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

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• 作者：Okada ; Yuichi ; Taniguchi ; Takanobu ; Akagi ; Yoshio ; Muramatsu ; Ikunobu
• 关键词：Acid extrusion ; Microphysiometer ; P2Y purinoceptor ; Na⁺/H⁺ exchanger
• 刊名：European Journal of Pharmacology
• 出版年：2002
• 出版时间：November 22, 2002
• 年：2002
• 卷：455
• 期：1
• 页码：19-25
• 全文大小：170 K

文摘

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 pEC₅₀ 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 P2Y₂ or P2Y₂-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 (pIC₅₀: 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 Ca²⁺ 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 Ca²⁺/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 α₁-adrenoceptor.