Nitric oxide increases carbon monoxide production by piglet cerebral microvessels

Abstract

Carbon monoxide (CO) and nitric oxide (NO) can be involved in the regulation of cerebral circulation. Inhibition of production of either one of these gaseous intercellular messengers inhibits newborn pig cerebral arteriolar dilation to the excitatory amino acid glutamate. Glutamate can increase NO production. Therefore, the present study tests the hypothesis that NO, which is increased by glutamate, stimulates the production of CO by cerebral microvessels. Experiments used freshly isolated cerebral microvessels from piglets that express only heme oxygenase-2 (HO-2). CO production was measured by gas chromatography-mass spectrometry. Although inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine (l-NNA) did not alter basal HO-2 catalytic activity or CO production, l-NNA blocked glutamate stimulation of HO-2 activity and CO production. Furthermore, the NO donor sodium nitroprusside mimicked the actions of glutamate on HO-2 and CO production. The action of NO appears to be via cGMP because 8-bromo-cGMP mimics and 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) blocks glutamate stimulation of CO production and HO-2 catalytic activity. Inhibitors of neither casein kinase nor phosphotidylinositol 3-kinase altered HO-2 catalytic activity. Conversely, inhibition of calmodulin with calmidazolium chloride blocked glutamate stimulation of CO production and reduced HO-2 catalytic activity. These data suggest that glutamate may activate NOS producing NO that leads to CO synthesis via a cGMP-dependent elevation of HO-2 catalytic activity. These results are consistent with the findings in vivo that either HO or NOS inhibition blocks cerebrovascular dilation to glutamate in piglets.

Figure 1

Effect of glutamate (10⁻⁴ M) on CO production by piglet cerebral microvessels from endogenous and exogenous heme (5x10⁻⁶ M) in the absence and presence of LNA (10⁻³ M). means±SEM. *P<0.05 compared to preceding bar. N=4.

Figure 2

Effect of sodium nitroprusside (SNP) on CO production and HO-2 catalytic activity (exogenous heme, 5x10⁻⁶ M) by piglet cerebral microvessels. means±SEM. *P<0.05 compared to no SNP. +P<0.05 compared to exogenous heme with no SNP. N=4.

Figure 3

Effect of ODQ (5x10⁻⁴M) on CO production from endogenous and exogenous heme (5x10⁻⁶ M) by piglet cerebral microvessels. means±SEM. *P<0.05 compared to control. +P<0.05 compared to heme without ODQ. N=6.

Figure 4

Effect of ODQ (5x10⁻⁴M) on glutamate (10⁻⁴ M) stimulation of CO production by piglet cerebral microvessels. means±SEM. *P<0.05 compared to control. +P<0.05 compared to ODQ without glutamate. N=6.

Figure 5

Effect of 8 br-cGMP (10⁻⁵ M) on CO production by piglet cerebral microvessels without and with glutamate (10⁻⁴ M) and/or LNA (10⁻³M). means±SEM. *P<0.05 compared to LNA alone. N=8.

Figure 6

Effect of calmidizolium Cl (CzCl) (2x10⁻⁵M) on glutamate (10⁻⁴M) stimulation of CO production by piglet cerebral microvessels. means±SEM. *P<0.05 compared to control. N=7.