Actions of ADP, but not ATP, on cytosolic free Ca²⁺ in single rat hepatocytes mimicked by 2‐methylthioATP

Abstract

Aequorin‐injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin‐injected hepatocytes to ADP and ATP. The effects of the phosphorothioate analogue of ATP, 2‐methylthioATP (2‐meSATP), have been examined on single rat hepatocytes. This analogue is believed to be the most potent agonist at the P2Y1 subclass of purinoceptor. The [Ca2+]i transients induced by 2‐meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. At high concentrations, 2‐meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2‐meSATP‐ and ADP‐induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP‐induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i. ATP‐induced transients were specifically potentiated by the co‐addition of α,β‐methyleneATP, whereas 2‐meSATP‐ and ADP‐induced transients were unaffected by this treatment. We conclude that 2‐meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from the receptor(s) mediating the effects of ATP.

Establishing the stochastic nature of intracellular calcium oscillations from experimental data

“…Single hepatocytes were isolated from fed, male Wistar-strain rats (150-250g) by collagenase perfusion as described previously [45]. Briefly, the hepatic portal vein was cannulated and an initial calcium-free perfusion was followed by perfusion with collagenase (0.04% w/v) and Ca 2+ (3.8mM) for 15 minutes.…”

Regulation of Rat Hepatocyte Function by P2Y Receptors: Focus on Control of Glycogen Phosphorylase and Cyclic AMP by 2-Methylthioadenosine 5′-Diphosphate

“…Hepatocytes were isolated from fed, male Wistar-strain rats (150 -250 g) by collagenase perfusion as described previously (Dixon et al, 1995). Briefly, the rat was killed by cervical dislocation in accordance with institutional guidelines, and the hepatic portal vein was cannulated.…”

“…In common with many other cell types, rat hepatocytes are known to express multiple P2Y subtypes, although these have not been conclusively identified (Dixon et al, 1990(Dixon et al, , 1995(Dixon et al, , 2003Keppens 1993). Reverse transcriptasepolymerase chain reaction (RT-PCR) has demonstrated the presence in primary rat hepatocytes of mRNA transcripts encoding all four of the cloned rat P2Y subtypes investigated (P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 ; Dixon et al, 2000).…”

“…Extracellular nucleotides play a well established role in the regulation of this key function in rat hepatocytes through the activation of P2Y receptors (Okajima et al, 1987;Keppens et al, 1992Keppens et al, , 1993Keppens, 1993), a family of G-protein-coupled receptors responding to the native nucleotides ADP, ATP, UDP, UTP, and UDP-glucose (Boarder and Hourani, 1998;Abbracchio et al, 2003). Curiously, stimulation of P2Y receptors on rat hepatocytes leads to both increases in [Ca 2ϩ ] c and inhibition of adenylyl cyclase; these responses will stimulate and limit, respectively, activation of glycogen phosphorylase (Okajima et al, 1987;Dixon et al, 1990Dixon et al, , 1995Dixon et al, , 2000Keppens et al, 1992;1993;Keppens, 1993;Edgecombe et al, 1999). The mechanism underlying the regulation of glycogen phosphorylase by cyclic AMP is well established, with increased cyclic AMP levels leading to phosphorylation and activation of the enzyme, and with dephosphorylation by protein phosphatase 1 as cyclic AMP levels fall.…”

Regulation of Human Hepatocytes by P2Y Receptors: Control of Glycogen Phosphorylase, Ca2+, and Mitogen-Activated Protein Kinases

“…Rat hepatocytes were isolated from fed, male Wistar strain rats (150 -250 g) by collagenase perfusion as described previously (Dixon et al, 1995). The cells were harvested and cultured as described above for human cells.…”