K S R Cuthbertson

Modelling receptor-controlled intracellular calcium oscillators

This paper presents mathematical models for the hepatocyte calcium oscillator which follow the concepts in a class of informal models developed to account for the striking dependence on the receptor type of several features of the calcium oscillations, in particular the shape and duration of the free calcium transients. The essence of these models is that the transients should be timed by a build-up of activated GTP-binding proteins, which, combined with positive feedback processes and perhaps with cooperative effects, leads to a sudden activation of phospholipase C (PLC), followed by negative feedback processes which switch off the calcium rise and lead to a fall in free calcium back to resting levels. These models predict pulsatile oscillations in inositol (1,4,5)P3 as well as in free calcium. We show that receptor-controlled intracellular calcium oscillators involving an unknown positive feedback pathway onto PLC and negative feedback from protein kinase C (PKC) onto G-proteins and receptors, or negative feedback by stimulation of GTPase activity can simulate many of the features of observed intracellular calcium oscillations. These oscillators exhibit a dependence of frequency on agonist concentration and a dependence of transient duration on receptor and G-protein type. We also show that a PLC-dependent GTPase activating factor (GAF) could provide explanations for some otherwise puzzling features of intracellular calcium oscillations.

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.

Thapsigargin induces cytoplasmic free Ca2+ oscillations in mouse oocytes

The mechanisms of calcium signalling in mammalian oocytes during maturation and fertilization are controversial. In this study we measured intracellular free Ca2+ concentrations ([Ca2+]i) with the photoprotein aequorin microinjected into immature mouse oocytes. Immature mouse oocytes typically produced [Ca2+]i responses to muscarinic acetylcholine (ACh) stimulation with two types of component. The first component consisted of a broad transient rise in [Ca2+]i lasting about 1 min. The second component consisted of pulsatile oscillations which could occur before, during or after the broad transient, but typically occurred on the rising phase of the broad transient, with a duration of about 5 s. Removal of external Ca2+ ([Ca2+]o) abolished the Ca2+ responses to ACh. Exposure of oocytes to the specific microsomal Ca(2+)-ATPase inhibitors thapsigargin (TG) and cyclopiazonic acid unexpectedly produced sustained oscillations in [Ca2+]i which were sensitive to the concentration of Ca2+ in the external milieu. The frequency of these oscillations was slow, and ceased, sometimes after several cycles, when Ca2+o was removed. Raised [Ca2+]o significantly increased the frequency in cells oscillating to TG and stimulated nonoscillating cells to begin oscillating. The majority of responsive oocytes which did not produce oscillations to ACh alone (70%), did so after TG treatment. Detailed data analysis indicated that these oscillations were identical to those generated by TG alone, with a similar sensitivity to changes in [Ca2+]o. Exposure of oocytes to ryanodine did not inhibit oscillatory behaviour. These results suggest that immature mouse oocytes possess a store which is insensitive to both TG and ryanodine and is capable of supporting [Ca2+]i oscillations.