Effects of extracellular nucleotides and their hydrolysis products on regulatory volume decrease of trout hepatocytes

In trout hepatocytes, hypotonic swelling is followed by a compensatory shrinkage called regulatory volume decrease (RVD). It has been postulated that extracellular ATP and other nucleotides may interact with type 2 receptors (P-2) to modulate this response. In addition, specific ectoenzymes hydrolyze ATP sequentially down to adenosine, which may bind to type 1 receptors (P-1) and also influence RVD. Accordingly, in this study, we assessed the role of extracellular nucleoside 5'-tri- and diphosphates and of adenosine on RVD of trout hepatocytes. The extent of RVD after 40 min of maximum swelling was denoted as RVD40, whereas the initial rate of RVD was called v(RVD). In the presence of hypotonic medium (60% of isotonic), hepatocytes swelled 1.6 times followed by v(RVD) of 1.7 min(-1) and RVD40 of 60.2%. ATP, UTP, UDP, or ATPgammaS (P-2 agonists; 5 muM) increased v(RVD) 1.5-2 times, whereas no changes were observed in the values of RVD40. Addition of 100 muM suramin or cibacron blue (P-2 antagonists) to the hypotonic medium produced no effect on v(RVD) but a 53-58% inhibition of RVD40. Incubation of hepatocytes in the presence of either 5 muM [gamma-P-32]ATP or [alpha-P-32]ATP induced the extracellular release of [gamma-P-32]P-i (0.21 nmol.10(-6) cells(-1). min(-1)) and [alpha-P-32]P-i (similar to8 x 10(-3) nmol.10(-6) cells(-1).min(-1)), suggesting the presence of ectoenzymes capable of fully dephosphorylating ATP. Concerning the effect of P-1 activation on RVD, 5 muM adenosine, both in the presence and absence of 100 muM S-(4-nitrobenzil)-6-tioinosine (a blocker of adenosine uptake), decreased RVD40 by 37-44%, whereas 8-phenyl theophylline, a P-1 antagonist, increased RVD40 by 15%. Overall, results indicate that ATP, UTP, and UDP, acting via P-2, are important factors promoting RVD of trout hepatocytes, whereas adenosine binding to P-1 inhibits this process.