Extracellular ATP activates P2X purinoceptors, a class of receptors that form Ca2+-permeable channels. It has previously been observed that primary cultures of human renal proximal tubule cells release ATP in response to cell swelling, and that this ATP may then activate P2X receptors leading to a rise in intracellular Ca2+ (Wilson et al. 1999). A previous study has also reported that single proximal tubule cells isolated from frog undergo an extracellular Ca2+-dependent regulatory volume decrease (RVD) in response to hyposmotic challenge (Robson & Hunter, 1994). The aim of the following study was to investigate whether release of ATP and activation of P2X receptors may play a role in RVD in the renal proximal tubule.
Frogs were killed humanely by cervical dislocation and single proximal tubule cells isolated from the kidneys by enzyme digestion (Hunter, 1989). Cell length was measured using an optical technique (Robson & Hunter, 1994). The bath contained a high Na+, low K+ amphibian Ringer solution that contained 89 mM mannitol. This Ringer solution was made hypotonic by the removal of 40 mM mannitol. RVD was examined under the control circumstance and in the presence of (i) 3 units ml-1 ADP/ATP apyrase, (ii) 2 mM brilliant blue G (BBG) (a P2X7 antagonist) and (iii) 120 nM KN-62 (a P2X7 antagonist). Data are expressed as means ± S.E.M. Statistical analysis was performed using ANOVAs and significance was assumed at the 5 % level.
Under the control circumstance cell length was 21.23 ± 0.34 mm (n = 40). This increased by 0.8 ± 0.04 mm on exposure to a hypotonic shock and subsequently decreased by 0.68 ± 0.06 mm on RVD. At steady state the length was 0.13 ± 0.05 mm above the initial control level. In the presence of ATP/ADP apyrase, BBG or KN-62 RVD was inhibited. Steady-state lengths were 0.52 ± 0.12 (n = 7), 0.46 ± 0.08 (n = 9) and 0.36 ± 0.1 mm (n = 24) above the control level for apyrase, BBG and KN-62, respectively.
In summary, these data support the hypothesis that swelling of renal proximal tubule cells leads to the release of ATP and subsequent activation of purinoceptors. The inhibitory effect of BBG and KN-62, both of which are P2X7 antagonists, supports a role for P2X7 in the RVD response. Activation of these P2X receptors may provide the entry pathway for extracellular Ca2+ during RVD in the renal proximal tubule.
This work was supported by The Wellcome Trust.
All procedures accord with current UK legislation.