Tonic protein kinase C-mediated vasoconstriction is unmasked when nitric oxide synthase is inhibited in cerebral microvessels

Recent evidence indicates that nitric oxide participates in the modulation of vascular tone in a variety of vascular beds, including the parenchymal microvasculature of the brain. The present study examined the role of protein kinase activity in the induction and maintenance of the contractile response when endogenous nitric oxide production is inhibited in parenchymal microvessels of the rat hippocampus. Microvessels in in vitro slices of the hippocampus were monitored using computer-assisted video microscopy. The effects of inhibitors of two kinases, protein kinase C and calcium/calmodulin-dependent protein kinase, on the vasoconstrictor response to N(G)-nitro-L-arginine (L-NNA) were investigated. The resting luminal diameter of the microvessels examined in this study ranged from 9 to 29 μm. Addition of 100 μM L-NNA to the medium superfusing the slice constricted microvessels by 38.8 ± 0.6%. The addition of protein kinase inhibitors reversed this constriction in a dose-dependent manner. H-7 (50 μM), a relatively non-selective protein kinase C inhibitor, elicited an 81.4 ± 10.0% reversal of the L-NNA-induced constriction. Bisindolylmaleimide (5 μM), a selective protein kinase C inhibitor, reversed the constriction by 69.1 ± 13.7%. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II, elicited a smaller yet statistically significant reversal of 17.1 ± 5.1%. Pretreatment with H-7 or bisindolylmaleimide blocked the LNNA-induced constriction entirely, while KN-62 did not significantly inhibit the response. These findings indicate that the contractile response observed upon removal of endogenous nitric oxidergic vasodilation is mediated by protein kinase activity, and the contribution of protein kinase C to this effect is greater than that of calcium/calmodulin-dependent protein kinase II. The results suggest that a tonic nitric oxidergic influence serves to mask the potential for protein kinase C-mediated vasoconstriction in cerebral microvessels.