Extracellular ATP activates P2X purinoceptors, a class of receptors that form Ca2+ permeable channels. A previous study has demonstrated that inhibitors of one of these, P2X7, block volume regulation in frog isolated proximal tubule cells (Davies & Robson, 2002). The aim of the following study was to identify whether whole cell ATP-activated currents observed in the frog proximal cells were mediated by P2X7.
Frogs were killed humanely by cervical dislocation and single proximal tubule cells were isolated from frog kidneys by enzyme digestion (Hunter, 1989). Standard patch clamp techniques were used to gain whole cell patches via the basolateral membrane. The pipette solution contained (mM): 100 NaCl, 2 MgCl2, 0.5 EGTA and 10 Hepes (NaOH). The bath solution contained (mM): 100 NaCl, 0.5 MgCl2, 0.5 CaCl2 and 100 Hepes (NaOH). Whole cell potential was held at -100 mV and was then ramped between -100 and +20 mV. To examine agonist potency, patches were exposed to either 500 µM ATP and 500 µM 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) or 500 µM ATP and 500 µM 2-methylthio-ATP (2MeSATP). Under both circumstances the order of exposure to the agonists was altered with each patch. Data are expressed as means ± S.E.M. Statistical analysis was performed using Student’s paired t test and significance was assumed at the 5 % level.
Addition of 500 µM ATP to the extracellular surface of patches increased outward conductance (Gout) and inward conductance (Gin) by 9.55 ± 1.61 and 5.45 ± 1.00 µS cm-2, respectively (n = 20). In paired patches, ATP increased Gout, while BzATP was without effect. The ATP-activated Gout was 7.11 ± 1.97 µS cm-2 (n = 12), while BzATP did not give a significant shift in Gout(0.14 ± 0.93 µS cm-2). In contrast, both ATP and 2-MeSATP increased Gout by 11.1 ± 1.35 µS cm-2 and 3.64 ± 0.89 µS cm-2, respectively (n = 25). The ATP-activated conductance was significantly greater than the 2-MeSATP activated conductance.
These data suggest that single proximal tubule cells isolated from frog kidney contain an ATP-activated conductance. The relative potencies of the P2X agonists ATP and BzATP suggest that the conductance is not due to P2X7. The relative potencies of ATP and 2-MeSATP suggest that the activated conductance may be mediated by either P2X4, P2X5 or P2X6. Further studies are needed to distinguish between these receptors.
This work was supported by the Wellcome Trust.