Kinase activation and smooth muscle contraction in the presence and absence of calcium.
OBJECTIVE
The intracellular signalling mechanisms that modulate the sustained vascular smooth muscle contractions that occur with vasospasm are not well understood. The purpose of this investigation was to examine cell signalling mechanisms that account for sustained vascular smooth muscle contraction, independent of increases in intracellular Ca2+ concentrations ([Ca2+]i).


METHODS
Fresh bovine carotid artery smooth muscles contractile responses were examined in a muscle bath. [Ca2+]i was depleted by use of the extracellular Ca2+ chelator, ethylene glycol-bis(beta-aminoethylether) N,N,N',N'-tetraacetic acid and the intracellular chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid.


RESULTS
In Ca(2+)-free conditions, depolarizing the membrane with high extracellular KCI failed to elicit a contraction. In addition, in Ca(2+)-free conditions the ([Ca2+]i) was less than 10 nmol/L as determined with the Ca(2+)-indicator, Fura 2. The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBu), induced slowly developing sustained contractions in bovine carotid artery smooth muscle, and the magnitude of the contractile response to PDBu (10 nmol/L to 10 mumol/L) was the same in the presence and absence of Ca2+. PDBu induced contractions in Ca(2+)-free conditions were not inhibited by the myosin light chain kinase inhibitor, ML-9 (50 mumol/L), but were inhibited by the PKC inhibitor, staurosporine (50 nmol/L).


CONCLUSIONS
These data suggest that vascular smooth muscle contractions can occur under conditions where the [Ca2+]i is low and fixed and that these contractions may be mediated by PKC.
Whitney
G
G
Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta 30912, USA.
Throckmorton
D
D
Isales
C
C
Takuwa
Y
Y
Yeh
J
J
Rasmussen
H
H
Brophy
C
C
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002339:Carotid Arteries / Q000502:physiology; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D019311:Staurosporine; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D000470:Alkaloids; D011450:Prostaglandin Endoperoxides, Synthetic; D014662:Vasoconstrictor Agents; D012701:Serotonin; D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D019311:Staurosporine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium