Insulin inhibits voltage-dependent calcium influx into rod photoreceptors

Abstract

Insulin inhibits the ERG b-wave and modulates L-type calcium currents (I(Ca)) in various preparations. We therefore examined insulin's effects on I(Ca) and depolarization-evoked [Ca2+]i increases in rod photoreceptors. Insulin inhibited I(Ca) and caused a dose-dependent reduction in the depolarization-evoked Ca2+ influx with an EC50 of 2.1 nM. Tyrosine kinase inhibitors, lavendustin A (100 nM) and genistein (10 microM), prevented insulin from reducing the depolarization-evoked Ca2+ increase in rods. Their less active analogues, lavendustin B and daidzein, had similar effects. An insulin receptor-specific tyrosine kinase inhibitor, HNMPA-(AM)3 (50 microM), prevented insulin (30 nM) from reducing the depolarization-evoked Ca2+ increase in rods. The results suggest that insulin inhibits Ca2+ influx through voltage-dependent I(Ca) in rod photoreceptors via tyrosine kinase activity.

Fig. 1

(a) Current–voltage relationships of rod I_Ca illustrating inhibition by insulin (100 nM). Current–voltage relationship of I_Ca obtained in control superfusate (thin trace) is overlaid with one obtained after perfusion for 3 min with insulin (thick trace). The cell was held at −70 mV and the voltage was ramped from −90 to +60 mV at 0.5 mV/ms. (b) Bar graph showing the mean reduction in I_Ca amplitude by insulin (100 nM). (c) The effect of insulin (100 nM) on Fura-2 ratio changes in a rod inner segment from the retinal slice produced by 1 min applications of elevated (50 mM) [K⁺]_o. (d) Concentration-dependent inhibition of the K⁺-evoked [Ca²⁺]_i increase in rods from retinal slice preparations by insulin. Fura-2-loaded slices were stimulated with elevated [K⁺]_o, in the presence of different concentrations of insulin (0.001–1 μM). Inhibition of K⁺-evoked [Ca2⁺]_i increase produced by insulin were normalized to prior control responses to high K⁺ and expressed as percent inhibition. Each point represents the mean ± s.e.m. Number of experiments is shown in parentheses. * p<0.05 compared with control response.

Fig. 2

Pretreating retinal slices for 1 h with 50 μM HNMPA-(AM)₃, an inhibitor of insulin receptor tyrosine kinase, prevented insulin-mediated inhibition of depolarization-evoked [Ca²⁺]i changes.

Fig. 3

Effects of tyrosine kinase inhibitors on insulin-mediated inhibition of [Ca²⁺]_i changes in photoreceptor inner segments produced by 1 min applications of elevated (50 mM) [K⁺]_o. (a) Genistein (10 μM). (b) The less active analogue of genistein, daidzein (10 μM). Genistein and daidzein were bath applied for 30 min prior to insulin application; shorter 3–5 min applications of genistein were ineffective at preventing the insulin-mediated reduction of K⁺-evoked [Ca²⁺]_i increase. (c) Lavendustin A (100 nM). (d) The less active analogue of lavendustin A, lavendustin B (100 nM). Lavendustin A and B were bath applied for 3 min prior to application of high K⁺ solution or high K⁺ solution plus insulin.