this study, we examined the inhibitory mechanism of monensin on high K+-induced contraction in guinea-pig urinary bladder. The relaxant effect of monensin (0.001 - 10 microM) was more potent than those of NaCN (100 microM - 1 mM) and forskolin (3 - 10 microM). Monensin (0.1 microM), NaCN (300 microM), or forskolin (10 microM) inhibited high K+-induced contraction without decreasing [Ca2+]i level. Monensin and NaCN remarkably decreased creatine phosphate and ATP contents. Monensin and NaCN inhibited high K+-induced increases in flavoprotein fluorescence, which is involved in mitochondrial respiration. Forskolin increased cAMP content but monensin did not. Monensin increased Na+ content at 10 microM but not at 0.1 microM that induced maximum relaxation. In the alpha-toxin-permeabilized muscle, forskolin significantly inhibited the Ca2+-induced contraction, but monensin did not affect it. These results suggest that the relaxation mechanism of monensin in smooth muscle of urinary bladder may be an inhibition of oxidative metabolism.     

Agonist-dependent difference in the mechanisms involved in Ca2+ sensitization of smooth muscle of porcine coronary artery

This study was undertaken to explore possible signal-transduction mechanisms involved in the Ca2+-sensitizing effects of carbachol and endothelin-1 (ET-1) by using beta-escin-skinned smooth muscle of porcine coronary artery. Pretreatment with C3 exoenzyme of Clostridium botulinum, which selectively inactivates rho p21 by adenosine diphosphate (ADP) ribosylation, resulted in a significant inhibition of ET-1-induced Ca2+ sensitization, but had no effect on carbachol-induced Ca2+ sensitization. Whereas the protein kinase C (PKC) inhibitors calphostin C and staurosporine did not affect the Ca2+-sensitizing effect of carbachol, the tyrosine kinase inhibitors genistein and tyrphostin 25 greatly but incompletely suppressed it. In contrast, the Ca2+-sensitizing effect of ET-1 was significantly inhibited by either calphostin C or genistein. Although the inhibitory effect of calphostin C on ET-1-induced Ca2+ sensitization was less than that of genistein, the effects of calphostin C and genistein were additive. The genistein-sensitive component of ET-1-induced Ca2+ sensitization appeared to include the C3-sensitive one. However, a substantial enhancement by ET-1 of the Ca2+-induced contraction was observed even in the presence of the two inhibitors. In beta-escin-skinned smooth muscle of rabbit mesenteric artery, ET-1-induced Ca2+ sensitization was marginally affected by C3 pretreatment, calphostin C, and genistein. We conclude that, although PKC activation and rho p21 protein-dependent and -independent tyrosine phosphorylation each plays an important role in an increase in myofilament Ca2+ sensitivity, the contributions of these signaling pathways to Ca2+ sensitization are different depending on receptor agonists and tissues used. Furthermore, these data suggest the existence of an as yet undefined signal-transduction mechanism involved in Ca2+ sensitization caused by receptor agonists.     

Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle.

Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.     

Effects of a water-soluble forskolin derivative (NKH477) and a membrane-permeable cyclic AMP analogue on noradrenaline-induced Ca2+ mobilization in smooth muscle of rabbit mesenteric artery.

1. Effects were studied of 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative and of dibutyryl-cyclic AMP, a membrane-permeable cyclic AMP analogue on noradrenaline (NA)-induced Ca2+ mobilization in smooth muscle strips of the rabbit mesenteric artery. The intracellular concentration of Ca2+ ([Ca2+]i), isometric force and cellular concentration of inositol 1,4,5-trisphosphate (InsP3) were measured. 2. NA (10 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force in a solution containing 2.6 mM Ca2+. NKH477 (0.01-0.3 microM) attenuated the phasic and the tonic increases in both [Ca2+]i and force induced by 10 microM NA, in a concentration-dependent manner. 3. In Ca(2+)-free solution containing 2 mM EGTA with 5.9 mM K+, NA (10 microM) produced only phasic increases in [Ca2+]i and force. NKH477 (0.01 microM) and dibutyryl-cyclic AMP (0.1 mM) each greatly inhibited these increases. 4. NA (10 microM) led to the production of InsP3 in intact smooth muscle strips and InsP3 (10 microM) increased Ca2+ in Ca(2+)-free solution after a brief application of Ca2+ in beta-escin-skinned smooth muscle strips. NKH477 (0.01 microM) or dibutyryl-cyclic AMP (0.1 mM) modified neither the NA-induced synthesis of InsP3 in intact muscle strips nor the InsP3-induced Ca2+ release in skinned strips. 5. In Ca(2+)-free solution, high K+ (40 and 128 mM) itself failed to increase [Ca2+]i but concentration-dependently enhanced the amplitude of the increase in [Ca2+]i induced by 10 microM NA with a parallel enhancement of the maximum rate of rise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cilostazol, a selective cAMP phosphodiesterase inhibitor on the contraction of vascular smooth muscle

The effects of cilostazol (OPC-13013, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quin olinone) on cyclic nucleotide metabolism and Ca2+-induced contraction of intact and skinned rabbit arterial smooth muscles were investigated. The concentrations of cilostazol producing 50% inhibition of cyclic adenosine monophosphate phosphodiesterase and Ca2+-dependent cyclic nucleotide phosphodiesterase were 0.4 microM and above 100 microM, respectively. This compound has no significant effect on adenylate cyclase in concentrations of up to 100 microM. Addition of cilostazol increased significantly the cAMP content without significant effect on cyclic guanosine monophosphate level of rabbit thoracic aorta in the presence of forskolin. Moreover, the ED50 value of cilostazol in relaxation of rabbit mesenteric arterial strips was decreased selectively by addition of 0.01 microM forskolin, which alone at this concentration has no effect on vascular contraction. Cilostazol of up to 30 microM did not suppress the Ca2+-induced contraction of the chemically skinned rabbit mesenteric artery. Therefore, cilostazol may produce the relaxation of intact vascular smooth muscle by its inhibition of cyclic adenosine monophosphate hydrolysis.     

Involvement of Rho kinase and protein kinase C in carbachol-induced calcium sensitization in beta-escin skinned rat and guinea-pig bladders

1. The signal transduction pathways involved in carbachol (CCh)-induced calcium sensitization in beta-escin permeabilized rat and guinea-pig bladder smooth muscles were investigated and the results were compared with guinea-pig taenia caecum. 2. Calcium contractions elicited cumulatively (pCa 7.5-5) in the presence of calmodulin were significantly increased in all three tissues when CCh (50 microM) was added to the medium. 3. Under constant [Ca2+]i conditions (pCa 6), calmodulin (1 microM) and then GTP (100 microM) initiated significant contractions. CCh (50 microM) added to the bath caused a further contraction in all three tissues - calcium sensitization. This sensitization was significantly inhibited by atropine (50 microM). 4. The incubation of the tissues with the IP3-receptor blocker 2-APB (30 microM) reduced the subsequent development of calcium sensitization by CCh in rat bladder but did not affect it in guinea-pig bladder and taenia ceacum. 5. The Rho kinase (ROK) inhibitor Y-27632 (5 microM) added in the presence of CCh reversed the calcium sensitization in rat bladder, whereas a transient contraction followed by a relaxation to a level not significantly different from the CCh contraction was seen in both guinea-pig bladder and taenia caecum. Y-27632 (1 microM) continuously present significantly inhibited the CCh-induced Ca2+ sensitization in rat bladder but not in guinea-pig bladder or taenia caecum. 6. In the presence of cyclopiazonic acid (CPA) (1 microM) and calmodulin (1 microM), Y-27632 (5 microM) did not change the calcium response curve (3 x 10(-7)-10(-5) M) in rat bladder but increased the contractile responses significantly in both guinea-pig bladder and taenia caecum. 7. The protein kinase C (PKC) inhibitor GF 109203X (5 microM) added in the presence of CCh inhibited the calcium sensitization induced by this muscarinic agonist in all three tissues in different ratios. 8. In conclusion, muscarinic receptor activation induces calcium sensitization in rat and guinea-pig detrusor smooth muscles but there are differences in their pathways.     

Effects of pinacidil on contractile proteins in high K(+)-treated intact, and in beta-escin-treated skinned smooth muscle of the rabbit mesenteric artery.

1. The effects of pinacidil were investigated on changes in cellular Ca2+ concentration ([Ca2+]i) and tension in intact and chemically skinned smooth muscle strips of the rabbit mesenteric artery. 2. High K+ (128 mM) produced a large phasic followed by a tonic increase in [Ca2+]i and tension in intact muscle strips. Pinacidil at 10 microM but not 1 microM, inhibited the phasic and tonic contractions induced by 128 mM K+ without a corresponding change in [Ca2+]i. 3. In beta-escin-treated skinned smooth muscle, the minimum Ca2+ concentration that produced contraction was 0.1 microM and the maximum contraction was obtained at 10 microM. Pinacidil at 10 microM but not 1 microM, shifted the pCa-tension relation curve to the right and also inhibited the maximum contraction induced by Ca2+. The concentrations of Ca2+ required for half maximal tension were 0.9 microM in control and 1.5 microM in the presence of 10 microM pinacidil. Calmodulin (2 microM) increased the contraction induced by 0.3 microM Ca2+ (but not by 10 microM Ca2+) in the skinned strips. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM or 10 microM Ca2+ in the presence of 2 microM calmodulin. 4. Noradrenaline (NA, 10 microM) with guanosine triphosphate (GTP, 3 microM), guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 3 microM) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) all enhanced the contraction induced by 0.3 microM Ca2+. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM Ca2+ more strongly in the presence of the above agents than in their absence.(ABSTRACT TRUNCATED AT 250 WORDS)