CALCIUM-ION REQUIREMENT FOR ACETYLCHOLINE-STIMULATED BREAKDOWN OF TRIPHOSPHOINOSITIDE IN RABBIT IRIS SMOOTH-MUSCLE

Abstract

Addition of acetylcholine (ACh) or norepinephrine to 32P-labeled rabbit iris smooth muscle significantly increases the breakdown of triphosphoinositide (TPI), and these stimulatory effects are blocked by atropine and phentolamine, respectively. The effect of Ca2+ on the ACh-stimulated breakdown of TPI (TPI effect) was demonstrated in this tissue. Paired iris smooth muscles were prelabeled with 32Pi for 30 min at 37.degree. C in Ca2+-free iso-osmotic salt medium. The prelabeled irises were then washed and incubated for 10 min in nonradioactive Ca2+-free medium which contained 10 mM 2-deoxyglucose under various conditions. The phospholipids were isolated by 2-dimensional TLC, and their radioactivities were determined. In the absence of Ca2+, 50 .mu.M ACh increased TPI breakdown and phosphatidic acid (PA) labeling by 16 and 38%, respectively. In the absence of ACh, 0.75 mM Ca2+ increased TPI breakdown and PA labeling by 11 and 20%, respectively. When ACh and Ca2+ were added, the increase in TPI breakdown and PA labeling rose to 32 and 74%, respectively. The labeling of phosphatidylinositol was insensitive to the presence of Ca2+. Ca2+ was determined in the iris smooth muscle, and it contained 3.13 .mu.mol of Ca2+/g of tissue. This was reduced by 80% after the muscle was washed and incubated in a medium which contained 0.25 mM ethyleneglycol bis(.beta.-aminoethyl ether)-N,N''-tetraacetic acid (EGTA). The TPI effect was abolished by 0.25 mM EGTA and restored when excess Ca2+ (1.25 mM) was added. Concentrations of Ca2+ as low as 50 .mu.M provoked a TPI effect. Sr2+ (2 mM), but not Ba2+ or Mn2+, partially substituted for Ca2+. Ionophore A-23187 (20 .mu.M) increased the breakdown of TPI and labeling of PA by 11 and 24%, respectively. High concentrations of Ca2+ (20 mM) exerted similar effects. The increase in TPI breakdown and PA labeling in response to these agents, in contrast to the TPI effect in response to ACh, was not blocked by atropine. Apparently these effects are not caused by the release of endogenous ACh from the muscle. A possible interpretation for these observations on the role of Ca2+ in the TPI effect at the postsynaptic membrane of the iris smooth muscle could be: ACh + receptor .fwdarw. ACh .sbd..sbd..sbd. receptor .fwdarw. Ca2+int .uparw. .fwdarw. TPI breakdown .fwdarw. changes in membrane permeability .fwdarw. response.