C J Dixon

The hepatocyte calcium oscillator

Hepatocytes stimulated with calcium-mobilising agonists generate free Ca transients whose frequency is modulated by hormone concentration. Importantly, the time-course of individual free Ca transients is independent of agonist dose but does change with agonist species. A receptor-controlled model in which protein kinase C provides negative feedback directed against the different receptors, or receptor-specific G proteins, has been proposed in order to explain the agonist-specificity of the falling phase of the free Ca spikes. Here we show further evidence, from mixing of hormones and from the effects of elevated cAMP, of receptor-specific information within the spikes.

Modulation of free Ca oscillations in single hepatocytes by changes in extracellular K+, Na+ and Ca2+

Single rat hepatocytes, microinjected with the calcium-sensitive photoprotein aequorin, when stimulated with either phenylephrine or arg8-vasopressin exhibit agonist-specific oscillations in cytosolic free calcium levels (free Ca). In the majority of the cells examined adding excess potassium chloride, sodium chloride or choline chloride abolished transient behaviour. However, in cells that continued to oscillate the transient parameters were subtly modified by these treatments. In experiments using phenylephrine as the agonist, adding excess potassium chloride to the superfusate significantly reduced transient length, increased the rate of transient rise and reduced the smoothed peak free Ca level without significantly altering the intertransient resting free Ca level or the falling time constant. The possible mechanisms by which these alterations may occur are discussed.

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

1 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. 2 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. 3 The [Ca2+]i transients induced by 2‐meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. 4 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. 5 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. 6 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. 7 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.