Ca2+ mobilization in dispersed smooth muscle cells of the porcine coronary artery was investigated using the fluorescent Ca2+ indicator, quin2. The resting [Ca2+]i was 113 +/- 8 nM (a mean +/- SE), and was independent of intracellular quin2 concentrations. Acetylcholine (ACh; over 10 nM) or caffeine (over 3 mM) transiently increased the intensity of fluorescence, thereby reflecting the elevation of intracellular free Ca2+ (Ca2+ transient), while excess K+ gradually increased and maintained the intensity of fluorescence. Application of EGTA reduced the resting intensity of the fluorescence and blocked the K+-induced Ca2+ transient, but did not suppress the ACh- or caffeine-induced ones. Nisoldipine (0.1 microM) did not affect the resting intensity of the fluorescence. This agent blocked the K+ induced but not the ACh- or caffeine- induced Ca2+ transient. Thus, sources of Ca2+ contributing to the K+ -induced Ca2+ transient differ from those evoked by other agents. The amount of Ca2+, as estimated from the increased Ca2+ transient by caffeine or ACh, was increased in proportion to the excess K+-induced influx of Ca2+.

SUMMARY1. To clarify the role of protein kinase C in the mechanical response, the effects of 12-o-tetradecanoylphorbol-13-acetate (TPA), an activator ofprotein kinase C, were investigated on intact and skinned smooth muscle preparations of the rabbit mesenteric artery.2. TPA (0-1 uM) showed dual actions (initial enhancement followed by inhibition during long exposure) on the K+-induced contraction. The enhancement was marked in the presence of 39 mM-K+ but inhibition was the predominant effect in the presence of 128 mM-K+.3. Addition of2-6 mM-Ca2+ to a Ca2+-free solution containing 2 mm-EGTA following application of A23187 (1 /LM), produced contraction. TPA showed the same dual actions on this Ca2+-induced contraction.4. In chemically skinned muscles, TPA increased the amplitude of Ca2+-induced contractions evoked by low concentrations of Ca2+ (0-1-0{3 /LM), but reduced those evoked by high concentrations of Ca2+ (1-10 /M). Both actions of TPA were facilitated in the presence of phosphatidylserine (PS).5. TPA with PS had no effect on the Ca2+-independent contraction evoked in relaxing solution containing 10 mM-EGTA and 4 mM-Mg ATP following application of adenosine-5-o-3-thiotriphosphate (ATPYS) and 0-3 ,uM-Ca2 .6. The amount of Ca2+ stored in cells estimated from the amplitude of the caffeine-induced contraction was not modified by application of TPA with PS in skinned or intact muscle tissues.7. The effects of TPA were investigated on the Ca2+ transient measured from the intensity of fluorescence of quin-2 in dispersed cell suspensions prepared from the porcine coronary artery. TPA had no effect on the Ca2+ transient in high K+ but enhanced the amplitude of the contraction.8. Amplitudes of the tonic response evoked by 39 mM-K+ in intact muscle tissues and the contraction induced by 0 3 /sM-Ca2+ in skinned muscle were much the same.TPA with PS enhanced the amplitudes of both contractions to the same extent. 9. From the above results, we concluded that TPA shows dual actions on the contractile machinery and may act on the regulatory systems of contractile proteins. 17-2 T. ITOH AND OTHERSBoth excitatory and inhibitory actions ofTPA depended on the concentration ofCa2 .However, the physiological action of protein kinase C as estimated from the action of TPA seems to be related to an excitatory action on the contractile machinery.

No abstract

1 The effects of procaterol, a P2-adrenoceptor agonist, on smooth muscle cells of the dog trachea were investigated by use ofmicroelectrode and isometric tension recording methods, and by measurement of Ca transients as estimated fromthe fura-2 fluorescence, adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and breakdown of phosphatidylinositols. 2 Procaterol hyperpolarized the membrane and increased the ionic conductance (above 10 nM) in a dose-dependent manner. These actions were inhibited by propranolol. 3 Procaterol inhibited the mechanical responses evoked by acetylcholine (ACh), histamine or 5-hydroxytryptamine (5-HT), in the presence or absence of Ca2+ in the bath solution, but not that evoked by high concentrations of ACh (1 JiM). The ID_,0 value of procaterol for the peak amplitude of the ACh-induced contraction (30 nM) was 0.3 nM. The equivalent values for the histamine-induced phasic and tonic responses (10I1M) were 0.15 and 0.01 nM), respectively. 4 Procaterol (over 1 nM) increased the amount ofcyclic AMP in a dose-dependent manner which was blocked by prior application of propranolol. 5 Procaterol did not alter the changes in the amounts of phosphatidylinositol 4,5-bisphosphate (PI-P2) and phosphatidic acid (PA) induced by ACh, histamine or 5-HT. Thus, the synthesis of inositol 1,4,5-trisphosphate is not affected by stimulation of the P2-adrenoceptor.6 ACh increased the free Ca2" concentration to a greater extent than that produced by histamine or 5-HT. These changes were reduced by procaterol, except for those induced by high concentrations of ACh (over I1LM).7 It is concluded that procaterol relaxes tissues precontracted by various agonists due to a reduction in the free Ca2+. This inhibitory action may be due to an increase in the amount ofcyclic AMP but does not result from an inhibition of the hydrolysis of phosphatidyl inositols. The hyperpolarization induced by procaterol may partly contribute to the observed relaxation.

The action of procaine on pharmaco-mechanical coupling activated by application of acetylcholine (ACh) was investigated using collagenase-treated dispersed intact and skinned smooth muscle cells and intact muscle tissues of the porcine coronary artery. ACh reduced stored 45