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M P Walsh

Publications and source records attributed to M P Walsh.

At least 19 recordsLinked to original sources
PubMed1992

A comparison of the effects of calponin on smooth and skeletal muscle actomyosin systems in the presence and absence of caldesmon.

Thiosphosphorylated smooth muscle myosin and skeletal muscle myosin, both of which express Ca(2+)-independent actin-activated MgATPase activity, were used to examine the functional effects of calponin and caldesmon separately and together. Separately, calponin and caldesmon inhibited the actin-activated MgATPase activities of thiophosphorylated smooth muscle myosin and skeletal muscle myosin, calponin being significantly more potent in both systems. Calponin-mediated inhibition resulted from the interaction of calponin with actin since it could be reversed by increasing the actin concentration. Caldesmon had no significant influence on the calponin-induced inhibition of the smooth muscle actomyosin ATPase, nor did calponin have a significant effect on caldesmon-induced inhibition. In the skeletal muscle system, however, caldesmon was found to override the inhibitory effect of calponin. This difference probably reflects the lower affinity of skeletal muscle actin for calponin compared with that of smooth muscle actin. Calponin inhibition of skeletal muscle actin-activated myosin MgATPase was not significantly affected by troponin/tropomyosin, suggesting that the thin filament can readily accommodate calponin in addition to the troponin complex, or that calponin may be able to displace troponin. Calponin also inhibited acto-phosphorylated smooth muscle heavy meromyosin and acto-skeletal muscle heavy meromyosin MgATPases. The most appropriate protein preparations for analysis of the regulatory effects of calponin in the actomyosin system therefore would be smooth muscle actin, tropomyosin and thiophosphorylated myosin, and for analysis of the kinetic effects of calponin on the actomyosin ATPase cycle they would be smooth muscle actin, tropomyosin and phosphorylated heavy meromyosin, due to the latter's solubility.

Actins↗
PubMed1992

The mechanism of inhibition of the actin-activated myosin MgATPase by calponin.

Calponin inhibits the actin-activated ATPase of smooth muscle myosin and thus has been proposed as a thin filament-based regulatory component in smooth muscle. To obtain information on the mechanism of inhibition by calponin we have used chemical modification of actin and cross-linking of actin and subfragment 1. Modification of Lys 61 of actin had no effect on the inhibition by calponin of acto-heavy meromyosin ATPase, i.e. different from tropomyosin-troponin. In addition, modification of the acidic N-terminal region of actin did not impair the ability of calponin to bind to F-actin. Finally, calponin was effective in inhibiting ATPase activity of cross-linked acto-subfragment 1. Therefore the mechanism of inhibition by calponin is distinct from troponin-tropomyosin and caldesmon in that it does not involve either the N-terminal acidic region of actin nor the area around Lys 61 and does not fit a simple steric blocking model.

Actins↗
PubMed1992

Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function.

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.

Actins↗
PubMed1992

Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells.

Many neurohormones alter the force of cardiac contraction by variations in the intracellular Ca2+ concentration. alpha 1-Adrenergic and muscarinic stimulations, rather, modify the sensitivity of contractile proteins to Ca(2+)-calmodulin-myosin light-chain kinase (MLCK) complex induces a large increase in Ca2+ sensitivity (0.14 pCa unit) of these easily accessible myofilaments. This increase is further enhanced by up to 0.19 pCa unit when protein kinase C (PKC) is added together with MLCK. Similarly, the Ca2+ ATPase activity of skinned cells in suspension is increased in the presence of MLCK and further in the presence of both kinases. 32P-labelling and SDS/PAGE show that these changes are associated with light-chain 2 (LC2) phosphorylation together with phosphorylation of troponin I and troponin T when PKC is added. Although to a smaller extent than in smooth muscle, phosphorylation of cardiac myosin LC2 may be involved in the modulation of heart contractility.

Adenosine Triphosphatases↗
PubMed1992

Phosphorylation of the C-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II.

The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines. A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo. cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger [Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280]. We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone. cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase. The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of the expressed protein. Control and experimental pEX-1-Na+/H+ exchanger protein was purified on a p-aminophenyl beta-D-thiogalactopyranoside-agarose column. Purified Ca2+/calmodulin-dependent protein kinase II readily phosphorylated the Na+/H+ exchanger protein in a Ca(2+)- and calmodulin-dependent manner in vitro, but this region of the protein was not a substrate for purified protein kinase C or for the catalytic subunit of cyclic AMP-dependent protein kinase. Control-expressed beta-galactosidase was phosphorylated to a maximal level of 0.77 +/- 0.17 mol of Pi/mol (mean +/- S.E.M., n = 6) whereas the fusion protein was phosphorylated to a maximal level of 4.09 +/- 0.39 mol of Pi/mol (n = 6), suggesting one site of phosphorylation in beta-galactosidase and three in the C-terminal domain of the Na+/H+ exchanger. Examination of the deduced amino acid sequence of this part of the exchanger reveals three consensus sequences for Ca2+/calmodulin-dependent protein kinase II. These results suggest that the exchanger may be directly regulated in vivo by calmodulin-dependent protein kinase II but not by protein kinase C or cyclic AMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1992

Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres.

The role of protein kinase C (PKC) in regulating the contractile state of smooth muscle was investigated using the constitutively active catalytic fragment of PKC (PKM) with skinned (demembranated) chicken gizzard fibres. PKM attenuated a submaximal contraction in gizzard smooth muscle skinned fibres, but not in rabbit cardiac skinned fibres. PKM-mediated relaxation of submaximal contractions of smooth muscle was accompanied by a reduction in the rate of ATP hydrolysis in the fibre and by phosphorylation of the 20 kDa light chain of gizzard myosin at the PKC sites (serine-1, serine-2 and threonine-9). In addition, several other endogenous proteins were phosphorylated by PKM. However, the inhibitory effects on tension and ATPase are consistent with the biochemical effects of PKC-catalysed phosphorylation of myosin, i.e. reduction of the actin-activated MgATPase activity of myosin prephosphorylated at serine-19 by myosin light chain kinase. Pretreatment of skinned fibres with PKM and ATP gamma S in the absence of Ca2+ had no inhibitory effect on the subsequent submaximal Ca(2+)-activation of force. Consistent with this observation, PKC was not able to utilize ATP gamma S as a substrate, confirming that the observed effects were the result of PKM-catalysed protein phosphorylation. We suggest that PKC may have two distinct effects on smooth muscle contraction: translocation of PKC to the sarcolemma on stimulation results in phosphorylation of a protein(s) other than myosin and a slow, sustained contraction; in some circumstances PKC may undergo proteolysis to PKM resulting in myosin phosphorylation at PKC-specific sites, a reduction in ATPase activity and relaxation of the muscle.

Adenosine Triphosphatases↗
PubMed1992

Contraction of single vascular smooth muscle cells by phenylephrine at constant [Ca2+]i.

The mechanism of alpha-adrenergic agonist-mediated force generation was investigated in single hyperpermeable vascular smooth muscle cells. By use of a previously described method, force was recorded from individual ferret aortic cells made hyperpermeable by exposure to saponin. When the cells were clamped at pCa 7, addition of protein kinase M (PKM), the constitutively active form of protein kinase C (PKC), caused a sustained increase in force, which was reversible upon addition of the PKC pseudosubstrate inhibitor peptide (PSSI) corresponding to residues 19-31 of PKC. Phenylephrine at pCa 7 caused a dose-dependent increase in contractile force of the hyperpermeable cells, which was reversible on addition of phentolamine. The phenylephrine contraction could also be inhibited by the same concentration of PSSI that was effective toward the PKM-induced contraction. The response of the cells to a constant [phenylephrine] in different Ca buffers showed a lack of Ca dependence between pCa 8.6 and 7.0. The addition of PSSI to unstimulated cells caused a small but significant drop in basal tone. Taken together, these results suggest that a fraction of the basal tone, as well as the phenylephrine contraction that occurs in the skinned cells at constant intracellular free Ca2+ concentration, is the result of activation of a Ca-independent isozyme of PKC.

Animals↗
PubMed1992

Protein kinase C of smooth muscle.

The primary mechanism of regulation of smooth muscle contraction involves the phosphorylation of myosin catalyzed by Ca2+/calmodulin-dependent myosin light chain kinase. However, additional mechanisms, both Ca(2+)-dependent and Ca(2+)-independent, can modulate the contractile state of smooth muscle. Protein kinase C was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. Protein kinase C occurs in at least four Ca(2+)-dependent (alpha, beta I, beta II, and gamma) and four Ca(2+)-independent (delta, epsilon, zeta, and eta) isoenzymes. Only the alpha, beta, epsilon, and zeta isoenzymes have been identified in smooth muscle. Both classes of isoenzymes have been implicated in the regulation of smooth muscle contraction. However, the physiologically important protein substrates of protein kinase C have not yet been identified. Specific isoenzymes may be activated by different contractile agonists, and individual isoenzymes exhibit some degree of substrate specificity. Prolonged activation of protein kinase C can result in its proteolysis to the constitutively active catalytic fragment protein kinase M, which would dissociate from the sarcolemma and phosphorylate proteins such as myosin that are inaccessible to membrane-bound protein kinase C. Protein kinase M induces relaxation of demembranated smooth muscle fibers contracted at submaximal Ca2+ concentrations. We suggest that protein kinase C plays two distinct roles in regulating smooth muscle contractility. Stimuli triggering phosphoinositide turnover or phosphatidylcholine hydrolysis induce translocation of protein kinase C (probably specific isoenzymes) to the sarcolemma, phosphorylation of protein, and a slow contraction. Prolonged association of the kinase with the membrane may lead to proteolysis and release into the cytosol of protein kinase M, resulting in myosin phosphorylation and relaxation.

Amino Acid Sequence↗
PubMed1991

The major endogenous bovine brain protein kinase C inhibitor is a heat-labile protein.

A crude cytosolic fraction prepared from bovine brain contained protein kinase C, as shown by immunoblotting, but its activity was undetectable, suggesting the presence of interfering factors. Phosphatase, ATPase and protease activities did not account for the absence of detectable protein kinase C activity. The major contributing factor was found to be a heat-labile protein which was separated from the kinase by ion-exchange chromatography. The contribution to the total inhibitory activity of heat-stable proteins was relatively minor, suggesting that they may not function physiologically as protein kinase C inhibitors.

Animals↗
PubMed1991

Chemical modification of the sole histidine residue of smooth muscle caldesmon.

Caldesmon was stoichiometrically N-carbethoxylated specifically at the only histidine residue (His-610) with diethylpyrocarbonate. Carbethoxylation of a 1:1 molar complex of caldesmon and calmodulin in the presence of Ca2+ resulted in the stoichiometric N-carbethoxylation of His-610 of caldesmon and His-107 of calmodulin. Carbethoxy-caldesmon, like the unmodified protein, bound to immobilized calmodulin (in the presence of Ca2+) and to immobilized tropomyosin (at low ionic strength). The affinity of F-actin for carbethoxy-caldesmon (Kd = 1.29 x 10(-6)M) was similar to that for unmodified caldesmon (Kd = 0.88 x 10(-6)M), and the modified protein was as effective as control caldesmon in the inhibition of the actin-activated MgATPase of skeletal muscle myosin. We conclude that the predicted basic amphiphilic alpha-helical sequence (Arg-593-His-610) does not represent the calmodulin-binding site of caldesmon. Furthermore, His-610 does not play a major role in the interaction of caldesmon with F-actin or tropomyosin.

Adenosine Triphosphate↗
PubMed1991

Characterization of a novel zinc binding site of protein kinase C inhibitor-1.

The zinc-binding properties of an endogenous protein inhibitor of protein kinase C was studied. Equilibrium gel penetration revealed that 1 mol of this protein binds 0.97 mol of zinc with a dissociation constant of 4.3 microM. The site of zinc-binding, MVVNEGSDGGQSVYHVHLHVLGGR, was identified by a multi-step process consisting of tryptic digestion, fragment isolation, transfer to nitrocellulose, and hybridization with 65ZnCl2. Binding of 65ZnCl2 to selected synthetic fragments further localized the site of interaction to the sequence QSVYHVHLHVL. This region contains 3 closely positioned histidine residues and represents a novel zinc-binding site.

Amino Acid Sequence↗
PubMed1991

The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction.

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).

Actin Cytoskeleton↗
PubMed1991

Protein kinase C activation does not stimulate lung liquid clearance in anesthetized sheep.

Although active transport of ions could play an important role in the resolution process of pulmonary edema, the exact mechanism regulating this process is still unknown. In this study, we investigated the effect of phorbol myristate acetate (PMA) on lung liquid clearance in anesthetized, ventilated sheep to evaluate the possible role of protein kinase C. To study lung liquid and protein clearance, we measured the removal of 100 ml of autologous serum from the air spaces of anesthetized sheep. Either serum alone or serum mixed with PMA (10(-7) M) was instilled. After 4 h, the residual lung water was 76.8 +/- 9.2 ml when serum alone was instilled and 79.5 +/- 15.7 when serum with PMA (10(-7) M) was instilled. The lack of effect of PMA (10(-7) M) on lung liquid clearance cannot be explained by increased movement of liquid from the vascular space to the air space since we did not have any evidence of increased pressure or increased permeability in the lung. This lack of effect of PMA (10(-7) M) is not due to an absence of stimulation of protein kinase C since instillation of BSA and PMA (10(-7) M) in rat lung produced a translocation of protein kinase C activity from the cytosolic fraction to the membrane fraction 2 h after the instillation. These results were confirmed in two sheep experiments, which demonstrated clear activation of protein kinase C after 4 h. These data suggest that activation of protein kinase C does not stimulate lung liquid clearance. However, a possible role of protein kinase C in modulating lung liquid clearance has not been excluded.

Anesthesia, Intravenous↗
PubMed1990

Amino acid sequence of a 12-kDa inhibitor of protein kinase C.

The complete primary structure of a bovine-brain-derived inhibitor of protein kinase C has been established. Fragments of the purified protein were obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease, trypsin and chymotrypsin. Subsequent analysis of the resulting fragments by fast-atom-bombardment mass spectrometry and Edman degradation revealed a calculated molecular mass of 11,779 Da with the following 107-amino-acid sequence: [sequence: see text] This inhibitor does not share significant primary structural identity with any other known protein.

Amino Acid Sequence↗