FIG. 4.

Calcium signaling functions of TSHRs in pituitary corticotrophs and melanotrophs: single-cell recording. (A–C) Effects of TSH on calcium signaling in corticotrophs (A, B) and melanotrophs (C). Cells were bathed in calcium-containing medium, and arrows indicate the moment of application of hormones. TSH (20 nM) induced calcium transients in quiescent cells (A, B) or increased the amplitude and frequency of spontaneous calcium transients in spontaneously active cells (C). Corticotrophs were identified by stimulatory effects of CRH in physiological concentrations and inhibitory effects in pharmacological concentrations on calcium influx, as well as by arginine VP-induced calcium mobilization (A). TSH effect on calcium influx was not affected by 500 nM antalarmin, a CRH receptor antagonist, in contrast to CRH effects (B). Melanotrophs were identified by blockade of TSH-stimulated calcium transients by dopamine and the lack of TRH response on calcium signaling (C). (D) Stimulatory effect of CRH on calcium signaling was mimicked by application of forskolin, an adenylyl cyclase allosteric activator. (E–G) Calcium signaling function of TSHR in corticotrophs was dependent on calcium influx. Blue areas indicate duration of application of Ca²⁺-deficient medium (to exclude Ca²⁺ influx from extracellular medium), and horizontal bars indicate duration of TSH application in three concentrations. Arrows indicate the moment of CRH application, which was added to identify corticotrophs. Notice abolition of spontaneous calcium transients by removal of bath Ca²⁺. All experiments were performed 20 hours after cell dispersion. CRH, corticotropin-releasing hormone; VP, vasopressin. Color images are available online.