The time course and extracellular Ca2+ involvement of growth hormone (GH) releasing factor-induced GH secretion in perifused dispersed rat pituitary cells.

Abstract

The time course of GH secretion in response to hpGRF and its dependency on the extracellular Ca2 + concentration were studied in perifused dispersed anterior pituitary cells. The onset of GH secretion in response to 1 nM hpGRF was relatively rapid (within 5 s) but removal of hpGRF after 10-min application further increased the rate of secretion (off-response). The threshold and maximum concentrations of hpGRF in stimulatory secretion were 10 -12 and 10 -8 M respectively. Between these two concentrations, the responses showed dose dependency. A reduction in the extracellular Ca2 + concentration to 0.25 mM or to nominally zero reduced hpGRF-induced GH secretion to 64.4% or to 1.9%, respectively, of the control response in the presence of 2.5 mM Ca2 + . Two mM Co2 +, known as a strong calcium channel blocker, completely suppressed hpGRF-induced GH secretion. The removal of Ca2 + from the perifusion buffer immediately after the offset of 1 min-applied 1 nM hpGRF accelerated the falling phase of GH secretion, which is parallel to the decline in [Ca2+]o in the perifusion chamber. Under nominal Ca2+-free conditions, hpGRF produced no increase in GH secretion. However, 10 min after the offset of 1 min-applied hpGRF under Ca2 + -free conditions, the introduction of normal buffer containing 2.5 mM Ca2 + substantially restored GH secretion, although after 20 min the introduction of normal buffer produced only a slight increase in GH secretion. In perifusion experiment of 106 cells, intracellular cyclic AMP (CAMP) content was raised from the basal value of 4 to 26 pmol by 2-min application of 1 nM hpGRF. After cessation of hpGRF application, cAMP content decreased to 8.7 pmol at 11 min and returned to the basal value by 20 min. The same tendency was observed in Ca2 +-free buffer. In conclusion, the extracellular Ca2 + was essential for hpGRF-induced GH secretion. This indicates the importance of the influx of Ca2 + in response to hpGRF. The time course of hpGRFinduced rise and fall in CAMP content was roughly parallel to the GH secretion. The possible explanations of the off-response and the re-

Ghrelin‐Induced Growth Hormone Release From Isolated Rat Anterior Pituitary Cells Depends on Intracellullar and Extracellular Ca2+ Sources

“…This finding supports the findings of Kato et al . (28) and is compatible with previously reported findings concerning [Ca 2+ ] i evoked by GHRH treatment. This finding also shows that the perifusion system used in this study works effectively.…”

“…Based on the results of our previous study, we determined the concentrations of ghrelin (10 )8 M) and GHRH (10 )8 M) used in this study. Modified Ca 2+ concentration in perifusion medium B was supplemented with EGTA, as previously reported (28). Ca 2+ -free solution and/or thapsigargin (10 )6 M, WAKO, Osaka, Japan) and nifedipine (10 )5 M, Sigma, St Louis, MO, USA) were preincubated for 45 min and 30 min, respectively, before GHRH or ghrelin stimulation.…”

Flow cytometric analysis of functional anterior pituitary cells from female rats

“…The failure of St John et al (1986) to demonstrate repro¬ ducible changes in labelling when stimulatory and inhibitory factors were added may have been due to the addition of the factors 1 h before primary anti¬ body labelling, i.e. the effect on secretion is short lived, which is born out by superfusion experiments using pulsed stimulatory and inhibitory factors (Kato & Suzuki, 1986;Watanabe & Orth, 1987) and there¬ fore was no longer evident at the time of labelling. Recent work by Lewis, Goodman, St John & Barker (1988) confirms the findings of St John et al (1986) in that cell-surface prolactin labelling was only present on 40-50% of prolactin cells (as judged by intra¬ cellular staining) and they therefore postulated an autoreceptor for prolactin on some lactotrophs.…”

Pituitary growth hormone network responses are sexually dimorphic and regulated by gonadal steroids in adulthood

“…GHRH-induced GH secretion depends on calcium entry (15,26,27), which in endocrine cells occurs during action potential firing (12,23,28). Trains of calcium spikes, as induced by GHRH in both male and female pituitary slices, are considered one of the most effective patterns of episodic calcium entry in triggering exocytosis in various neuroendocrine/endocrine tissues (29,30), including pituitary cells recorded in situ (31).…”