Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Abstract

Electrophysiological recordings on retinal rod cells, horizontal cells and on‐bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L‐arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.

Purified retinal nitric oxide synthase enhances ADP‐ribosylation of rod outer segment proteins

“…Free Ca 2+ concentrations were adjusted by Ca2+/EGTA buffer mixtures calculated with a calcium buffer program as previously described [26]. Functional coupling of retNOS and soluble guanylyl cyclase was determined a described [6], but Zaprinast was used as a phosphodiesterase inhibitor instead of 3-isobutyl-l-methylxanthine (IBMX). Neuronal NADPH diaphorase activity was assayed with 0.5 mM Nitro blue tetrazolium and 1 mM NADPH exactly as described [27].…”

“…There is also evidence from recent studies that NO is involved in several different processes in the vertebrate retina, for example it influences light responses of amphibian rods [4,5], it activates a soluble guanylyl cyclase and modulates a Ca 2+ channel and a voltage-independent conductance in inner segments of photoreceptors [6,7]. Effects of NO were also recorded on hemi-gap junction channels in horizontal cells [8], on cGMP-gated cation channels in retinal ganglion [9] and on-bipolar cells [10].…”

“…Most authors describe staining of subpopulations of amacrine and ganglion cells, but in some reports labelling of photoreceptor inner segments and cone outer segments was also demonstrated [6,7,17,19]. Additionally, NOS activities have been determined biochemically in crude retinal extracts [22,23] and in a photoreceptor preparation of outer segments attached to parts of the inner segment (OS-IS), where the NOS activity was almost *Corresponding author.…”

“…exclusively found in the inner segment [6]. Enzymatic properties indicate a relationship between the retinal enzyme and the neuronal NOS type I from mammalian brain [24,25].…”

Novel signals mediating the functions of somatostatin: The emerging role of NO/cGMP

“…The role of NO as a neuronal messenger and neuromodulator in retina is well established (Goldstein et al, 1996). As in other systems, it activates a soluble GC (Koch et al, 1994;Ding and Weinberg, 2007) leading to an increase in cGMP production. cGMP is an important regulator of retinal physiology, since it influences a series of biochemical substrates including ion channels (Meriney et al, 1994).…”