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 RVD(40), 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 RVD(40) of 60.2%. ATP, UTP, UDP, or ATPgammaS (P(2) agonists; 5 microM) increased v(RVD) 1.5-2 times, whereas no changes were observed in the values of RVD(40). Addition of 100 microM suramin or cibacron blue (P(2) antagonists) to the hypotonic medium produced no effect on v(RVD) but a 53-58% inhibition of RVD(40). Incubation of hepatocytes in the presence of either 5 microM [gamma-(32)P]ATP or [alpha-(32)P]ATP induced the extracellular release of [gamma-(32)P]P(i) (0.21 nmol.10(-6) cells(-1).min(-1)) and [alpha-(32)P]P(i) ( approximately 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 P(1) activation on RVD, 5 microM adenosine, both in the presence and absence of 100 microM S-(4-nitrobenzil)-6-tioinosine (a blocker of adenosine uptake), decreased RVD(40) by 37-44%, whereas 8-phenyl theophylline, a P(1) antagonist, increased RVD(40) 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.
Pafundo
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Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
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Pérez Recalde
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González-Lebrero
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Fachino
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Krumschnabel
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Schwarzbaum
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D000241:Adenosine / Q000096:biosynthesis; D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D000465:Algorithms; D000818:Animals; D048429:Cell Size / Q000187:drug effects; D005110:Extracellular Space / Q000502:physiology*; D006054:Goldfish / Q000502:physiology; D022781:Hepatocytes / Q000187:drug effects*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology*; D017686:Oncorhynchus mykiss / Q000502:physiology*; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018047:Receptors, Purinergic P1 / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000502:physiology; D013806:Theophylline / Q000031:analogs & derivatives* / Q000494:pharmacology
D009711:Nucleotides; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D013806:Theophylline; C028322:8-phenyltheophylline; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D000241:Adenosine