CHRONIC HYPOXIA REDUCES ADENOSINE A(2A) RECEPTOR-MEDIATED INHIBITION OF CALCIUM CURRENT IN RAT PC12 CELLS VIA DOWN-REGULATION OF PROTEIN-KINASE-A

1. Adenosine has been shown to decrease Ca2+ current (I-Ca) and attenu ate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+] (i)) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These eff ects are mediated via the adenosine A(A2) receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+](i) duri ng chronic hypoxia. 2. Whole cell patch-clamp studies revealed that th e effect of adenosine on I-Ca was significantly reduced when PC12 cell s were exposed to hypoxia (10 % O-2) for 24 and 48 h. 3. Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2 +](i) was significantly enhanced when PC12 cells were exposed to 10% O -2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+](i) was significantly blunted in PC 12 cells exposed to hypoxia for 48 h. 4. Northern blot analysis reveal ed that mRNA for the A(2A) receptor, which is the only adenosine recep tor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [H-3]CGS21680, a selective A(2A) receptor ligand, showed that the number of adenosine A(2A) recep tor binding sites was similarly increased during exposure to 10% O-2 f or 48 h.5. PKA enzyme activity was significantly inhibited when PC12 c ells were exposed to 10% O-2 for 24 and 48 h. However, we found that h ypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity Chronic hypoxia also did not alter t he immunoreactivity level of the G protein G(s alpha), an effector of the A(2) signalling pathway. 6. Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on I-Ca was sig nificantly attenuated during 48 h exposure to 10% O-2. 7. We conclude therefore that the reduced effect of adenosine on I-Ca and [Ca2+](i) i n PC12 cells exposed to chronic hypoxia is due to hypoxia-induced down regulation of PKA. This mechanism may serve to reduce the negative fee dback on I-Ca and [Ca2+](i) by adenosine and therefore maintain enhanc ed membrane excitability of PC12 cells during long-term hypoxia.