MORPHOLOGIC EVIDENCE FOR L-CITRULLINE CONVERSION TO L-ARGININE VIA THE ARGININOSUCCINATE PATHWAY IN PORCINE CEREBRAL PERIVASCULAR NERVES

Results from biochemical and pharmacologic studies suggest that Lcitru lline is taken up by cerebral perivascular nerves and is converted to Larginine for synthesizing nitric oxide (NO). The current study was de signed using morphologic techniques to determine whether Lcitrulline i s taken up into axoplasm of perivascular nerves and to explore the pos sibility that conversion of Lcitrulline to Larginine in these nerves i s through the argininosuccinate pathway in porcine cerebral arteries. Results from light and electron microscopic autoradiographic studies i ndicated that dense silver grains representing L-[H-3] citrulline upta ke were found in cytoplasm of perivascular nerves, smooth muscle cells , and endothelial cells. The neuronal silver grains were significantly decreased in arteries pretreated with glutamine, which has been shown biochemically to block neuronal uptake of Lcitrulline. Results from l ight and electron microscopic immunohistochemical and histochemical st udies indicate that dense nitric oxide synthase-immunoreactive (NOS-I) , argininosuccinate synthetase-immunoreactive (ASS-I), and argininosuc cinate lyase-immunoreactive (ASL-I) fibers were found in the adventiti a of cerebral arteries. NOS-, ASS-, and ASL-immunoreactivities fibers were found in the axoplasm and in the endothelium. In whole-mount prep arations, the NOS-I, ASS-I, and ASL-I fibers were completely coinciden t with NADPH diaphorase fibers, suggesting that axoplasmic ASS, ASL, a nd NOS were colocalized in the same neurons. These studies provide the first morphologic evidence indicating that Lcitrulline is taken up in to cytoplasm of cerebral perivascular nerves and that the axoplasmic e nzymes catalyzing the conversion of Lcitrulline to Larginine (for synt hesizing NO) by argininosuccinate pathway always are co-localized in s ame neurons. These results support the hypothesis that Lcitrulline, th e by-product of NO synthesis, is recycled to form Larginine for synthe sizing NO in perivascular nerves to mediate cerebral neurogenic vasodi lation. Results of the current morphologic studies also support the pr esence of Lcitrulline-Larginine cycle in cerebral vascular endothelium .