CALCIUM WAVES AND DYNAMICS VISUALIZED BY CONFOCAL MICROSCOPY IN XENOPUS-OOCYTES EXPRESSING CLONED TRH RECEPTORS

Laser scanning confocal microscopy was used to analyse changes in free cytosolic calcium (Ca2+(i)) in Xenopus laevis oocytes expressing th e cloned rat TRH receptor in response to TRH. In oocytes expressing TR H 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, chlordiazep oxide, markedly inhibited this calcium wave. The origins of calcium in volved in this biphasic response were investigated using a variety of intra- and extra-cellular calcium antagonists. The intracellular calci um antagonists thapsigargin and TMB-8 reduced the initial and to a les ser 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 sli ghtly reduced. These results indicate that the release of intracellula r calcium is predominantly responsible for the initial phase of the ca lcium wave while the influx of extracellular calcium is mainly involve d in the secondary phase. Qualitative changes in the patterns of calci um release induced by TRH were observed following pretreatment with in tracellular calcium antagonists. Following pretreatment with these com pounds, TRH induced spiral or regenerative calcium waves. Addition of EGTA to the extracellular medium did not alter these responses confirm ing the importance of intracellular calcium in the generation of these spiral calcium waves. This study demonstrates the nature and multipli city of regulating mechanisms of Ca2+(i) following activation of TRH receptors expressed in Xenopus oocytes.