Two-phase response of acid extrusion triggered by purinoceptor in Chinese hamster ovary cells.
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 alpha,beta-methyleneATP (alpha,beta-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 alpha(1)-adrenoceptor.
Okada
Yuichi
Y
Department of Pharmacology, School of Medicine, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan.
Taniguchi
Takanobu
T
Akagi
Yoshio
Y
Muramatsu
Ikunobu
I
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects; D014544:Uridine Triphosphate / Q000494:pharmacology*
D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D017923:Sodium-Hydrogen Exchangers; D014544:Uridine Triphosphate