The effects of a GH secretagogue, L-692,585 (L-585), and human GH-releasing hormone (hGHRH) on calcium transient and GH release were investigated in isolated porcine pituitary cells using calcium imaging and the reverse hemolytic plaque assay (RHPA). Somatotropes were functionally identified by the application of hGHRH. All cells that responded to hGHRH responded to L-585 application. Perfusion application of 10 microM hGHRH and L-585 for 2 min resulted in an increase in intracellular calcium concentrations ([Ca(2+)](i)) of 53+/-1 nM (mean+/-S.E.M.) (P < 0.01) and 68+/-2 nM (P < 0.01) respectively. The L-585 response was characterized by an initial increase in [Ca(2+)](i) followed by a decline to a plateau level above the baseline. Concurrent calcium imaging with RHPA indicated that the L-585-evoked increase in [Ca(2+)](i) coincided with GH release. L-585 significantly increased the percentage of plaque-forming cells (24+/-3 vs 40+/-6%; P < 0.05) and mean area of plaques (1892+/-177 vs 3641+/-189 micro m(2); P < 0.01) indicating increased GH release. Substance P (SP) analogue ([d -Arg(1),d -Phe(5),d -Trp(7,11)]-SP) blocked, and the hGHRH receptor antagonist ((Phenylac-Tyr(1),d -Arg(2), p-chloro-Phe(6), Homoarg(9), Tyr (Me)(10), Abu(15), Nle(27),d -Arg(28), Homoarg(29))-GRF (1-29) amide) decreased the stimulatory effect of hGHRH. These failed to block the stimulatory effect of L-585, suggesting a different receptor for L-585 from the GHRH receptor. The hGHRH-induced calcium transients and initial peak increase induced by L-585 were significantly decreased by removal of calcium from the bathing medium or the addition of nifedipine, an L-calcium channel blocker. The plateau component of L-585-induced calcium change was abolished by removal of calcium and nifedipine. These results suggest an involvement of calcium channels in GH release. Either SQ-22536, an adenylate cyclase inhibitor, or U73122, a phospholipase C (PLC) inhibitor, blocked the stimulatory effects of hGHRH and L-585 on [Ca(2+)](i) transient, indicating the involvement of adenylate cyclase-cAMP and PLC-inositol triphosphate pathways. These results further suggested that calcium mobilization from internal stores during the first phase of the L-585 response induced an increase in [Ca(2+)](i) whereas calcium influx during the second phase is a consequence of somatotrope depolarization.
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A Glavaski-Joksimovic, K Jeftinija, A Jeremic, LL Anderson, and S Jeftinija
LL Anderson, DL Hard, LS Carpenter, EK Awotwi, MA Diekman, AH Trenkle, and J Cho
Progesterone secretion is crucial for maintaining pregnancy to parturition in mammalian species, and in cattle the corpus luteum is the primary source of this hormone. This study determined the roles of prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) in the luteotropic process in beef heifers hypophyseal stalk-transected (HST, n=7) or sham operated (sham operated controls, SOC, n=9) during midgestation. The main finding was that endogenous PRL and GH maintained progesterone secretion in HST heifers in a similar manner to that in SOC throughout pregnancy. Serum PRL averaged 37 vs 187 and GH 2 vs 4 ng/ml in HST heifers compared with SOC, whereas LH abruptly decreased to undetectable levels after HST compared with a modest 0.4 ng/ml in SOC heifers. The second finding was that parturition and lactation occurred in HST heifers with calf delivery induced to occur at the same time as SOC. Milk production in HST animals was severely limited, and postpartum estrus obliterated compared with SOC. The suckling stimulus sustained milk ejection in HST heifers in spite of diminished PRL, GH, thyroid stimulating hormone, thyroxine and tri-iodothyronine secretion. The results suggest that PRL, GH and possibly placental lactogen are luteotropic during pregnancy in cattle.