Calcium Channel Activation Facilitated By Nitric Oxide In Retinal Ganglion Cells. Kazuyuki Hirooka1, Dmitri E. Kourennyi2 and Steven Barnes1. 1Departments of Physiology & Biophysics and Ophthalmology, Dalhousie University, Halifax, Nova Scotia; and 2Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH.
APStracts 6:0491N, 1999.
We investigated the modulation of voltage-gated Ca channels by nitric oxide (NO) in isolated salamander retinal ganglion cells with the goals of determining the type of Ca channel affected and the signaling pathway by which modulation might occur. The NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 1 mM) and S-nitroso-cysteine (SNC, 1 mM) induced modest increases in the amplitude of Ca channel currents recorded with ruptured- and permeabilized-patch techniques by causing a subpopulation of the Ca channels to activate at more negative potentials. The Ca channel antagonists (-conotoxin GVIA and nisoldipine each reduced the Ca channel current partially but only (-conotoxin GVIA blocked the enhancement by SNAP. The SNAP-induced increase was blocked by oxadiazolo-quinoxaline (ODQ, 50 然), suggesting that the NO generated by SNAP acts via a soluble guanylyl cyclase to raise levels of cGMP. The membrane-permeant cGMP analogue CPT-cGMP also enhanced Ca channel currents and 8-Br-cGMP (1 mM) occluded enhancement by SNAP. Consistent with these results, isobutyl-methyl-xanthine (IBMX, 10 然), which can raise cGMP levels by inhibiting phosphodiesterase activity, increased Ca channel current by the same amount as SNAP and occluded subsequent enhancement by SNAP. Neither IBMX, the cGMP analogues, nor SNAP itself, led to activation of cGMP-gated channels. H-8 (2 然), a broad spectrum inhibitor of protein kinase activity, KT5823 (1然), a specific protein kinase G (PKG) inhibitor, and a peptide inhibitor of PKG (200然) blocked SNAP enhancement, as did AMP-PNP (1.5 mM), a nonhydrolyzable ATP analogue that prevents protein phosphorylation. A peptide inhibitor of protein kinase A (10 nM) did not block the facilitory effects of SNAP. Okadaic acid (1 然), a phosphatase inhibitor, had no effect by itself but increased the enhancement of Ca channel current by SNAP. These results suggest that NO modulates retinal ganglion cell N-type Ca channels by facilitating their voltage dependent activation via a mechanism involving guanylyl cyclase/PKG dependent phosphorylation. This effect could fine-tune neural integration in ganglion cells or play a role in ganglion cell disease by modulating intracellular calcium signaling.
Received 23 April 1999; accepted in final form 17 September 1999.
APS Manuscript Number J333-9.
Article publication pending Journal of Neurophysiology.
ISSN 1080-4757 Copyright 1999 The American Physiological Society.
Published in APStracts on 21 December 1999