Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

Abstract

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.

Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation

“…Fig. 2 indicate that there is indeed a signal interpreted by the microelectrode at a vasoactive [NO] within the vessel lumen as judged by in vivo studies by various laboratories (1,2,9,10,25,44,45). In Fig.…”

“…Total nitrosothiol concentration in plasma is a controversial issue, as Stamler's group (19,39) has reviewed, but could be at a low micromolar concentration. This is cause for concern because direct measurements of in vivo vessel wall [NO] predict a range of 300 -1,000 nM [NO] in a variety of tissues (10), arterioles (1,2,46), and small mesenteric arteries (47). (25).…”

“…NO-sensitive microelectrodes were made from 6-to 8-m-diameter carbon fibers (Fibre Glast, Brookville, OH) sealed in glass micropipettes, calibrated the same as published recently (2,34), and tested for reactions with all chemicals to be used. Only the open tip of the microelectrode can respond to NO because of the insulation provided by the glass envelope.…”

“…A key issue in this area is whether formation of nitrosothiols, particularly with hemoglobin, are so rapid and pervasive that NO made in vessel walls would be rapidly removed to and released at downstream locations. Direct measurements of in vivo vessel wall NO concentration ([NO]) by multiple laboratories indicate [NO] in the range of 300 -1,000 nM in a variety of tissues and small mesenteric arteries, of which a few are now referenced (1,2,10,25,46,47). In these studies, activation of endothelial nitric oxide synthase (eNOS) by appropriate receptor agonists increased the measured [NO], and suppression of nitric oxide synthases with nitroarginine compounds dramatically lowered the measured [NO], giving credence that a form of NO is measured by the microelectrode.…”

Contemporary Approaches to Modulating the Nitric Oxide–cGMP Pathway in Cardiovascular Disease

“…Cavtratin decreases eNOS activity by mimicking the CAV1 inhibitory clamp (Figure 2B) and does not affect the function of other NOSs in vivo and targets eNOS to a significant extent 53 . Cavtratin reduces microvascular hyperpermeability and blocks tumor progression 52, 54 , restores tight junctions in C-C motif chemokine 2 (CCL2) treated brain microvascular endothelial cells (BMECs) 55 , inhibits matrix metalloproteinase-2/9 (MMP2/9) and cyclooxygenase-2 (COX2), which could lead to stabilization of atherosclerotic plaques and protective effects for intracerebral hemorrhages 56, 57 .…”