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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

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

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 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 ATPS (P₂ agonists; 5 µM) increased v_RVD 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 v_RVD but a 53–58% inhibition of RVD₄₀. Incubation of hepatocytes in the presence of either 5 µM [-³²P]ATP or [-³²P]ATP induced the extracellular release of [-³²P]P_i (0.21 nmol·10^–6 cells^–1·min^–1) and [-³²P]P_i (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

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