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

Abstract

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_RVDof 1.7 min⁻¹and RVD₄₀of 60.2%. ATP, UTP, UDP, or ATPγS (P₂agonists; 5 μM) increased v_RVD1.5–2 times, whereas no changes were observed in the values of RVD₄₀. Addition of 100 μM suramin or cibacron blue (P₂antagonists) to the hypotonic medium produced no effect on v_RVDbut a 53–58% inhibition of RVD₄₀. Incubation of hepatocytes in the presence of either 5 μM [γ-³²P]ATP or [α-³²P]ATP induced the extracellular release of [γ-³²P]P_i(0.21 nmol·10⁻⁶cells⁻¹·min⁻¹) and [α-³²P]P_i(∼8 × 10⁻³nmol·10⁻⁶cells⁻¹·min⁻¹), suggesting the presence of ectoenzymes capable of fully dephosphorylating ATP. Concerning the effect of P₁activation 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 RVD₄₀by 37–44%, whereas 8-phenyl theophylline, a P₁antagonist, increased RVD₄₀by 15%. Overall, results indicate that ATP, UTP, and UDP, acting via P₂, are important factors promoting RVD of trout hepatocytes, whereas adenosine binding to P₁inhibits this process.