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Am J Physiol Regul Integr Comp Physiol 287: R833-R843, 2004. First published June 24, 2004; doi:10.1152/ajpregu.00199.2004
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

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

D. E. Pafundo,1 P. Mut,1 M. Pérez Recalde,1 R. M. González-Lebrero,1 V. Fachino,1 G. Krumschnabel,2 and P. J. Schwarzbaum1

1Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina; and 2Institut für Zoologie, Abteilung für Ökophysiologie, Universität Innsbruck, A-6020 Austria

Submitted 25 March 2004 ; accepted in final form 15 June 2004

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 (P2) to modulate this response. In addition, specific ectoenzymes hydrolyze ATP sequentially down to adenosine, which may bind to type 1 receptors (P1) 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 vRVD. In the presence of hypotonic medium (60% of isotonic), hepatocytes swelled 1.6 times followed by vRVD of 1.7 min–1 and RVD40 of 60.2%. ATP, UTP, UDP, or ATP{gamma}S (P2 agonists; 5 µM) increased vRVD 1.5–2 times, whereas no changes were observed in the values of RVD40. Addition of 100 µM suramin or cibacron blue (P2 antagonists) to the hypotonic medium produced no effect on vRVD but a 53–58% inhibition of RVD40. Incubation of hepatocytes in the presence of either 5 µM [{gamma}-32P]ATP or [{alpha}-32P]ATP induced the extracellular release of [{gamma}-32P]Pi (0.21 nmol·10–6 cells–1·min–1) and [{alpha}-32P]Pi (~8 x 10–3 nmol·10–6 cells–1·min–1), suggesting the presence of ectoenzymes capable of fully dephosphorylating ATP. Concerning the effect of P1 activation on RVD, 5 µM adenosine, both in the presence and absence of 100 µM S-(4-nitrobenzil)-6-tioinosine (a blocker of adenosine uptake), decreased RVD40 by 37–44%, whereas 8-phenyl theophylline, a P1 antagonist, increased RVD40 by 15%. Overall, results indicate that ATP, UTP, and UDP, acting via P2, are important factors promoting RVD of trout hepatocytes, whereas adenosine binding to P1 inhibits this process.

fish; ectonucleoside triphosphate diphosphohydrolases; extracellular adenosine 5'-triphosphate; cyprinids


Address for reprint requests and other correspondence: P. J. Schwarzbaum, Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina (E-mail: pablos{at}qb.ffyb.uba.ar)




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