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.1998-1999 Mathematics in Biology
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