We investigated the mechanism of EDHF-mediated dilation to bradykinin (BK) in piglet pial arteries. Topically applied BK (3 micromol/l) induced vasodilation (62 +/- 12%) after the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which was inhibited by endothelial impairment or by the BK(2) receptor antagonist HOE-140 (0.3 micromol/l). Western blotting showed the presence of BK(2) receptors in brain cortex and pial vascular tissue samples. The cytochrome P-450 antagonist miconazole (20 micromol/l) and the lipoxygenase inhibitors baicalein (10 micromol/l) and cinnamyl-3,4-dyhydroxy-alpha-cyanocinnamate (1 micromol/l) failed to reduce the BK-induced dilation. However, the H(2)O(2) scavenger catalase (400 U/ml) abolished the response (from 54 +/- 11 to 0 +/- 2 microm; P < 0.01). The ATP-dependent K(+) (K(ATP)) channel inhibitor glibenclamide (10 micromol/l) had a similar effect as well (from 54 +/- 11 to 16 +/- 5 microm; P < 0.05). Coapplication of the Ca(2+)-dependent K(+) channel inhibitors charybdotoxin (0.1 micromol/l) and apamin (0.5 micromol/l) failed to reduce the response. We conclude that H(2)O(2) mediates the non-nitric oxide-, non-prostanoid-dependent vasorelaxation to BK in the piglet pial vasculature. The response is mediated via BK(2) receptors and the opening of K(ATP) channels.

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. N-methyl-D-aspartate-induced vasodilation is mediated by endothelium-independent nitric oxide release in piglets. Am J Physiol Heart Circ Physiol 282: H1404-H1409, 2002. First published November 23, 2001 10.1152/ajpheart. 00523.2001.-N-methyl-D-aspartate (NMDA) elicits pial arteriolar dilation that has been associated with neuronal nitric oxide (NO) production. However, endothelial factors or glial P-450 epoxygenase products may play a role. We tested whether NMDA-induced pial vasodilation 1) primarily involves NO diffusion from the parenchyma to the surface arterioles, 2) involves intact endothelial function, and 3) involves a miconazole-sensitive component. Arteriolar diameters were determined using closed cranial window-intravital microscopy in anesthetized piglets. NMDA (10-100 M) elicited virtually identical dose-dependent dilations in paired arterioles (r ϭ 0.94, n ϭ 15). However, NMDA-but not bradykinin (BK)-induced dilations of arteriolar sections over large veins were reduced by 31 Ϯ 1% (means Ϯ SE, P Ͻ 0.05, n ϭ 4) compared with adjacent sections on the cortical surface. Also, 100 M NMDA increased cerebrospinal fluid levels of NO metabolites from 3.7 Ϯ 1.0 to 5.3 Ϯ 1.2 M (P Ͻ 0.05, n ϭ 6). Endothelial stunning by intracarotid injection of phorbol 12,13-dibutyrate did not affect NMDA-induced vasodilation but attenuated vascular responses to hypercapnia and BK by ϳ70% (n ϭ 7). Finally, miconazole (n ϭ 6, 20 M) pretreatment and coapplication with NMDA did not alter vascular responses to NMDA. In conclusion, NMDA appears to dilate pial arterioles exclusively through release and diffusion of NO from neurons to the pial surface in piglets. cerebral circulation; miconazole; pial arterioles; bradykinin PREVIOUS STUDIES suggest that stimulation of N-methyl-D-aspartate (NMDA) receptors on cortical neurons results in dose-dependent pial arteriolar dilation via a mechanism involving neuronal nitric oxide (NO) synthase (nNOS) activation and subsequent NO release in newborn pigs (11,29). Because cerebral resistance vessels and cortical astroglia lack NMDA receptors, cerebral vasodilation to NMDA must be initiated by substances released from activated neurons (30,38). Similar involvement of NO in NMDA-induced pial arteriolar vasodilation or increased cerebral blood flow (CBF) have been observed in many other species (14,32,40). Glutamate receptor activation either by nervous or pharmacological stimulation results in increased blood flow in a variety of regions, such as the cerebral cortex (25, 33, 41), striatum (10), hippocampus (18), cerebellum (3), and medulla (16) in rats. In all these regions, NO seems to be involved in the mechanism of CBF increase, and the major glutamate receptor subtype appears to be the NMDA receptor, except in the cerebellum (3, 40). However, many of the details of this relationship between activation of NMDA receptors and cerebrovascular dilation are unclear. Whereas there is agreement that NO is essential for NMDAinduced arteriolar dilation, it is unclear whether parenchymal-derived N...

Background and Purpose-Production of NO by endothelial NO synthase (eNOS) plays a protective role in cerebral ischemia. We studied the effects of transient focal ischemia on eNOS expression. Methods-Wistar rats (nϭ72) underwent reversible filament occlusion of the right middle cerebral artery for 75 minutes.After 6, 24, 72, or 168 hours of reperfusion, brains were removed and coronal sections cut for eNOS immunohistochemistry, eNOS-alkaline phosphatase costaining, and hematoxylin-eosin staining. Samples for eNOS immunoblots were taken from corresponding striatum and overlying parietal cortex bilaterally. Results-eNOS protein occurred in virtually all blood vessels and was consistently increased in microvessels in the ischemic striatum after 24 to 168 hours of reperfusion but not at 6 hours. eNOS upregulation in the parietal cortex was only present in animals with evidence of cortical infarcts documented on adjacent HE-stained sections. Costaining of endogenous alkaline phosphatase and eNOS demonstrated eNOS expression in all segments of cerebral microvessels. Quantitative analysis of eNOS immunostaining and immunoblots showed no attenuated increase in animals that were treated with indomethacin (5 mg/kg IP), NS398 (20 mg/kg IP), or L-arginine-methyl ester (10 mg/kg IP). In contrast to eNOS, levels of brain NOS did not increase after ischemia. Conclusion-eNOS protein is upregulated in pre-and postcapillary microvessels and upregulation appears slower after transient compared with permanent ischemia. Cyclooxygenase and NOS products do not play a major role in postischemic eNOS induction.