Hypoxia and cobalt stimulate lactate dehydrogenase (LDH) activity in vascular smooth muscle cells

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Abstract
O2 plays a dominant role in the metabolism and viability of cells; changes in O2 supply lead to many physiological responses in the cell. Recent reports have shown that hypoxia induces the transcription of a number of genes, among them those for the glycolytic enzymes. We have investigated signalling events that may lead to enhanced activity of lactate dehydrogenase (LDH) in cultured vascular smooth muscle (VSM) cells derived from rat aorta, grown under hypoxic conditions (1% versus 20% O2). LDH was chosen because this enzyme exhibits one of the largest increases in activity among the glycolytic enzymes after hypoxic stimulation of cells. Hypoxic exposure of VSM cells for 24 h resulted in a 2-fold increase in LDH activity and in a 2.5-fold increase in intracellular cAMP levels. Agents that activate adenylate cyclase, such as forskolin, cholera toxin and 1-methyl-3-isobutylxanthine (IBMX), and thus increase cAMP production, significantly induced LDH activity. Moreover, induction of LDH activity by hypoxia was prevented in the presence of the protein kinase A inhibitor N-[2-(methyl-amino)ethyl]-5-isoquinolinsulphonamide dihydrochloride (H-8), and the cyclooxygenase inhibitor indomethacin. In contrast to the cAMP-stimulating agents, stable cGMP analogues (dibutyryl-cGMP, 8 bromo-cGMP), activators of protein kinase C [12-O tetradecanoylphorbol-13-acetate (TPA), and 1-oleoyl-2 acetyl-glycerol (OAG), and the calcium ionophore ionomycin did not alter LDH activity in VSM cells kept at 20% O2. A dose-dependent increase in LDH activity was also observed in normoxic cells exposed to cobalt chloride (50–200 μM), indicating that a metal binding protein might be involved in this signalling cascade. This transition metal does not seem to act by interfering with cellular oxidative phosphorylation, because 10−5–10−4M cyanide, a potent inhibitor of cell respiration, had no effect on LDH activity, as has been also shown for the production of erythropoietin (EPO). Thus, we suggest that the phosphorylation potential is not crucial to the O2-sensing mechanism regulating LDH activity and EPO production. Our results suggest that the “metabolic indicator” leading to an enhanced LDH activity under hypoxic conditions in VSM cells is represented by cAMP.