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Title: A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle

Abstract

Phorbol esters can induce contraction of vascular smooth muscle and potentiate calcium-induced contractions of permeabilized smooth muscle strips. The authors have used a synthetic peptide inhibitor based on residues 19-31 of PKC (PKC-I) to determine the importance of PKC in the PDBu potentiation of calcium-induced contractions in permeabilized coronary artery smooth muscle. Although peptides similar to PKC-I have been shown to also inhibit MLCK in vitro, MLCK was presumably not inhibited in our system since 30 {mu}M PKC-I alone did not alter the calcium-induced contractions. However, the potentiation of these contractions by 1 {mu}M PDBu was reduced by about 50% in the presence of 10 {mu}M PKC-I, and the potentiation was completely abolished by 30 {mu}M PKC-I. These data indicate that, in this system, PKC is not involved in calcium-induced contractions but that activation of PKC may be the mechanism by which PDBu potentiates calcium-induced contractions in permeabilized coronary artery smooth muscle.

Authors:
;  [1]
  1. (Vanderbilt Univ., Nashville, TN (United States))
Publication Date:
OSTI Identifier:
5374034
Report Number(s):
CONF-9104107--
Journal ID: ISSN 0892-6638; CODEN: FAJOE
Resource Type:
Conference
Resource Relation:
Journal Name: FASEB Journal (Federation of American Societies for Experimental Biology); (United States); Journal Volume: 5:4; Conference: 75. annual meeting of the Federation of American Societies for Experimental Biology (FASEB), Atlanta, GA (United States), 21-25 Apr 1991
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; MUSCLES; CONTRACTION; PEPTIDES; BIOCHEMICAL REACTION KINETICS; PHORBOL ESTERS; BIOLOGICAL EFFECTS; CALCIUM; CORONARIES; INHIBITION; PERMEABILITY; ALKALINE EARTH METALS; ARTERIES; BLOOD VESSELS; BODY; CARCINOGENS; CARDIOVASCULAR SYSTEM; ELEMENTS; ESTERS; KINETICS; METALS; ORGANIC COMPOUNDS; ORGANS; PROTEINS; REACTION KINETICS 560300* -- Chemicals Metabolism & Toxicology

Citation Formats

Sullivan, T.S., and Wells, J.N. A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle. United States: N. p., 1991. Web.
Sullivan, T.S., & Wells, J.N. A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle. United States.
Sullivan, T.S., and Wells, J.N. 1991. "A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle". United States. doi:.
@article{osti_5374034,
title = {A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle},
author = {Sullivan, T.S. and Wells, J.N.},
abstractNote = {Phorbol esters can induce contraction of vascular smooth muscle and potentiate calcium-induced contractions of permeabilized smooth muscle strips. The authors have used a synthetic peptide inhibitor based on residues 19-31 of PKC (PKC-I) to determine the importance of PKC in the PDBu potentiation of calcium-induced contractions in permeabilized coronary artery smooth muscle. Although peptides similar to PKC-I have been shown to also inhibit MLCK in vitro, MLCK was presumably not inhibited in our system since 30 {mu}M PKC-I alone did not alter the calcium-induced contractions. However, the potentiation of these contractions by 1 {mu}M PDBu was reduced by about 50% in the presence of 10 {mu}M PKC-I, and the potentiation was completely abolished by 30 {mu}M PKC-I. These data indicate that, in this system, PKC is not involved in calcium-induced contractions but that activation of PKC may be the mechanism by which PDBu potentiates calcium-induced contractions in permeabilized coronary artery smooth muscle.},
doi = {},
journal = {FASEB Journal (Federation of American Societies for Experimental Biology); (United States)},
number = ,
volume = 5:4,
place = {United States},
year = 1991,
month = 3
}

Conference:
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  • The biological receptor for tumor-promoting phorbol esters has been identified as the CaS /phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular CaS is allowed to rise. Since cellular CaS in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of TH-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attemptsmore » to assay the enzyme directly as phospholipid-activated TSP incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition.« less
  • A-10 cells which are derived from embryonic rat thoracic aorta contain a high density of vasopressin receptors and relatively fewer ..beta..-adrenergic receptors. The effects of vasopressin binding to these cells are two-fold: a) inhibition of isoproterenol-stimulated cAMP accumulation, and; b) stimulation of phosphatidyl inositol turnover. Incubation of these cells with phorbol dibutyrate leads to an attenuation of the responses mediated by ..beta..-adrenergic agonist as well as vasopressin. This effect of phorbol ester is concentration- and time-dependent and can be observed as early as five minutes. The inactive phorbol ester (4 ..cap alpha.. phorbol 12,13-didecanoate) is ineffective in inhibiting ..beta..-adrenergic agonistmore » and vasopressin-mediated responses. Since present evidence indicates that the enzyme protein kinase C (PK-C) is involved in both short-term and long-term regulatory processes such as secretion, smooth muscle contraction and cell growth, these data suggest that both ..beta..-adrenergic and vasopressin receptors and/or some mediator(s) of ..beta..-adrenergic and/or vasopressin responses may be phosphorylated by protein kinase C resulting in an attenuated response of these two hormones.« less
  • Marked changes in the shape of vascular smooth muscle cells (VSMC) occur during early development, repair of the vascular wall, and formation of atherosclerotic plaques. Yet, surprisingly little is known about mechanisms which regulate the shape of VSMC. Since protein kinase C (PKC) is involved in regulation of multiple cellular functions including interactions between contractile and cytoskeletal proteins, the authors suspected it might also regulate VSMC shape. Accordingly, the authors studied the influence of a known activator of PKC, phorbol 12-myristate 13-acetate (PMA), on the shape of cultured canine carotid arterial BSMC. PMA produced time and concentration dependent changes frommore » normal elongated shape to pronounced circular forms. Cells recovered normal shape within 24 hrs even though exposure to PMA was continued. Analogs of PMA which do not activate PKC did not alter shape, whereas phorbol 13, 14 diacetate, an analog which activates PKC, did produce changes in shape similar to those produced by PMA. Cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an inhibitor of mRNA synthesis, did not alter PMA-induced changes in morphology. In contrast, however, recovery of normal shape after prolonged exposure to PMA was blocked by either cycloheximide or actinomycin D. These results suggest activation of PKC produces changes in VSMC shape that are independent of transcription or translation, whereas recovery is dependent on both transcription and translation. The results also suggest PKC may modulate in vivo changes in VSMC shape occurring during different pathophysiological states.« less
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  • Studies from this laboratory have shown that HSV-1 infection suppresses matrix protein synthesis by endothelial cells in vitro. In this study the authors have investigated the effects of HSV-1 infection on SMC. Monolayers of SMC were infected with HSV-1 at a multiplicity of infection (MOI) ranging from 0.1 to 20. Viral replication and release to the medium was measured by plaque assay in Vero cells. At an MOI of 0.1, 10 or 20, viral replication occurred and maximum virus titers were achieved by 24 hrs. post-infection. Virus release in the medium began during the first 12 hrs. post-infection and reachedmore » maximum at 24 hrs. Infected and uninfected cultures of SMC were pulse labeled with either (/sup 14/C)proline or (/sup 35/S)-methionine at different hrs. post-infection. Incorporation of radioactivity into non-dialyzable protein was determined in fluorograms following SDS-PAGE of the cell-matrix or medium fractions. The synthesis of fibronectin and collagen Types I and III was suppressed and the degree of suppression was dependent on the duration of infection and on the virus dose. These data suggest that SMC can support HSV-1 replication in vitro and that such infection can lead to altered extracellular matrix synthesis.« less