Spontaneous and Receptor-controled Pacemaker Activity and Growth Hormone Secretion in Pituitary Somatotrophs

Wednesday, February 17, 1999 - 2:00pm - 3:00pm
Keller 3-180
Stanko Stojilkovic (National Institutes of Health)
Joint work with M. Tomic, D. Zivadinovic, D. Yuan, and F. Van Goor.

Immortalized somatotrophs exhibit periods of spontaneous firing of action potentials accompanied with fluctuations in cytosolic calcium concentration ([Ca2+]i transients). Here we demonstrate that pituitary somatotrophs also exhibit an endogenous pacemaker activity associated with [Ca2+]i transients highly comparable to that observed in GH cell lines. The transitions from silent to active and from active to silent state and modulation of frequency of [Ca2+]i transients were frequently observed in the same cells. Spontaneous [Ca2+]i transients in single somatotrophs were inhibited by nifedipine, an L-type Ca2+ channel blocker, but not by thapsigargin, an inhibitor of endoplasmic reticulum (Ca2+)ATPase. Basal GH secretion in perifused pituitary cells was also inhibited by nifedipine. The majority of cells also expressed functional ETA receptors. Their activation by ET-1 lead to a triphasic change in electrical activity, Ca2+ signaling, and GH secretion. Initially, ET-1 induced an extracellular Ca2+-independent transient increase in [Ca2+]i, cessation of electrical activity in single somatotrophs, and a transient stimulation of GH secretion in perifused pituitary cells. This was followed by a prolonged (10-30 min) hyperpolarization of cells, leading to inhibition of pacemaker activity, and a significant decrease in GH secretion. The pacemaker activity was re-established in cells continuously exposed to ET-1, frequently with enhanced capacity for Ca2+ influx. The inhibitory effects of ET-1 on electrical activity, Ca2+ signaling, and GH secretion were mimicked by nifedipine, a blocker of L-type voltage-gated calcium channels, as well as by somatostatin (SRIF). The unique triphasic action of ET-1 was transformed into a biphasic mode by the treatment of cells with pertussis toxin overnight; the Ca2+-mobilizing phase was preserved, but was immediately followed by facilitated voltage-gated Ca2+ influx. This led to a biphasic pattern of Ca2+ signaling and GH secretion, the extracellular Ca2+-independent spike phase and the extracellular Ca2+-dependent and nifedipine sensitive plateau phase. As in SRIF-treated cells, ET-1 inhibited cAMP production and stimulated an inward rectifier potassium channel (Kir), both in a pertussis toxin-sensitive manner. In contrast to SRIF, ET-1 did not inhibit L-type channels, as documented by the ability of Bay K 8644 and high potassium to reactivate the pacemaker in the presence of agonist. Inhibitory effects of ET-1 on pacemaker activity and GH secretion were also observed in 8-bromo cAMP-treated cells, suggesting that cAMP-protein kinase A pathway is not responsible for inhibition of endogenous electrical activity and associated Ca2+ transients. These results indicate that somatotrophs express Ca2+-mobilizing ETA receptors, which are cross-coupled to Gi/Go pathway. Such cross-coupling accounts for the transient dissociation of early Ca2+ mobilization phase from the accompanied facilitation of voltage-gated Ca2+ influx phase, and is mediated by the activation of Kir channels in a cAMP-independent manner.