The Effect of Various Corticotropin-Releasing Factor Trains on the Release of Adrenocorticotropin,β-Endorphin, andβ-Lipotropin from Perifused Ovine Pituitary Cells*

Abstract

The dynamics of the release of proopiomelanocortin-derived hormones from ovine anterior pituitary cells in response to varying pulse characteristics of ovine corticotropin-releasing factor (CRF) were investigated with an in vitro automated 15-column simultaneous perifusion system. Columns of cells were stimulated continuously or with trains of CRF pulses of varying pulse length (2-16 min), pulse period (20-160 min), and concentration, for 500 min. Ovine ACTH, beta-lipotropin, and beta-endorphin immunoreactivity were measured by unextracted RIA. Each pulse of CRF stimulated clearly defined and highly correlated (r greater than 0.9) pulses of the three pituitary hormones, suggesting similar mechanisms controlling release. In dose-response experiments, the minimum concentration of CRF in a 10-min pulse required to significantly raise the output of ACTH was 200 pM, and initial responses had not attained maximal levels with concentrations of CRF increased to 2 microM. Responses to pulsed CRF stimulation decreased with time with all stimulation patterns selected, although previously unstimulated control columns retained the initial capacity to respond. Multiple linear regression analysis showed that hormone output per pulse of CRF (43 nM) increased with increasing pulse period and pulse length. Output of pituitary hormones per unit of CRF applied decreased with pulse length but increased with pulse period. In summary, the responses of proopiomelanocortin derivatives were shown to be sensitive to abrupt increases in CRF, to reduce output under continued stimulation, and to have an inherent time lag before responding maximally to subsequent pulsed stimulation.

Mechanisms of desensitization of the adrenocorticotropin response to arginine vasopressin in ovine anterior pituitary cells

“…Dispersed ovine anterior pituitary cells were prepared as previously described (Evans et al 1985, Le Beau & Mason 1994. Briefly, whole pituitary glands were removed from sheep shortly after slaughter and placed into cold, sterile HEPES buffer (25 mM HEPES, 137 mM NaCl, 5 mM KCl, 10 mM glucose and 0·002% phenol red, pH 7·3) containing antibiotic and antimycotic agents (100 U penicillin/ml, 100 mg streptomycin/ml and 0·25 mg amphotericin B/ml).…”

“…The multicolumn perifusion system, as described previously and with modifications (Evans et al 1985(Evans et al , 1996, allows for the simultaneous perifusion of up to 15 cell chambers or columns. Through the use of a solenoid switching system, various trains of peptide hormones and pharmacologic agents can be applied to the cells.…”

“…ACTH radioimmunoassay was performed as previously described (Evans et al 1985). The antiserum used (rabbit antiporcine ACTH) was a gift from Dr Richard Donald, Christchurch Hospital (Christchurch, New Zealand), and the highly purified ovine ACTH, used for 125 Iradioiodination and assay standards, was a gift from Dr C H Li, Hormone Research Laboratory, University of California (San Francisco, CA, USA).…”

Atrial natriuretic factor in hypertension: bioactivity at normal plasma levels.

“…Samples for plasma renin activity (PRA), 11 aldosterone, 12 cortisol (enzyme-linked radiosorbant assay), catecholamines, 13 hematocrit, and serial standard automated multianalyzer plasma biochemistry profiles were taken hourly at -60, 0, +60, +120, + 180, +240, and then +270 minutes. Plasma samples for arginine vasopressin, 14 and adrenocorticotropic hormone (ACTH) 15 were taken at 0, +180 and +270 minutes. Plasma volume was measured by the Evans Blue method 16 within the 30 minutes before completion of active and placebo infusions (n=5).…”

Desensitization of the adrenocorticotrophin responses to arginine vasopressin and corticotrophin-releasing hormone in ovine anterior pituitary cells

“…The multi-column perifusion system, as described previously and with modifications (McIntosh & McIntosh 1983, Evans et al 1985, 1988, 1996, allows for the simultaneous perifusion of up to 15 cell chambers, or columns. Through the use of a solenoid switching system various trains of peptide hormones and pharmacological agents can be applied to the cells.…”