© 1997 American Heart Association, Inc.
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Modulation by pHo and Intracellular Ca2+ of Na+-H+ Exchange in Diabetic Rat Isolated Ventricular Myocytes
the Laboratoire de Physiologie Cellulaire (K. Le P., D.F.), CNRS ERS 100, Universite Paris XI, Orsay, France, and INSERM-Groupe DRT (D.L-G.), Faculte de Pharmacie, Rennes, France.
Correspondence to Dr D. Lagadic-Gossmann, INSERM-Groupe DRT, 2, avenue Pr. Leon Bernard, Faculte de Pharmacie, 35 043 Rennes cedex, France.
We have previously shown that diabetes is associated with a decrease in Na+-H+ exchange activity in rat cardiac papillary muscle. The present work has been carried out in order to elucidate the factors responsible for such an alteration. Thus, we have studied the effects of pHo and intracellular Ca2+ on Na+-H+ exchange in ventricular myocytes isolated from streptozotocin-induced diabetic rat hearts. pHi was recorded using carboxy-seminaphthorhodafluor (SNARF-1). The NH4+ (10 mmol/L) prepulse method was used to induce an acid load in order to activate Na+-H+ exchange in HEPES-buffered Tyrode's solution. Whereas diabetes did not change intracellular buffering power, it significantly decreased acid efflux through Na+-H+ exchange (acid efflux, 4.32±0.4 [n=32, normal cells] versus 2.5±0.2 [n=43, diabetic cells] meq/L per minute at pHi 6.9; P<.02). Upon changes of pHo (at a range of 8.0 to 6.8), acid efflux similarly varied in normal and diabetic cells, thus pointing to an unchanged pHo sensitivity of Na+-H+ exchange. Buffering of intracellular Ca2+ by pretreatment of the cells with BAPTA-AM (25 µmol/L, Ca2+-chelator) resulted in a decrease by 
58% of acid efflux in the diabetic group. This decrease was even more marked in normal cells (by 74%). Interestingly, the pHi dependence of the acid efflux carried by Na+-H+ exchange then became identical in both groups of cells, thus pointing to a role for intracellular Ca2+ in the diabetes-related alterations of the exchange. Inhibition of calmodulin (by 1.5 µmol/L calmidazolium) and of Ca2+/calmodulin-dependent protein kinase II (by 2 µmol/L 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine [KN-62]) significantly slowed down pHi recovery in both normal and diabetic cells. However, the effect of KN-62 was significantly lower in diabetic cells (efflux decreased by 17%) compared with normal cells (decrease by 45%). In conclusion, these data, in light of recent observations showing a decreased [Ca2+]i associated with diabetes in isolated ventricular myocytes, suggest that changes in intracellular Ca2+ may play an important role in altering Na+-H+ exchange activity in diabetic ventricular myocytes. They also point to diabetes-related alterations in the Ca2+/calmodulin protein kinase II–dependent phosphorylation of Na+-H+ exchange.
Key Words: Na+-H+ exchange • ventricular myocyte • diabetes • intracellular Ca2+ • pHo
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