The receptors and signal transduction mechanisms of gonadotropin- releasing hormone (GnRH) were studied in cultured pituitary gonadotrophs, in which gonadotropin secretion is stimulated by GnRH and other factors including oxytocin and endothelin. Endothelin (ET) was found to bind to high affinity receptors of the ET-A subtype in the pituitary gland, and to cause rapid increases in phosphoinositide hydrolysis, cytoplasmic calcium, and LH secretion. The action of ET on gonadotrophs was notable for its rapid desensitization, which is concomitant with internalization of the ET-receptor complex. The mechanism of the calcium signaling response in GnRH-stimulated gonadotrophs, which is manifested in single cells as prominant dose- dependent oscillatory to biphasic cytoplasmic calcium elevations, was further analyzed by patch clamping and fluorimetric assay of calcium responses in individual gonadotrophs. Most gonadotrophs exhibited spontaneous low-amplitude calcium fluctuations that were due to intermittent firing of nifedipine-sensitive action potentials. The activity of this membrane oscillator was determined by GnRH, which is initiated high-amplitude cytoplasmic calcium oscillations and concomitant changes in membrane potential (Vm). The onset of the agonist-induced calcium oscillations was associated with hyperpolarization of the plasma membrane, interrupted by regular waves of depolarization with firing action of potentials at the peak of each wave. The phases of hyperpolarization were synchronous with the agonist- induced cytoplasmic calcium transients, such that Vm maxima alternated with peak elevations of calcium. The Vm oscillations were found to result from repetitive activation of apamine-sensitive potassium channels by the elevation of cytoplasmic calcium. The synchronization between electrical activity and calcium spiking shows that the operation of the cytoplasmic oscillator can be integrated with that of a plasma- membrane oscillator to maintain the prolonged calcium signal during sustained agonist stimulation. In addition to its effects on pituitary hormone secretion, ET was found to act on cultured hypothalamic neurons and immortalized GT1 neuronal cells to stimulate inositol phosphate production. This response was mediated by ET-A receptors and was accompanied by stimulation of GnRH release in both normal and transformed neurons. Since ET is also produced by cultured hypothalamic cells, the locally formed peptide could participate in the hypothalamic control of gonadotropin secretion. Both cultured hypothalamic neurons and GT1 cells exhibited spontaneous pulsatile release of GnRH, comparable to that observed in vivo. This spontaneous activity was dependent on calcium entry through voltage-sensitive channels. Thus, pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks, and may underlie the activity of the pulse generator in vivo.
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