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Abstract
Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na(+)/Ca(2+) exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I(NCX)) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Delta562-679)) via adenoviral gene transfer. I(NCX) was recorded by whole-cell patch clamp as the Ni(2+)-sensitive current at 37 degrees C with intracellular Ca(2+) buffered. Insulin (1 microm) increased I(NCX) (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Delta562-679) the response was eliminated (with 100 microm XIP included to suppress any native guinea pig I(NCX)). The insulin effect on I(NCX) was not inhibited by wortmannin, a nitric-oxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4,5-bisphosphate-induced activation of I(NCX). The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca(2+) balance in the heart.
Highlights
Insulin is an essential hormone for the control of blood glucose concentration
The Insulin Response and the Role of the f-Loop—Membrane currents were evoked by depolarizing pulses between ϩ80 and Ϫ80 mV from a holding potential of Ϫ40 mV in guinea pig myocytes at 37 °C at baseline (Fig. 1A, Control) in the presence of insulin (1 mol/liter, Fig. 1B) and after the addition of NiCl2 (10 mmol/liter, Fig. 1C)
Concluding Remarks— several mechanistic questions remain to be answered regarding the details of the Na؉/Ca2؉ exchanger (NCX) f-loop interactions leading to the activation of NCX current in response to insulin, the results strongly implicate protein kinase C, and not PI3-kinase, in the response
Summary
Insulin is an essential hormone for the control of blood glucose concentration. In the heart, insulin affects many physiological and pathological functions, including energy metabolism, contractility [1, 2], ion transport [3], cardiac protection [4], hypertrophy [5], and cardiomyopathy in diabetes [6]. Ion exchange and transport functions are associated with the transmembrane segments, whereas exchanger regulation appears to be mediated by the large (520 amino acids) cytoplasmic domain f-loop. This intracellular loop is essential for regulation of NCX activity by a number of intracellular factors Previous evidence from our laboratory indicated that f-loop residues 562– 679 contain sites involved in the regulation of the NCX by the endogenous XIP region [22] Deletion of these amino acids eliminated XIP-induced inhibition of NCX in either non-cardiac cell lines or in adult guinea pig cardiomyocytes [22]. The aims of the present study were to evaluate the effects of insulin on the native NCX current (INCX) and to examine the role of the 562– 679 f-loop deletion on the insulin response in intact adult myocytes
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