Inhibitory effect of forskolin on myosin phosphorylation-dependent and independent contractions in bovine tracheal smooth muscle

In bovine tracheal smooth muscle, carbachol (CCh, 1 microM) and high K+ (72.7 mM) induced sustained increases in cytosolic Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10 microM) inhibited the CCh-induced increase in [Ca2+]i, MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K(+)-induced contraction and MLC phosphorylation without changing [Ca2+]i. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), CCh (10 microM) and caffeine (20 mM) induced transient increase in [Ca2+]i and contractile force by releasing Ca2+ from cellular store. FK strongly inhibited the CCh-induced Ca2+ transient, but failed to inhibit the caffeine-induced Ca2+ transient. In the absence of external Ca2+, 12-deoxyphorbol 13-isobutylate (DPB, 1 microM) induced sustained contraction without increase in [Ca2+]i and MLC phosphorylation. FK inhibited this contraction without changing [Ca2+]i. In permeabilized muscle, Ca2+ induced contraction in a concentration-dependent manner. FK (10 microM) and cAMP (1-100 microM) shifted the Ca(2+)-force curve to the higher Ca2+ levels. CCh with GTP, GTP gamma S or DPB enhanced contraction in the presence of constant level of Ca2+. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca2+. cAMP (300 microM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca2+ influx and Ca2+ release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.     

Human platelets possess tyrosylprotein sulfotransferase (TPST) activity

Phosphorylation of myosin light chain kinase by a Ca(2+)-dependent protein kinase increases the concentration of Ca2+/calmodulin required for half-maximal activation. The Ca2+ concentrations required for myosin light chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin light chain phosphorylation. Both GTP gamma S and carbachol increase the Ca2+ sensitivity of myosin light chain kinase phosphorylation as well as light chain phosphorylation. It is proposed that a similar G-protein mediated mechanism regulates the Ca(2+)-dependent phosphorylation of these two contractile proteins in smooth muscle.     

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle

To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca2+] ([Ca2+]i) and phasic force showed that Wortmannin (1-4 microM) abolishes force with little or no change in [Ca2+]i and electrical activity. High-K+-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent - at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40-50%. It also significantly increased the delay between the Ca2+ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca2+ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca2+ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered.     

Effect of stress-like concentrations of cortisol on gonadotroph function in orchidectomized sheep

The effects of the small noncatalytic subunit of myosin light chain phosphatase (MLCPsr) on the Ca2+-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. The full-length recombinant chicken MLCPsr obtained by the bacterial expression system induced an additional contraction at a constant [Ca2+]i and shifted the [Ca2+]i-force relation curve to the left. A deletion mutant containing the N-terminal 78 amino acids of MLCPsr retained the full action, compared with the full-length MLCPsr, while the deletion of this region completely abolished its effect. The process of relaxation was also delayed by the fragment containing the N-terminal 78 amino acids. These results indicated that MLCPsr increases the Ca2+ sensitivity of the contractile apparatus while the N-terminal 78 amino acids are responsible for this effect in vascular smooth muscle.     

Polarity of epithelial cells of the liver. Cellular and molecular mechanisms, and pathologic changes

Smooth muscle contraction is primarily regulated not only by changes in cytosolic Ca2+ concentrations ([Ca2+]i) but also by changes in the force/[Ca2+]i ratio. The use of membrane-permeabilization technique facilitated demonstration of an increase in the level of force at constant [Ca2+]i (Ca2+ sensitization). It was clarified that Rho-associated kinase (Rho-kinase) is a novel mediator of Ca2+ sensitization of the smooth muscle contraction, by introducing the recombinant catalytic domain of Rho-kinase into the cytosol of vascular smooth muscle permeabilized with beta-escin. This review article focuses on novel mechanisms, by which activation of receptor-coupled G-protein(s) increases Ca2+ sensitivity of the contractile apparatus in smooth muscle: Rho-kinase and protein kinase C.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca2+], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22 degrees C. In the maximally thiophosphorylated muscle neither OA (10 microM) nor Ca2+ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca2+] in the presence of 0.5 microM calmodulin, both force and the maximal shortening velocity (Vmax) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, Vmax was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca2+] and at varied [Ca2+]. The relation between force and Vmax when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca2+]. The results show that Ca2+ and OA do not influence force or Vmax in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining Vmax. The finding that the relationship between force and Vmax was similar when activation was altered with OA and Ca2+ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres.     

Effects of SCN substitution for Cl- on tension, [Ca2+]i, and ionic currents in vascular smooth muscle

Substitution of thiocyanate ions (SCN-) for chloride ions (Cl-) in the extracellular medium of aortic rings and strips causes a biphasic contractile response; initial relaxation followed by sustained contraction. Alterations in these responses are sex-specific, and may elucidate fundamental differences in vascular function between males and females. In order to investigate the role of changes in intracellular Ca2+ ([Ca2+]i) in these changes in tension, we investigated effects of SCN- on [Ca2+]i and ionic currents in vascular smooth muscle cells (VSMC). Extracellular substitution of SCN- for Cl- caused a biphasic change in [Ca2+]i. Initially, [Ca2+]i decreased, reaching a minimum within 1-2 min, subsequently returned to original levels within 4-5 min, and then increased to a higher plateau over the next 10 minutes. This pattern of change in [Ca2+]i is identical to the pattern of tension changes in aortic rings, but it occurs somewhat faster. Partial substitution of SCN- for Cl- elicited increased, but no preceding decrease in [Ca2+]i. In the absence of external Ca2+, anion substitution elicited the decrease in [Ca2+]i but not the subsequent increase. Verapamil (1 microM) blocked the increased [Ca2+]i phase but not the decreased [Ca2+]i phase; whereas, R+ verapamil (up to 5 microM for 20 min), an inactive enantiomer, caused no change. Ionic current measurements obtained using whole cell patch and current clamp techniques revealed two responses to anion substitution: (a) a rapid, transient outward shift in holding current, and (b) a sustained increase in peak current and a hyperpolarizing shift in voltage sensitivity of Ca2+ channels. The calcium channel blocker PN200-110 blocked SCN(-)-enhanced current but had no effect on the changes in holding current. S- verapamil, but not R+ verapamil, reduced SCN(-)-enhanced current. In current clamp mode, SCN- caused an initial hyperpolarization followed by a slow depolarization punctuated by spikes. Thus, SCN- causes changes in vascular smooth muscle [Ca2+]i that could underlie both phases of its effects on tension in isolated aortas and may be explained by the following model: an initial outward shift in current causes hyperpolarization with a consequent decrease in cell excitability, and the somewhat slower increase in Ca2+ channel excitability eventually leads to enhanced calcium influx and tension. These data shed light on possible mechanisms underlying gender-related differences in VSMC physiology.     

Diversity and attention deficit hyperactivity disorder

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin triggered by an increase in sarcoplasmic free Ca2+ concentration ([Ca2+]i). Contraction can, however, be modulated by other signal transduction pathways, one of which involves the thin filament-associated protein calponin. The h1 (basic) isoform of calponin binds to actin with high affinity and is expressed specifically in smooth muscle at a molar ratio to actin of 1:7. Calponin inhibits (i) the actin-activated MgATPase activity of smooth muscle myosin (the cross-bridge cycling rate) via its interaction with actin, (ii) the movement of actin filaments over immobilized myosin in the in vitro motility assay, and (iii) force development or shortening velocity in permeabilized smooth muscle strips and single cells. These inhibitory effects of calponin can be alleviated by protein kinase C (PKC)-catalysed phosphorylation and restored following dephosphorylation by a type 2A phosphatase. Three physiological roles of calponin can be considered based on its in vitro functional properties: (i) maintenance of relaxation at resting [Ca2+]i, (ii) energy conservation during prolonged contractions, and (iii) Ca(2+)-independent contraction mediated by phosphorylation of calponin by PKC epsilon, a Ca(2+)-independent isoenzyme of PKC.     

Signal transduction in gastrointestinal smooth muscle

Signal transduction in gastric and intestinal smooth muscle is mediated by receptors coupled via distinct G proteins to various effector enzymes, including PI-specific PLC-beta 1 and PLC-beta 3, and phosphatidylcholine (PC)-specific PLC, PLD and PLA2. Activation of these enzymes is different in circular and longitudinal muscle cells, generating Ca(2+)-mobilizing (IP3, AA, cADPR) and other (DAG) messengers responsible for the initial and sustained phases of contraction, respectively. IP3-dependent Ca2+ release occurs only in circular muscle. Ca2+ mobilization in longitudinal muscle involves a cascade initiated by agonist-induced transient activation of PLA2 and formation of AA, AA-dependent depolarization of the plasma membrane and opening of voltage-sensitive Ca2+ channels. The influx of Ca2+ induces Ca2+ release by activating sarcoplasmic ryanodine receptor/Ca2+ channel and stimulates cADPR formation which enhances Ca(2+)-induced Ca2+ release. The initial [Ca2+]i transient in both muscle cell types results in Ca2+/calmodulin-dependent activation of MLC kinase, phosphorylation of MLC20 and interaction of actin and myosin. The sustained phase is mediated by a Ca(2+)-independent isoform of PKC, PKC-epsilon DAG for this process is generated by PLC- and PLD-mediated hydrolysis of PC. Relaxation is mediated by cAMP-and/or cGMP-dependent protein kinase which inhibit the initial [Ca2+]i transient and reduce the sensitivity of MLC kinase to [Ca2+]i. Relaxation induced by the main neurotransmitters, VIP and PACAP, involves two cascades, one of which reflects activation of adenylyl cyclase. A distinct cascade involves G-protein-dependent stimulation of Ca2+ influx leading to Ca2+/calmodulin-dependent activation of a constitutive eNOS in muscle cells; the generation of NO activates soluble guanylyl cyclase. The resultant activation of PKA and PKG is jointly responsible for muscle relaxation.     

Effect of phorbol ester, 12-deoxyphorbol 13-isobutylate (DPB), on muscle tension and cytosolic Ca2+ in rat anococcygeus muscle

Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10(-6) M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or beta-escin in the anococcygeus muscle. 3) In the beta-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca(2+)-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10(-5) M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.     

Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.     

Intracellular alkalinization by NH4Cl increases cytosolic Ca2+ level and tension in the rat aortic smooth muscle

Intracellular pH (pHi) is elucidated to be an important regulator of various cell functions, but the role of pHi in smooth muscle contraction remains to be clarified. The purpose of the present study is to examine the effects of cell alkalinization by exposure to NH4Cl on cytosolic Ca2+ level ([Ca2+]i) and on muscle tone. We attempted simultaneous measurements of both [Ca2+]i and contractile force in rat isolated thoracic aorta from which the endothelium was removed. NH4Cl (10-80 mM) increased both [Ca2+]i and muscle tone in the presence of external Ca2+. These responses were reproducible. The removal of Ca2+ from the nutrient solution partially inhibited the rise in [Ca2+]i and the smooth muscle contraction induced by NH4Cl. In addition, the Ca2+ channel blocker verapamil also partially attenuated the responses to NH4Cl. The NH4Cl-induced responses were gradually reduced as NH4Cl was repeatedly added in a Ca(2+)-free solution. Norepinephrine (NE, 1 microM) induced a transient increase in [Ca2+]i and sustained contraction in the absence of external Ca2+, and the subsequent application of NE had little effect on [Ca2+]i. After internal Ca2+ stores were depleted by exposure to NE, the subsequent application of NH4Cl induced increases in [Ca2+]i and tension of the aorta in a Ca(2+)-free solution. These results suggest that NH4Cl mainly evokes Ca2+ release from the internal Ca2+ stores that are not linked with adrenergic alpha-receptor and causes Ca2+ influx through voltage-dependent Ca2+ channels in the vascular smooth muscle.     

The role of myosin light chain kinase-dependent phosphorylation of myosin light chain in phorbol ester-induced contraction of rabbit aorta

We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca2+ ([Ca2+]i). Phosphorylation on Ser19 of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 +/- 5.1% and 22.3 +/- 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca2+-free, EGTA solution, [Ca2+]i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca2+-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser19 were inhibited. The phospholipase A2 inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca2+-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca2+]i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca2+-independent regulation of contractile proteins.     

Rho-associated kinase of chicken gizzard smooth muscle

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKalpha isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rho-kinase was stimulated 1.5-2-fold by guanosine 5'-O-3-(thio)triphosphate.RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 microM) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.     

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.     

Role of tyrosine phosphorylation in excitation-contraction coupling in vascular smooth muscle

Increasingly it is recognized that tyrosine phosphorylation plays an important part in the regulation of function in differentiated contractile vascular smooth muscle. Tyrosine kinases and phosphatases are present in large amounts in vascular smooth muscle and have been reported to influence a number of processes crucial to contraction, including ion channel gating, calcium homeostasis and sensitization of the contractile process to [Ca2+]i. This review summarizes current understanding regarding the role of tyrosine phosphorylation in excitation-contraction coupling in blood vessels.     

Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.     

Thy-1 and AvGp50 signal transduction complex in the avian nervous system: c-Fyn and G alpha i protein association and activation of signalling pathways

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of approximately 600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the approximately 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of approximately 600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the approximately 600 kDa protein regulates catch.     

Effects of semotiadil fumarate (SD-3211) and its enantiomer, SD-3212, on the changes in cytosolic Ca2+ and tension caused by KCl and norepinephrine in isolated rat aortas

Semotiadil fumarate (SD-3211), a Ca2+ channel blocker of benzothiazine derivative and its (S)-(-)-enantiomer (SD-3212), inhibited K(+)- and norepinephrine (NE)-induced contractions in isolated rat aortas. Inhibition of NE contraction induced by both drugs was greater than that induced by diltiazem or bepridil, whereas inhibition of K(+)-contraction was similar to that induced by diltiazem or bepridil. Semotiadil and SD-3212 (10 microM) inhibited the increase in cytosolic Ca2+ ([Ca2+]i) induced by 65.4 mM K+ in fura-2-loaded preparations as well as diltiazem and bepridil (10 microM). On the other hand, semotiadil and SD-3212 (10 microM) inhibited only the early phase of increase in [Ca2+]i induced by 1 microM NE. After 5 min, no significant effect on [Ca2+]i was observed with these compounds despite the significant decrease in the contraction. In contrast to these compounds, diltiazem and bepridil 10 microM affected neither the increase in [Ca2+]i nor the contraction induced by NE. Semotiadil and SD-3212 inhibited the transient contraction induced by 1 microM NE in the absence of external Ca2+. Both compounds partially but significantly inhibited the NE-induced contraction in nifedipine-treated muscles. These results suggest that semotiadil and SD-3212 inhibit contractions of vascular smooth muscle (VSM) not only through blockade of voltage-dependent Ca2+ channels but also through other mechanisms, such as inhibition of Ca2+ release from Ca2+ stores or decrease in sensitivity of the contractile elements to Ca2+.     

Mentoring: a professional commitment

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca2+ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca2+]i, with half-maximal effect (EC50) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ETA receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca2+ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca2+]i to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i response to these peptides. ETs exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization of the Ca2+ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca2+ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by these peptides is due to the activation of ETA receptors and subsequently the release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)