Calcium Waves and Dynamics Visualized by Confocal Microscopy in Xenopus Oocytes Expressing Cloned TRH Receptors

Abstract

Laser scanning confocal microscopy was used to analyse changes in free cytosolic calcium ([Ca²⁺]_i) in Xenopus laevis oocytes expressing the cloned rat TRH receptor in response to TRH. In oocytes expressing TRH receptors, TRH invariably evoked a dose-dependent, biphasic calcium response. This response consisted of an initial transient planar wave of calcium propagating just below the surface of the membrane followed by a slower, secondary calcium phase. The TRH antagonist, chlordiazepoxide, markedly inhibited this calcium wave. The origins of calcium involved in this biphasic response were investigated using a variety of intra- and extra-cellular calcium antagonists. The intracellular calcium antagonists thapsigargin and TMB-8 reduced the initial and to a lesser extent the secondary phase of the planar calcium wave. In contrast, EGTA and the calcium channel blocker nifedipine produced a profound inhibition of the secondary phase while the initial phase was only slightly reduced. These results indicate that the release of intracellular calcium is predominantly responsible for the initial phase of the calcium wave while the influx of extracellular calcium is mainly involved in the secondary phase. Qualitative changes in the patterns of calcium release induced by TRH were observed following pretreatment with intracellular calcium antagonists. Following pretreatment with these compounds, TRH induced spiral or regenerative calcium waves. Addition of EGTA to the extracellular medium did not alter these responses confirming the importance of intracellular calcium in the generation of these spiral calcium waves. This study demonstrates the nature and multiplicity of regulating mechanisms of [Ca²⁺]_i following activation of TRH receptors expressed in Xenopus oocytes.