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Nitric oxide synthesis in retinal photoreceptor cells

Published online by Cambridge University Press:  02 June 2009

Akiko Yoshida ,
Nikolay Pozdnyakov ,
Loan Dang ,
Stephen M. Orselli ,
Venkat N. Reddy  and
Ari Sitaramayya
Akiko Yoshida
Affiliation:
Eye Research Institute, Oakland University, Rochester
Nikolay Pozdnyakov
Affiliation:
Eye Research Institute, Oakland University, Rochester
Loan Dang
Affiliation:
Eye Research Institute, Oakland University, Rochester
Stephen M. Orselli
Affiliation:
Eye Research Institute, Oakland University, Rochester
Venkat N. Reddy
Affiliation:
Eye Research Institute, Oakland University, Rochester
Ari Sitaramayya
Affiliation:
Eye Research Institute, Oakland University, Rochester
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Abstract

Nitric oxide (NO) is known to be synthesized in several tissues and to increase the formation of cyclic GMP through the activation of soluble guanylate cyclases. Since cyclic GMP plays an important role in visual transduction, we investigated the presence of nitric oxide synthesizing activity in retinal rod outer segments. Bovine rod outer segments were isolated intact and separated into membrane and cytosolic fractions. Nitric oxide synthase activity was assayed by measuring the conversion of L-arginine to L-citrulline. Both membrane and cytosolic fractions were active in the presence of calcium and calmodulin. The activity in both fractions was stimulated by the nitric oxide synthase cofactors FAD, FMN, and tetrahydrobiopterin and inhibited by the L-arginine analog, L-monomethyl arginine. The Km for L-arginine was similar, about 5 μM for the enzyme in both fractions. However, the two fractions differed in their calcium/calmodulin dependence: the membrane fraction exhibited basal activity even in the absence of added calcium and calmodulin while the cytosolic fraction was inactive. But the activity increased in both fractions when supplemented with calcium/calmodulin: in membranes from about 40 to 110 fmol/min/mg of protein and in the cytosol from near zero to about 350 fmol/min/mg of protein in assays carried out at 0.3 μM L-arginine. The two enzymes also responded differently to detergent: the activity of the membrane enzyme was doubled by Triton X-100 while that of the cytosolic enzyme was unaffected. These results show that NO is produced by cytosolic and membrane-associated enzymes with distinguishable properties. Investigations on the purity of isolated ROS showed that about 50% of the NOS activity is endogenous to the outer segments, and that the rest is due to membrane vesicles rich in Na, K-ATPase activity. If and how NO influences the rod outer segment physiology remains to be investigated.

Keywords

Nitric oxide synthaseNADPH-diaphoraseRod outer segmentsPhotoreceptor
Type
Research Articles
Information
Visual Neuroscience , Volume 12 , Issue 3 , May 1995 , pp. 493 - 500
Copyright
Copyright © Cambridge University Press 1995

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