RESUMEN
Acid extrusion responses to prostaglandin E2 were investigated in Chinese hamster ovary (CHO) cells heterologously expressing human EP1, EP2, and EP3I receptors (hEP1, hEP2 and hEP3I) by using a microphysiometer that detected small pH changes in the extracellular microenvironment. In the cells expressing hEP1, which is known to increase intracellular Ca2+, prostaglandin E2 (1 and 10 nM) slowly accelerated acid extrusion, but at higher concentrations an initial transient phase (approximately 5 times greater than the basal acidification) overlapped the slowly developing phase. In contrast, the cells expressing hEP2, which evokes cAMP production, showed dual responses to prostaglandin E2: an initial reduction followed by an acceleration of acid extrusion. In the cells expressing hEP3I, which is known to produce both a decrease in cAMP and a modest increase in intracellular Ca2+, acid extrusion was gradually accelerated by prostaglandin E2 and reached a plateau at around 2 min. Elimination of extracellular Ca2+ diminished the responses to prostaglandin E2 in hEP1 cells, but had little effect on the responses in hEP2 and hEP3I cells. Forskolin mimicked the dual effects of prostaglandin E2 observed in the hEP2 cells. Pretreatment with pertussis toxin inhibited the response to prostaglandin E2 in hEP3I cells, but the responses in hEP1 and hEP2 cells were not affected. Na+/H+ exchanger (NHE) inhibitors (EIPA and HOE642) suppressed all the responses induced by prostaglandin E2 in hEP1, hEP2, and hEP3I cells. These results suggest that EP receptor subtypes regulate acid extrusion mainly via NHE-1 through distinct signal transduction pathways in CHO cells.
Asunto(s)
Ovario/metabolismo , Receptores de Prostaglandina E/biosíntesis , Animales , Células CHO , Calcio/fisiología , Colforsina/farmacología , Cricetinae , Interpretación Estadística de Datos , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Toxina del Pertussis/farmacología , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP3 de Receptores de Prostaglandina ERESUMEN
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.