INTRACELLULAR EVENTS IN RESPONSE TO GNRH CAUSING GONADOTROPIN-RELEASEFROM PITUITARY-CELLS OF A CHANNID FISH, CHANNA-PUNCTATUS (BLOCH)

Addition of Channa punctatus GnRH (cGnRH, 2 mu g/well) to suspended pi tuitary cell (6 x 10(4) cells/well) with 2 mM Ca2+ caused about 14% in crease in cellular Ca2+ as compared to controls after 15 min. Ca2+ rem ained at the elevated level for a further 45 min. A concurrent decreas e of Ca2+ in the medium from 2 to 1.25 mM/well (400 mu l medium) was o bserved during the first 15 min of cGnRH incubation. Addition of verap amil (3 mu M/well) significantly inhibited cellular Ca2+ increase and medium Ca2+ decrease (p < 0.05). A sharp rise of gonadotropin (GtH) re lease from the pituitary cells in response to cGnRH plus Ca2+ occurred between 5 and 15 min. GtH remained steady for an additional 15 min an d was followed by a significant increase at 45 min (p < 0.01). cGnRH s timulated GtH synthesis transiently. Cycloheximide (20 mu g/well) bloc ked the increase of GtH release from 30 min onward but had no effect o n stimulated GtH release at 15 min in response to cGnRH. Addition of 0 .5 mM dibutyryl cAMP stimulated the release and synthesis of GtH from 45 min onward. cGnRH plus Ca2+ resulted in a peak of cellular cAMP at 15 min, which followed by a sharp decline after 30 min. Baseline level s were reached after 60 min. cGnRH plus Ca2+ lead to a significant inc rease in cellular calmodulin (CaM). Stimulation of phosphodiesterase ( PDE) activity could only be detected at 30 min, but was increased sign ificantly at 45 min. There is a clear correlation between cAMP level, Ca2+, CaM, and PDE activity suggesting that a rise in cellular Ca2+ re sults in an increase in CaM, which in turn stimulates the PDE activity and this affects degradation of cAMP. cGnRH plus Ca2+ stimulated cell ular calmodulin-dependent protein kinase II (CaM-kinase II) activity a s early as 5 min. Increases in protein kinase A (PKA) activity were on ly noticed in the later (60 min) part of incubation. These results ind icate two distinct phases of events in murrel pituitary cells in respo nse to cGnRH: 1) cGnRH causes a rapid influx of extracellular Ca2+ wit h a concurrent rise in cellular CaM-kinase II activity, resulting in a n acute GtH release from the stored GtH, and 2) a sustained release of GtH, involving cAMP and PKA. (C) 1997 Elsevier Science Inc.