Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression.

Abstract

Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, NO-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by Nwnitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO. Nitric oxide (NO) appears to mediate the cerebral blood flow increase during moderate hypercapnia (5% CO2) (1). Inhibitors of NO synthase (NOS, EC 1.14.13.39) activity such as Nw-nitro-L-arginine (L-NNA) or Nw-nitro-L-arginine methyl ester (L-NAME) attenuate the regional cerebral blood flow (rCBF) increase during CO2 inhalation in most studies (2-7), and the attenuated response correlates with inhibition of brain NOS activity as measured ex vivo by the conversion of [3H]arginine to [3H]citrulline (3). NO may act via cGMP, as 8-bromo-cGMP as well as an NO donor restores the hypercapnic response after treatment with NOS inhibitors (8).At least three NO sources may contribute to the CO2-induced blood flow response-the endothelium, perivascular nerves, and parenchymal neurons (1). The relative contributions of these sources have been difficult to establish because L-NNA and L-NAME are nonselective, inhibiting neuronal NOS (nNOS) and endothelial NOS (eNOS) similarly (9).A mutant mouse strain (Kn) lacking expression of nNOS has been developed (10). Residual brain NOS activity, <5% of wild type, may derive from eNOS which is expressed in brains of the Kn mutants in neurons as well as blood vessels (11). We recently established a physiological monitoring system which permits continuous monitoring of arterial blood pressure, end-tidal CO2, rCBF (laser Doppler flowmetry), heart rate, and pial vessel diameter in the artificially ventilated mouse (12, 13). We now show that nNOS is a mediator of C02-dependent hyperemia in wild-type mice. Kn mice display normal CO2 responsiveness which is achieved by non-NO, cGMP-independent mechanisms, reflecting true physiological redundancy. METHODSImmunohistochemistry. Mice were perfused with phosphate-buffered saline followed by freshly depolymerized 4%The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 2-4 hr. Immunostaining for eNOS and nNOS was performed as described (14) with affinity-purified eNOS antiserum (1:50 d...

Nitric oxide in excitable tissues: physiological roles and disease.

“…This local vascular reflex to hypercapnia is NO-dependent, as it is acutely blocked by NOS inhibitors (12). Surprisingly, hypercapnic cerebral blood flow responses are intact in nNOS knockout mice, and NOS inhibitors do not block the response in the nNOS knockout mice (15). Therefore, the maintenance of hypercapnic blood flow response in the nNOS knockout mice is not due to upregulation of other NOS isoforms but instead is mediated by an NO-independent mechanism.…”

Cerebral Microvessel Responses to Focal Ischemia

“…The relative homogeneity of the distribution and density of striatal microvessels in the nonhuman primate make it a suitable setting for examining microvessel responses. It should be mentioned that these vascular relationships, and therefore their responses to ischemia, may also be influenced by genetic factors (Edvinsson et al, 1993;Irikura et al, 1995;Niwa et al, 2000;Cavaglia et al, 2001;Bale et al, 2002;Demchenko et al, 2002).…”

Attenuation of activity-induced increases in cerebellar blood flow in mice lacking neuronal nitric oxide synthase

“…Therefore, it would be important to provide independent evidence of the role of NO in cerebellar hyperemia by using an approach not based on pharmacological agents. Mice lacking selected isoforms of NOS have been useful in exploring the role of NO in cerebrovascular regulation (5,16). In the present study, we used nNOS-null mice (12) to more specifically investigate the role of nNOS in the mechanisms of functional hyperemia in the cerebellum.…”