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.

Background and Purpose-N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K ATP ) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K ATP channels (mitoK ATP ) are involved in the ischemic preconditioning-like effect of K ϩ channel openers. We determined whether the selective mitoK ATP channel opener diazoxide preserves the vascular dilation to NMDA after I/R. Methods-Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 mol/L diazoxide; and coapplication of 100 mol/L 5-hydroxydecanoic acid (5-HD), a K ATP antagonist with diazoxide. Results-NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 mol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 mol/L NMDA-induced vasodilation after I/R was 53Ϯ19% (meanϮSEM, nϭ8) in vehicle-treated controls versus 55Ϯ10%, 85Ϯ5%, and 99Ϯ15% in animals pretreated with 1, 5, and 10 mol/L diazoxide (nϭ8, nϭ8, and nϭ12, respectively) and 60Ϯ15% in the group treated with 5-HDϩdiazoxide (nϭ5). Key Words: cerebral ischemia, global Ⅲ N-methyl-D-aspartate Ⅲ potassium channels Ⅲ reperfusion injury Ⅲ pigs G lutamate elicits cerebral arteriolar vasodilation in piglets via a multistep process, involving activation of neuronal N-methyl-D-aspartate (NMDA) receptors, stimulation of nitric oxide (NO) production by neuronal NO synthase, and actions of NO on vascular smooth muscle cells. [1][2][3] This sequence of events may represent an important mechanism coupling local blood flow to metabolism and neuronal activity. Conclusions-TheNMDA-induced vasodilation is attenuated by hypoxia and ischemia/reperfusion (I/R) in a dose-and time-dependent manner. 4 -6 For example, 10 minutes of global ischemia followed by reperfusion reduces NMDA-induced vasodilation by Ϸ50%. However, arteriolar dilator responses to exogenously applied NO are intact, 5,6 thereby implying that the attenuation of the vascular response to NMDA is due to See Editorial Comment, page 2718 effects of ischemia at the level of the neurons. Furthermore, results from other laboratories as well as our own indicate that dysfunction of the NMDA receptor rather than of general neuronal injury is the primary reason for attenuated arteriolar responsiveness to NMDA. 5,7 The mechanisms involved in attenuated arteriolar dilation to NMDA are not known with certai...

. 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...

Anoxic stress attenuates NMDA-induced pial arteriolar dilation via a mechanism involving actions of cyclooxygenase (COX)-derived reactive oxygen species (ROS). We examined whether the selective COX-2 inhibitor NS398 would protect neuronal function after global hypoxia/ischemia (H/I) in piglets. Pial arteriolar responses to NMDA (10-100 micromol/l) were determined using intravital microscopy in anesthetized piglets before and 1 h after H/I. Study groups received vehicle, 0.3, 1, or 5 mg/kg NS398, or 0.3 mg/kg indomethacin (n = 7, 6, 6, 5 and 8, respectively) i.v. 20 min prior to H/I. H/I reduced NMDA- induced dilation to 44 +/- 6% (100 micromol/l NMDA, mean +/- s.e.m.) of the pre-ischemic response in vehicle animals (p < 0.05). However, NS398 dose-dependently protected arteriolar dilation to NMDA (77 +/- 8, 81 +/- 16, and 102 +/- 10% preservation at 0.3, 1 and 5 mg/kg, respectively). Indomethacin caused similar preservation. However, indomethacin but not NS398 reduced serum thromboxane B(2) levels to undetectable values. In conclusion, COX-2 appears to be a major source of ROS in the piglet cerebral cortex after H/I.