Coupling of GnRH and ET receptors with phospholipase C- and D-dependent intracellular signals was examined in pituitary cells. GnRH induced both initial and sustained IP3/DAG production, while ET-1 induced only a transient response, a profile that was comparable to the GnRH-induced DAG response in protein kinase C-depleted cells. GnRH- and ET-induced IP3/DAG and calcium responses were reduced by phospholipase C inhibitors, U73122 and neomycin. Those two phospholipase inhibitors also reduced agonist-induced LH release by cultured pituitary cells. In contrast to phospholipase C, phospholipase D activity was stimulated by agonist activation of GnRH but not ET receptors. Furthermore, GnRH- but not ET- induced DAG formation was reduced in the presence of the two phospholipase D inhibitors, ethanol and propanolol, and the fall in DAG production was accompanied by the inhibition of GnRH-induced c-fos expression. We have also examined the interactions between these receptor-mediated intracellular signals and plasma membrane electrical events. The results showed that pituitary cells are excitable and that each calcium spike is produced by the calcium entry during a single action potential (AP). The frequency of APs and calcium oscillations in gonadotrophs was modulated by depolarization/hyperpolarization pulses. Using these and other data on plasma membrane channel characterization in gonadotrophs provided earlier, a quantitative mathematical model predicting spatio-temporal calcium oscillations was developed. The model contains a set of Hodgkin-Huxley-like equations and the equation for calcium diffusion towards the cell center. It suggests that AP-induced calcium spiking is prominent only in a thin shell layer neighboring the cell surface. We also found that in GnRH-stimulated cells a brief depolarization pulse during the sustained phase can elicit a transient calcium rise similar to the endogenous cycle. In addition, calcium entry during a single depolarizing pulse was found to shift the phase of subsequent endogenous calcium oscillations, without affecting their frequency. The application of two consecutive depolarizing pulses showed that the size of the calcium rise evoked by the second pulse depended on the time lapsed between two consecutive pulses, indicating that each endogenous or evoked calcium rise cycle leaves the calcium release mechanism of the gonadotroph in a refractory state. Electrical activity may play an important role in the modulation of phospholipase C-induced calcium oscillations, including resetting the clock of the oscillator.
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