Mechanism of glutamate stimulation of CO production in cerebral microvessels

Abstract

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway. heme oxygenase; cerebrovascular circulation; endothelium; vascular smooth muscle; phosphorylation; calcium CARBON MONOXIDE (CO) is an important cellular signaling molecule in the cerebral circulation. In newborn pigs, the constitutive enzyme that produces CO, heme oxygenase-2 (HO-2), has very high expression in cerebral microvessels. Of the vascular tissues examined, by far the greatest production of CO is by cerebral microvessels (19). The excitatory amino acid glutamate stimulates CO production by piglet cerebral microvessels (30). Furthermore, inhibition of HO reduces cerebral arteriolar dilation in response to glutamate and glutamatergic seizures (19,25,29). Therefore, glutamate increases activity of the HO-2 pathway, and the CO produced appears to cause subsequent dilation.In this study, we asked which vascular cells produce CO in response to glutamate and how is this production increased? Both endothelial cells and smooth muscle cells of the cerebral circulation express HO-2 (8, 21, 31) and have glutamate receptors (17,26,34). We (28) have shown that isolated cerebral vascular endothelial cells respond to glutamate by increasing CO production, but it was not known whether smooth muscle cells do so as well.Rapid elevation of CO production in response to glutamate obviates the possibility of increased expression of HO-2 or induction of HO-1. Assuming that total cellular HO-2 does not change during the experimental time course, CO production could be controlled by regulation of substrate availability to HO-2 or the effective catalytic activity of HO-2. Catalytic activity includes the specific catalytic efficiency of the enzyme, as well as intracellular localization, to optimize substrate and cofactor proximity.We used freshly isolated piglet cerebral microvessels and endothelial and smooth muscle cells in primary cu...

Glutamate-induced calcium signals stimulate CO production in piglet astrocytes

“…In the present study, we observed that glutamate causes Ca 2ϩ transients at 10 CO production has been measured in neurons (16), vascular smooth muscle cells (21,29), endothelial cells (21), and astrocytes (this study). In the cerebral vasculature, glutamate stimulates CO production in astrocytes (26) and endothelial cells (21). Glutamate is an excitatory neurotransmitter.…”

“…After 1 h incubation at 37°C, incubations were terminated by placing the samples in hot water (75°C) for 4 min. Immediately, 80 l of headspace gas were collected for CO detection by gas chromatography/mass spectrometry (GC/MS) using a Varian Saturn 3 GC/MS as described before (20,21). The amount of CO in samples was calculated from the ratio of peak areas of mass-to-charge ratio 28 and 31.…”

Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications

“…Nathanson et al (1995) found that glutamate augmented endogenous CO production, which was abolished after inhibition of HO-2 and PKC. HO-2 is also activated by glutamate in cerebral vascular endothelium (Parfenova et al, 2001) and smooth muscle cells (Leffler et al, 2003), leading to increased CO production. This effect of glutamate, independent of cytosolic calcium, was abolished by inhibiting protein tyrosine kinase but potentiated by inhibiting protein tyrosine phosphatases (Leffler et al, 2003).…”

Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression

“…Heme oxygenase (HO), which converts intracellular heme to carbon monoxide (CO) and biliverdin, is highly expressed in the brain and in cerebral microvessels as a constitutive isoform (HO-2) (21,31). The inducible HO isoform (HO-1) is not expressed in the cerebral microcirculation under basal conditions (21,31).…”