Characterization of Ca2+release from heterogeneous Ca2+stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Abstract

Ca2+transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15–33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or45Ca2+Millipore filtration. Thapsigargin blocked active Ca2+uptake and induced a slow Ca2+release from actively loaded vesicles. Unidirectional45Ca2+efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 μM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+release was significantly smaller and more sensitive to inhibition by Mg2+and ruthenium red in arterial vesicles. They released more Ca2+in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca+2store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.Key words: calcium channels, smooth muscle, sarcoplasmic reticulum.