The mechanism of G protein-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ was studied in receptor-coupled a-toxin-permeabilized rabbit portal vein smooth muscle. To test the hypothesis that Ca2+ sensitization is due to inhibition of myosin light-chain (MLC) phosphatase activity, we measured the effect of guanosine 5'-[y-thio]triphosphate and phenylephrine on the rate of MLC dephosphorylation in muscles preactivated with Ca2+ and incubated in Ca2+-and ATP-free solution containing 1-(5-chloronaphthalene-l-sulfonyl)-lH-hexahydro-1,4-diazepine actomyosin ATPase and is the major regulatory mechanism of smooth muscle contraction (1). However, although the rise in cytoplasmic Ca2' and consequent activation of MLC kinase is the primary mechanism triggering MLC phosphorylation and contraction, the relationship between cytoplasmic Ca2+ and phosphorylation and force is not constant (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Thus, a variety of agonists can sensitize the contractile regulatory apparatus to Ca2+ to increase the force/Ca2+ ratio (9, 10), whereas desensitization to Ca2+ causes a decline in force, without, necessarily, any decrease in [Ca2+] (11,12).The sensitizing effect of agonists is mediated by a G protein, as shown by the ability of guanosine 5'-[-thio]triphosphate (GTP[yS]) to induce and guanosine 5'-[3-thio]diphosphate to inhibit Ca2+ sensitization (6-8, 10, 13).We had proposed that Ca2+ sensitization by agonists is mediated by the inhibition ofMLC phosphatase (14) and have supported this hypothesis by demonstrating that Ca2+ sensitization was accompanied by, and presumably the result of, increased MLC phosphorylation (8, 12). The major purpose of the present study was to determine whether the sensitization-induced increase in MLC phosphorylation is from inhibition of MLC phosphatase or from the alternate possibility, stimulation of MLC kinase. We used smooth muscle permeabilized with a-toxin because this preparation retains both coupled receptors and soluble intrinsic proteins (6, 8, 12) and a MLC kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine (ML-9) (15), under EXPERIMENTAL PROCEDURES Small strips (50-70 ,um thick, 200-350 ,um wide, and 2-3 mm long) of longitudinal muscle of rabbit portal vein were dissected and freed from connective tissue. Single (for only force measurements) or duplicate (for measurements of both force and phosphorylation) strips were tied with monofilament silk to the fine tips oftwo tungsten needles, one ofwhich was connected to a force transducer and mounted in a well on a bubble plate, as described (7).After measuring contractions induced by high (154 mM) K+ and by agonists, the strips were incubated at room temperature (21-22°C) in relaxing solution containing 4.5 mM MgATP and 1 mM EGTA for several minutes and treated for 30-60 min with 5,000-10,000 units of Staphylococcus aureus a-toxin (GIBCO/BRL) per ml at pCa 6.3 buffered with 10 mM EGTA. To deplete the sarcoplasmic reticulum ofcalcium and maint...

Mechanisms of Ca 2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca 2+ sensitizing effect of guanosine 5'-O-(v-thiotriphosphate) (GTP~/S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP~/S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13-dibutyrate (PDBu) or GTP~S at constant [Ca ~+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca ~+ concentrations. In an ATP-and Ca2+-free solution containing 1-(5-chloronaphthalene-l-sulfonyl)-lH-hexahydro-l,4-diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP~/S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP~/S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP~/S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca 2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.

Two patients, a 56-year-old man and an 81-year-old woman who were admitted to hospital because of anteroseptal acute myocardial infarction, were initially treated successfully with direct percutaneous transluminal coronary angioplasty. However, both patients later developed sudden cardiogenic shock due to cardiac tamponade caused by left ventricular free wall rupture (LVFWR). Prompt, life-saving pericardiocentesis was performed, then fibrin-glue was percutaneously injected into the pericardial space. After the procedure, there was no detectable pericardial effusion on echocardiography and the hemodynamic state became stable. The surgical treatment was the standard procedure for LVFWR, but percutaneous fibrin-glue therapy can also be considered for oozing type LVFWR.

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