Two-phase response of acid extrusion triggered by purinoceptor in Chinese hamster ovary cells

Abstract

The functional characteristics of purinoceptors in Chinese hamster ovary (CHO) cells were investigated using a microphysiometer which detects small metabolic changes to living cells in real-time as variations of pH in the extracellular microenvironment. Uridine 5′-triphosphate (UTP) increased the extracellular acidification rate biphasically, namely a transient and a steady response were observed. The transient phase reached a peak (four- to fivefold the basal extracellular acidification rate in amplitude) within 20 s and was followed by the steady phase which was sustained for more than 1 min at an amplitude less than twofold the basal extracellular acidification rate. Both phases showed a concentration-dependent increase in response to UTP. However, there was a significant difference in the pEC 50 value for UTP between the transient (4.8) and steady phases (6.1). Like UTP, ATP increased the extracellular acidification rate, but α,β-methyleneATP (α,β-MeATP), 2-methylthioATP (2-MeSATP), ADP, UDP and adenosine did not. This result suggests that the acid is extruded through a P2Y 2 or P2Y 2-like purinoceptor. 5-( N-ethyl- N-isopropyl) amiloride (EIPA) and 4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate (HOE642) suppressed both phases of the UTP-stimulated extracellular acidification rate response with high affinity (pIC 50: approximately 7.0). This result suggests that the Na +/H + exchanger 1 (NHE-1) predominantly mediates the UTP-induced acid extrusion response in CHO cells. Elimination of extracellular Ca 2+ or treatment with thapsigargin diminished both phases of the UTP-stimulated extracellular acidification rate. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7) also abrogated the two phases. These results are consistent with the involvement of NHE-1 which is activated via Ca 2+/calmodulin. Persistent exposure to UTP reduced both extracellular acidification rate phases, causing desensitization of the P2Y purinoceptor. This desensitization did not affect the acid extrusion response mediated by the α 1-adrenoceptor.