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Effects of extracellular nucleotides and their hydrolysis products on regulatory volume decrease of trout hepatocytes
D E Pafundo1, P Mut, M Pérez Recalde
1Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
Summary
Extracellular nucleotides like ATP promote trout hepatocyte volume regulation, while adenosine inhibits it. This study clarifies the roles of purinergic receptors in trout cell volume control.
Area of Science:
- Cell Biology
- Physiology
- Biochemistry
Background:
- Hypotonic swelling in trout hepatocytes triggers regulatory volume decrease (RVD).
- Extracellular nucleotides (ATP, UTP, UDP) and adenosine are implicated in modulating RVD via P2 and P1 receptors, respectively.
- Ectoenzymes contribute to extracellular nucleotide metabolism, influencing receptor activation.
Purpose of the Study:
- To investigate the specific roles of extracellular nucleoside 5'-tri- and diphosphates and adenosine in trout hepatocyte RVD.
- To elucidate the involvement of P2 and P1 purinergic receptors in regulating cell volume.
- To assess the impact of ectoenzymes on extracellular nucleotide signaling during RVD.
Main Methods:
- Trout hepatocytes were subjected to hypotonic swelling (60% isotonic).
- The effects of various purinergic agonists (ATP, UTP, UDP, ATPgammaS) and antagonists (suramin, cibacron blue) on RVD rate (v(RVD)) and extent (RVD(40)) were measured.
- Extracellular ATP hydrolysis was assessed using radiolabeled ATP ([gamma-(32)P]ATP, [alpha-(32)P]ATP).
- Adenosine's effects on RVD were studied with and without an adenosine uptake blocker (S-(4-nitrobenzil)-6-tioinosine) and with a P1 antagonist (8-phenyl theophylline).
Main Results:
- P2 agonists (ATP, UTP, UDP, ATPgammaS) significantly increased the initial rate of RVD (v(RVD)) but did not alter the final volume decrease (RVD(40)).
- P2 antagonists (suramin, cibacron blue) inhibited RVD(40) by 53-58% without affecting v(RVD).
- Extracellular release of inorganic phosphate ([P(i)]) from radiolabeled ATP confirmed ectoenzyme activity.
- Adenosine decreased RVD(40) by 37-44%, an effect not blocked by inhibiting adenosine uptake.
- The P1 antagonist 8-phenyl theophylline increased RVD(40) by 15%.
Conclusions:
- Extracellular ATP, UTP, and UDP act via P2 receptors to enhance the rate of RVD in trout hepatocytes.
- Adenosine, acting through P1 receptors, inhibits the extent of RVD in trout hepatocytes.
- Ectoenzymes contribute to the extracellular purinergic signaling environment influencing RVD.
- Purinergic signaling via both P1 and P2 receptors plays a crucial role in the volume regulatory response of trout hepatocytes.