Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle

The effect of phorbol ester-induced down-regulation of protein kinase C (PKC) on diacylglycerol (sn-1,2-dioctanoylglycerol, diC8)- and G-protein-coupled Ca2+ sensitization and on the relationship between phosphorylation of the regulatory myosin light chains (MLC20) and force during Ca2+ sensitization were investigated in rabbit portal vein (PV), femoral artery (FA) and ileum smooth muscle. The effects of phorbol dibutyrate (PDBu), guanosine 5´-[γ-thio]triphosphate (GTP[S]) and agonists on the membrane versus cytosolic distribution of PKC isoenzymes were also determined. Down-regulation of PKC abolished Ca2+ sensitization of force and the accompanying increases in MLC20 phosphorylation induced by PDBu, as well as Ca2+ sensitization of force by diC8, but not that by GTP[S], aluminum fluoride (AlF4-) or agonists (phenylephrine, endothelin or carbachol). Down-regulation also inhibited the PDBu-, but not the GTP[S]-induced increase in force under Ca2+-free conditions. In ileum, PDBu translocated PKCs α, β1, β2, ϵ and θ to the membrane fraction, and GTP[S] caused a small translocation of PKC-ϵ. Carbachol- and GTP[S]-induced Ca2+ sensitization remained unaffected in down-regulated ileum in which no cytosolic PKC-ϵ was detectable. We conclude that, although both phorbol ester-induced and G-protein-coupled Ca2+ sensitization of force are mediated by increased MLC20 phosphorylation, it is likely that PKCs α, β1, β2, ϵ and θ do not play an essential role in, although they may contribute to, the G-protein-coupled mechanism.

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Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.5.c1253 ◽

1998 ◽

Vol 274 (5) ◽

pp. C1253-C1260 ◽

Cited By ~ 34

Author(s):

Dorothee H. Bremerich ◽

Tetsuya Kai ◽

David O. Warner ◽

Keith A. Jones

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Phorbol Esters ◽

Isometric Force ◽

Calphostin C ◽

Regulatory Myosin Light Chain ◽

Kinase C ◽

The Relationship

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19—31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.

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Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.279 ◽

1997 ◽

Vol 8 (2) ◽

pp. 279-286 ◽

Cited By ~ 30

Author(s):

M C Gong ◽

H Fujihara ◽

L A Walker ◽

A V Somlyo ◽

A P Somlyo

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

G Protein ◽

Total Content ◽

Prolonged Treatment ◽

Down Regulation ◽

Kinase C ◽

G Alpha Q ◽

Gamma S

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.

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Inhibition of phorbol ester-induced contraction by calmodulin antagonists in rat aorta

AJP Cell Physiology ◽

10.1152/ajpcell.1991.261.4.c675 ◽

1991 ◽

Vol 261 (4) ◽

pp. C675-C684 ◽

Cited By ~ 20

Author(s):

J. K. Chuprun ◽

E. Bazan ◽

K. C. Chang ◽

A. K. Campbell ◽

R. M. Rapoport

Keyword(s):

Smooth Muscle ◽

Phorbol Ester ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Rat Aorta ◽

Prior Exposure ◽

Calmodulin Antagonists ◽

Enzyme Preparations ◽

Kinase C ◽

Inhibitory Mechanisms

The purpose of the present study was to investigate the relative roles of protein kinase C (PKC) and myosin light chain kinase (MLCK) in phorbol ester-induced contraction of vascular smooth muscle through the use of PKC and calmodulin antagonists. Prior exposure to PKC antagonists staurosporine (0.03 microM) and H-7 (10 microM) had relatively little effect on contractions to phorbol 12-myristate 13-acetate (PMA), while contractions to norepinephrine and KCl were greatly inhibited. Prior exposure to the calmodulin antagonists calmidazolium (3 and 10 microM) and W-7 (10 microM) inhibited contractions to PMA in the presence and absence of extracellular Ca2+, while contractions to norepinephrine and KCl remained relatively unaffected. Calmidazolium and W-7 were relatively weak relaxants when applied during the PMA contraction, and the magnitudes of relaxation were similar to those observed in norepinephrine- and KCl-contracted tissues. Calmidazolium partially inhibited the PMA-induced translocation of PKC. These results suggest that 1) the calmodulin antagonists inhibit the development of PMA-induced contraction, at least in part, through inhibition of PKC translocation; 2) the mechanisms of phorbol ester- and agonist-induced translocation of PKC are distinct; 3) the potencies and inhibitory mechanisms of these agents depend on whether the agents are added before or during the contraction; and 4) the selectivity of these agents, as evaluated in enzyme preparations, may not be consistent with their cellular actions.

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Phorbol ester-induced contraction in chemically skinned vascular smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1986.251.3.c356 ◽

1986 ◽

Vol 251 (3) ◽

pp. C356-C361 ◽

Cited By ~ 126

Author(s):

M. Chatterjee ◽

M. Tejada

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Phorbol Ester ◽

Light Chain ◽

Myosin Light Chain ◽

Phorbol Esters ◽

Myosin Light Chain Phosphorylation ◽

Kinase C

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

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