Abstract
The Ca2+ sensitivities of tonic (pulmonary and femoral artery) and phasic (portal vein and ileum) smooth muscles and the effects of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) and norepinephrine on Ca2+ sensitivity of force development and myosin light chain (MLC20) phosphorylation were determined in permeabilized preparations that retained coupled receptors and endogenous calmodulin. The Ca2+ sensitivity of force was higher (approximately 3-fold) in the tonic than in the phasic smooth muscles. The nucleotide specificity of Ca2+ sensitization was: GTP gamma S much greater than GTP greater than ITP much greater than CTP = UTP. Baseline phosphorylation (7% at pCa greater than 8) and maximal phosphorylation (58% at pCa 5.0) were both lower in portal vein than in femoral artery (20 and 97%). Norepinephrine and GTP gamma S increased phosphorylation at constant [Ca2+] (pCa 7.0-6.5). MLC20 phosphorylation induced by norepinephrine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S). In portal vein at pCa 5, GTP gamma S increased phosphorylation from 58%, the maximal Ca2(+)-activated value, to 75%, and at pCa greater than 8, from 7 to 13%. In femoral artery at pCa 5, neither phosphorylation (97%) nor force was affected by GTP gamma S, while at pCa greater than 8, GTP gamma S caused an increase in force (16% of maximum) with a borderline increase in MLC20 phosphorylation (from 20 to 27%). MLC20 phosphorylation (up to 100%) was positively correlated with force. The major results support the hypothesis that the G-protein coupled Ca2(+)-sensitizing effect of agonists on force development is secondary to increased MLC20 phosphorylation.
Highlights
The Ca” sensitivities of tonic (pulmonary and fem- tions inwhich a-adrenergic (Nishimuraet at., 1988; Kitazawa oral artery) anpdhasic smooth muscles and the effects of guanosine 5’-0-(y-thiotriphosphate) (GTPrS) and norepinephrine on Ca2+sensitivity of force development and myosin light chain (MLCzo)phosphorylation were determined in permeabilized preparations that retained coupled receptors and endogenouscalmodulin
After measurement of contractions induced by high K+ and by Portal Vein-In order to check our whole system ofMLC, phosagonists,thestrips were incubated a t 25 "C inrelaxingsolution phorylation measurements and compare the phosphorylated values containing 4.5 mM MgATP and 1 mM EGTA for several minutes, obtained from very small (20-SO pg) samples of permeabilized prepand treated for 25-35 min with 2-4 mg/ml of crude Staphylococcus arations with those in intact muscles, we first performedmeasureaurem a-toxin (Calbiochem) at pCa 6.7 or 6.3 buffered with 10 mM ments on nonpermeabilized guinea pig portal vein smooth muscle
The steady state pCa-force relationship was obtained in smooth muscles permeabilized with a-toxin, by cumulatively increasing Caz+, either in the absence or in the presence of saturating concentration (100 p ~ of) GTPrS
Summary
@ 1991 byThe American Societyfor Biochemistryand Molecular Biology, Inc. Vol 266, No., Issue of January 25, pp. 1708-1715,1391 Printed in U.S.A. After measurement of contractions induced by high K+ and by Portal Vein-In order to check our whole system ofMLC, phosagonists,thestrips were incubated a t 25 "C inrelaxingsolution phorylation measurements and compare the phosphorylated values containing 4.5 mM MgATP and 1 mM EGTA for several minutes, obtained from very small (20-SO pg) samples of permeabilized prepand treated for 25-35 min with 2-4 mg/ml of crude Staphylococcus arations with those in intact muscles, we first performedmeasureaurem a-toxin (Calbiochem) at pCa 6.7 or 6.3 buffered with 10 mM ments on nonpermeabilized guinea pig portal vein smooth muscle. Each 60 pl of homogenate alone increased phosphorylation of MLC, to 39 f 3.2% ( n = 4 ) and was applied to an isoelectricfocusingpolyacrylamide tube gel (1.5 force to 115 f4.2% a t 2 minafter stimulation.Thew results, obtained mm inner diameter) with 5% pH ampholytes 4.5/5.4 Software (EikonixCo., Bradford, MA) that permitted the subtraction of hackground obtained from regionsadjacent to the focused proteins
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