Calcium interactions with various subcellular membranes of the heart in the presence of insulin

358 CALCIUM INTERACTIONS WITH VARIOUS SUBCELLULAR MEMBRANES OF THE HEART IN THE PRESENCE OF INSULIN. G.N. Pierce, P,K, Ganguly, A. Dzurba, N-S. Dhalla. Department of Canada R3E OW3 Physiology, University of Manitoba, Winnipeg, Manitoba, At high concentrations, insulin has been shown to possess positive inotropic and chronotropic properties. Many studies have suggested that insulin may alter cellular The purpose no study has directly examined this problem. Ca*+ distribution; however, of the present study was to investigate if insulin (0.1 mU - 1000 mu/ml) may alter the in vitro function of the cardiac sarcoplasmic reticular (SR), mitochondrial or sarcolemmal membranes. For this purpose, relatively pure fractions of subcellular Insulin increased SR Ca2+organelles were isolated2pm the rat heart and employed. stimulated ATPase and Ca uptake activities particularly at the higher insulin conThis response was confirmed using two different SR centration examined (P < 0.05). Insulin did not affect mitochondrial Ca*+-uptake or membrane isolation procedures. insulin significantly increased sarcolemmal Mg*+-ATPase activity. On the other hand, Ca*+-stimulated ATPase activity at all concentrations examined (P < 0.05). Only lU/ ATPase activity as well ml insulin significantly enhanced sarcolemmal Ca*+ -dependent as both low and high affinity ATP-independent Ca*+-binding. These results suggest two subcellular sites of action for insulin located at the SR and sarcolemmal membranes and may provide some information regarding the mechanisms responsible for the (Supported by the Medical Research Council inotropic effect of insulin on the heart. of Canada). 357INSULIN ACTION ON CARDIAC GLUCOSE TRANSPORT: STUDIES ON THE ROLE OF SODIUM AND Diabetes Research Institute, POTASSIUM. Jijrgen Eckel, Anne Stocks, Hans Reinauer. DGsseldorP, Federal Republic of Germany. The interaction between monovalent ions and insulin-sensitive cardiac sugar transUsing isolated cardiac myocytes from adult rat we have now port is poorly understood. studied the effect of insulin on Rb+-uptake and on the intracellular content of Na+ and tried to correlate it to insulin's stimulatory action on glucose transand K+, port. Insulin treatment of cardiocytes resulted in an increase of the initial velocity of 3-D-methyl lucose transport by about 80 %. Under the same incubation conditions, uptake of 13' Rb + was found to be rapid with an initial phase up to 2 min and a under all conditions tested (addition of Ca* or Mg*, plateau by 40 min. However, measurement in the presence of 1.2 or 6 mfl K+,' 24 h fasted animals) no effect of insulin on Rb+-uptake could be detected. The content of Na+ and K+ was found to be 49 m m and 166 mN and remained unaltered in the presence of insulin. Treatment of cardioby 80 % and reduced the K+-content cytes with ouabain (10v3 N) inhibited Rb+ -uptake by 70 '$ while the Na+-content inGreased by 300 %. In contrast, both basal and insulin-stimulated 3-0-methylglucose transport remained unaffected after ouabain treatour data show that Na+ and @-ions are not involved in the ment. In conclusion, regulation of glucose transport in cardiac muscle.

358 EFFECT OF ATP ON THE APPARENT POTASSIUM STIMULATED UPTAKE OF CALCIUM BY PURIFIED BOVINE CARDIAC SARCOLEMMAL VESICLES(BSL).V.Flockerzi,R.Mewes,P.Ruth,F.Hofmann.Pharmakol.Institut,Universitlt Heidelberg,Im Neuenheimer Feld 366,D6900 Heidelberg,Germany. BSL were purified by differential and gradient density centrifugation.Na+/K+-ATPase activity(53umol/mg),specific Strophantin-(56pmol/mg)and+nitr$ndipine binding sites (0.7pmol/mg)were enriched 33.20 and 7-fold.Vesicular Na /Ca exchange was 6lnmol/mg. The intravesicular volume was 21 ul/mg.The major phosphopeptide of Mr 24.000 was phos phpfylated already in the presence of Mg.ATP.Its phosphorylation was stimu&ted by Ca /CaM and CAMP-kinase.The phosphorylation pattern in the presence of Cp+ /CaM was different from that observed with bovine SR.App.depolarization inducgd Ca uptake y$s Measured according to Bartschat et a1.(1980,J.Biol.Chem.255,1OO44).K stimulated Ca uptake measured 5sec after "depolarizationuwas about Snm~mg.This uptake was not stimulated by preincubation of BSL in the presence of Mg+ATP and CAMP-kinase.This value is in agreement with the theoretical amount of Ca whi$h can be accumulated by the BSL in the absence of a driving force other than the Ca gradient.The uptake was not blocked by the organic Ca blockers.Prolonged incubation of ATP with BSL in the presence of various &;Pase inhibitors led to complete hydrolysis of ATP.Pi liberated precipitated with Ca in the prpsence of LaCl, and resulted in an app.Ca uptakp, ranging from 50 to lOOOnmo1 Ca /mg.These results suggest that K+ stimulated Ca uptake of BSLis not enhanced by phosphorylation of a 24.000 dalton peptide.