Stimulatory effect of endogenous and exogenous growth hormone secretagogues on isolated porcine somatotropes.

摘要

This dissertation is an effort to further define sites and cellular mechanisms of action of exogenous and endogenous GH secretagogues (GHS) in porcine somatotropes.; The first study looked at the ability of benzolactam GHS - L-692,585 (L-585) to stimulate Ca2+ transients and GH secretion from cultured porcine somatotropes. Perfusion application of L-585 dose dependently increased intracellular calcium ([Ca2+]_i) in somatotropes. Using reverse hemolytic plaque assay (RHPA) it was shown that incubation of cultured cells with L-585 had a stimulatory effect of L-585 on GH secretion. With concurrent [Ca2+]_i measurements and RHPA and, it was shown that the increase in [Ca2+]_i evoked by L-585 coincides with GH secretion.; The second study using atomic force microscopy demonstrated for the first time the presence of the fusion pores at the plasma membrane of isolated porcine somatotropes. In the resting somatotropes were found ‘pits’ containing several ‘depressions’, or fusion pores, with diameter of 100–200 nm. After stimulation of secretion with L-585 ‘depressions’ enlargement for about 40% was observed. That ‘depressions’ are structures at plasma membrane of somatotropes involved in hormone secretion was confirmed by using gold-tagged GH-antibody. It is proposed that fusion pores may be common structures for all secretory cells, and that they are the sites where following stimulation membrane-bound secretory vesicles transiently dock and fuse to release vesicular content.; The third study investigated the effects of an endogenous GHS - ghrelin on [Ca2+]_i in cultured porcine somatotropes. The application of ghrelin resulted with changes in [Ca2+] _i similar to those induced by L-585. Our experiments have shown that rapid increase in [Ca2+]_i induced both by L-585 and ghrelin originated from the intracellular stores, while the plateau phase resulted from somatotropes depolarization and extracellular calcium entry. Simultaneous application or application in succession of L-585 and ghrelin has revealed antagonistic effects between these two agonists that may result from competition at the receptor level and/or downstream from GHS-R.; Collectively, the results from these studies indicate that stimulatory effect of ghrelin and synthetic GHS on [Ca2+]_i in cultured porcine somatotropes closely parallel each other, in a manner that is consistent with an increase of GH secretion.