Liliya Balabanova

Posttranslational regulation of cyclooxygenase by tyrosine phosphorylation in cerebral endothelial cells

Endothelium-derived cyclooxygenase (COX) products regulate cerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebral microvessels. We investigated the role of protein phosphorylation in the regulation of COX activity. The protein tyrosine phosphatase (PTP) inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acid rapidly stimulated COX activity, whereas the protein tyrosine kinase inhibitors, genistein and tyrphostins, inhibited it. Protein synthesis inhibitors did not reverse the stimulation of COX activity evoked by PTP inhibitors. Similar changes were observed in other vascular cells from newborn pigs that also express COX-1 and COX-2 (cerebral microvascular smooth muscle cells and aortic endothelial cells) but not in human umbilical vein endothelial cells or Swiss 3T3 fibroblasts that express COX-1 only. Tyrosine-phosphorylated proteins were immunodetected in endothelial cell lysates. COX-2 immunoprecipitated from 32P-loaded endothelial cells incorporated 32P that was increased by PTP inhibitors. COX-2, but not COX-1, was detected in endothelial fractions immunoprecipitated with anti-phosphotyrosine. These data indicate that tyrosine phosphorylation posttranslationally regulates COX activity in newborn pig vascular cells and that COX-2 is a substrate for phosphorylation.Endothelium-derived cyclooxygenase (COX) products regulate cerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebral microvessels. We investigated the role of protein phosphorylation in the regulation of COX activity. The protein tyrosine phosphatase (PTP) inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acid rapidly stimulated COX activity, whereas the protein tyrosine kinase inhibitors, genistein and tyrphostins, inhibited it. Protein synthesis inhibitors did not reverse the stimulation of COX activity evoked by PTP inhibitors. Similar changes were observed in other vascular cells from newborn pigs that also express COX-1 and COX-2 (cerebral microvascular smooth muscle cells and aortic endothelial cells) but not in human umbilical vein endothelial cells or Swiss 3T3 fibroblasts that express COX-1 only. Tyrosine-phosphorylated proteins were immunodetected in endothelial cell lysates. COX-2 immunoprecipitated from 32P-loaded endothelial cells incorporated 32P that was increased by PTP inhibitors. COX-2, but not COX-1, was detected in endothelial fractions immunoprecipitated with anti-phosphotyrosine. These data indicate that tyrosine phosphorylation posttranslationally regulates COX activity in newborn pig vascular cells and that COX-2 is a substrate for phosphorylation.

Regulation of CO production in cerebral microvessels of newborn pigs

Carbon monoxide (CO) is produced from heme by heme oxygenase-2 (HO-2) in cerebral blood vessels. Gas chromatography-mass spectrometry was used on piglet cerebral microvessels to address the hypothesis that CO production is regulated by heme delivery and HO-2 catalytic activity. CO production appears to be substrate limited because heme and its precursor aminolevulinate increase CO production. Ionomycin also increases CO production. However, CO production from exogenous heme was the same in Ca-replete medium, Ca-free medium with ionomycin, and Ca-replete medium with ionomycin. Phorbol myristate acetate increases CO production but does not change the catalytic activity of HO-2. Also, the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine has no effect on the HO-2 catalytic activity. Protein tyrosine kinase inhibition reduces HO-2 catalytic activity. Inhibition of protein tyrosine phosphatases increased HO-2 catalytic activity. Therefore, regulation of CO production by cerebral microvessels can include changing heme availability and HO-2 catalytic activity. HO-2 catalytic activity is stimulated by tyrosine phosphorylation.

cAMP production by piglet cerebral vascular smooth muscle cells: pHo, pHi, and permissive action of PGI2

In newborn pig pial arterioles and cocultures of cerebral microvascular endothelial and smooth muscle cells, hypercapnia increases cAMP. In the intact cerebral circulation, both the increase in cAMP and the accompanying vasodilation require the presence of PGI2. Using piglet cerebral microvascular smooth muscle in primary culture, we addressed the hypothesis that, in the presence of PGI2, hypercapnia-induced changes in extracellular pH cause increases in cAMP. The stable PGI2-receptor agonist iloprost did increase production of cAMP in response to combined extracellular pH and pHi (11 ± 6 vs. 32 ± 10% in the absence and presence of 10-10 M iloprost, respectively). However, there was no positive dose-response relationship between iloprost concentration and stimulation of cAMP production by acidosis (e.g., 58 ± 9 vs. 41 ± 5% in the presence of 10-12 and 10-9 M iloprost, respectively). Rapid decreases in pHi stimulate the cAMP production. Decreases in extracellular pH do not appear to contribute further. The G protein inhibitor pertussis toxin did not augment cAMP production in response to decreasing pHi. We conclude that PGI2 receptor activation permits another mechanism to enhance cAMP generation in response to intracellular, but not extracellular, acidosis and that the mechanism of the permissive effect of PGI2 does not involve inhibition of a pertussis toxin-sensitive G protein.

Mechanism of permissive prostacyclin action in cerebrovascular smooth muscle.

Prostacyclin permissively allows increased cAMP and cerebral vasodilation to hypercapnia in piglets. The prostacyclin receptor (IP) is coupled to phospholipase C (PLC) in piglet cerebral microvascular smooth muscle cells (SMC). We hypothesize that inhibition of PLC blocks the permissive action of IP receptor agonist, iloprost, and direct activation of PKC substitutes for the IP receptor agonist in SMC. SMC cAMP production was measured at normal pHi/pHo and with reduced pHi/pHo in the absence and presence of iloprost (100 pM). Half of the cells were pretreated with U73122, the PLC inhibitor, which decreased the basal IP3 and blocked the increase in IP3 caused by iloprost. Without iloprost, decreasing pHi/pHo increased cAMP production (40%). With iloprost, the cAMP response to acidosis increased to over 80%. U73122 prevented accentuation of the cAMP response by iloprost. Phorbol myristate acetate augmented the response to acidosis similarly to iloprost. These data suggest IP agonists augment the cAMP response to acidosis via coupling through PLC to activate PKC.