Gonadotropin releasing hormone stimulation of cyclic 3',5'-AMP in the pituitary cell of a teleost (Channa punctatus, Bloch) requires extracellular calcium: its relationship to gonadotropin release.
Pituitary cells were prepared by enzymatic dispersion and incubated in vitro. To observe the effect of gonadotropin releasing hormone (GnRH) and Ca2+ on the murrel pituitary cyclic 3',5'-AMP (cAMP), cells were dispersed by 0.3% collagenase plus 0.05% trypsin in Earle's minimum essential medium without Ca2+ and a considerably high yield of viable cells were obtained. Addition of a murrel, Channa punctatus, GnRH (cGnRH, 10 micrograms/incubation) to pituitary cell incubation (6 x 10(4) cells/well) containing 4 mM theophylline, a phosphodiesterase (PDE) inhibitor, stimulated cAMP accumulation in the pituitary cell 2.4-fold and its release into the medium about 2-fold as compared to control. The extent of stimulation was greatly increased on addition of Ca2+ (2 mM/incubation) with cGnRH: accumulation 5.8-fold and release 3.7-fold, respectively, in comparison to control. A time-course study with cGnRH (20 micrograms/incubation) plus Ca2+ (2 mM/incubation) on pituitary cell cAMP accumulation showed that the peak of cAMP level was reached at 15 min and remained at the same level until 60 min in the presence of theophylline; this peak was drastically reduced (5-fold) at 30 min in the absence of theophylline, indicating rapid hydrolysis of cAMP by PDE. Ca(2+)-augmented cGnRH stimulatory effect on cAMP accumulation and release could be significantly (P < 0.01) inhibited by verapamil (3 microM/incubation), a specific calcium channel blocker, suggesting requirement of extracellular Ca2+ influx in this process. Calmodulin (CaM), a Ca2+ carrier protein, addition to cGnRH and Ca2+ incubation further augmented the increase of cellular accumulation of cAMP and its release by 39.5 and 45%, respectively, in comparison to cGnRH and Ca2+ (both were statistically significant, P < 0.01). CaM effect could be blocked by calmidazolium (1 microM/incubation), a specific inhibitor of CaM, indicating specificity of the stimulatory action of CaM. Addition of radioiodinated 125I-CaM, in the presence of Ca2+ or cGnRH plus Ca2+ resulted in the binding of 125I-CaM to pituitary cells and to the pellet of the lysed cells. 125I-CaM specifically binds to pituitary cell plasma membrane preparation and saturation of 125I-CaM binding occurred at 9 ng of 125I-CaM. To investigate whether cGnRH plus Ca(2+)-stimulation of pituitary cells cAMP is linked to gonadotropin (GtH) release, similar protocols were followed. It was found that GtH release was augmented to 7-fold by cGnRH plus Ca2+, which was inhibited by verapamil and stimulated by CaM in a similar manner as observed in the case of cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)
Mukhopadhyay
B
B
Department of Zoology, Visva-Bharati University, West Bengal, India.
Biswas
R
R
Bhattacharya
S
S
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis*; D005399:Fishes / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology*; D006062:Gonadotropins / Q000378:metabolism*; D066298:In Vitro Techniques; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013268:Stimulation, Chemical; D013806:Theophylline / Q000494:pharmacology
D002147:Calmodulin; D006062:Gonadotropins; D005576:Colforsin; D007987:Gonadotropin-Releasing Hormone; D003994:Bucladesine; D013806:Theophylline; D000242:Cyclic AMP; D002118:Calcium