DOMESTIC ANIMAL ENDOCRINOLOGY
VoI. 7(1):35-42, 1990
INFLUENCE OF CATECHOLAMINES, PROSTAGLANDINS AND THYROID HORMONES ON GROWTH HORMONE SECRETION BY CHICKEN PITUITARY CELLS IN VITRO Dan J. Donoghue, 1 Frank M. Perez, 1,2, Bruce S. A. Diarnante, 1 Sasha Malamed 2 and Colin G. Scanes 1 1Department of Animal Sciences, Rutgers, The State University, New Brunswick, NJ 08903 and 2Department of Anatomy, Robert Wood Johnson Medical School UMDNJ, Piscataway, NJ 08854 Received April 4, 1989
ABSTRACT In young chickens plasma concentrations of growth hormone (GH) are depressed by prostaglandins (PG) El and E2, epinephrine, norepinephrine, 0.2 and [3agonists or thyroid hormones. A primary culture of chicken adenohypopyseal cells was used to examine the direct effects of these agents at the level of the pituitary as evaluated by GH release in the presence and absence of growth hormone releasing factor (GRF). Following collagenase dispersion and culture (preincubation, 48 hr) cells were exposed (incubation, 2 hr) to test agents, except for thyroid hormones which were added during the preincubation, and incubation period. Growth hormone release was increased (P95%) determined by the trypan blue exclusion method. The cells were aliquoted into 24 well plates (Falcon, Lincoln Park, NJ) at a concentration of 2XlO 5 cells/ml and maintained for 48 hr in a humidified incubator (37C) under 5% CO2 and air. Following the 48 hr preincubation period, media was aspirated and attached cells were washed with serum free M199. Cultured cells were then washed with fresh M199 and then exposed (2 hr) to M199 containing the test agents except for T3 and T4. Thyroid hormones were added during both the preincubation and incubation period, or, in a subsequent study, T3 ( 1 . 3 ) < lO-SM) was added during the preincubation or incubation period or both. Following the 2 hr incubation period, the media were collected, centrifuged and the supernatant solutions were retained for GH assay. Materials: Human pancreatic GRF (hpGRF 1-44 NH2), employed as the source of GRF, and clonidine were kindly donated by respectively Dr. T. Mowles (Hoffman La Roche) and Boehringer, prostaglandins El, E2, F2~, T3, T4, TRH, epinephrine, norepinephrine, dopamine, isoproterenol, and phenylephrine were obtained from SIGMA (St. Louis, M O ) . Cell culture medium was purchased from Gibco (Grand Island, NY). G r o w t h H o r m o n e RIA: Samples were assayed for chicken GH by the homologous chicken radioimmunoassay described previously in this laboratory
CHICKEN GH RELEASE IN VITRO
37
(23). All samples within experiments were assayed in a single assay and the coefficient of variation was less than 8%. Statistical Analysis: All data were analyzed by ANOVA and treatment means were partitioned by Fisher Least Squares difference with GRF, TRH, prostaglandins, catecholamine, T3 or I"4 as main effects. A probability of P
VoI. 7(1):35-42, 1990
INFLUENCE OF CATECHOLAMINES, PROSTAGLANDINS AND THYROID HORMONES ON GROWTH HORMONE SECRETION BY CHICKEN PITUITARY CELLS IN VITRO Dan J. Donoghue, 1 Frank M. Perez, 1,2, Bruce S. A. Diarnante, 1 Sasha Malamed 2 and Colin G. Scanes 1 1Department of Animal Sciences, Rutgers, The State University, New Brunswick, NJ 08903 and 2Department of Anatomy, Robert Wood Johnson Medical School UMDNJ, Piscataway, NJ 08854 Received April 4, 1989
ABSTRACT In young chickens plasma concentrations of growth hormone (GH) are depressed by prostaglandins (PG) El and E2, epinephrine, norepinephrine, 0.2 and [3agonists or thyroid hormones. A primary culture of chicken adenohypopyseal cells was used to examine the direct effects of these agents at the level of the pituitary as evaluated by GH release in the presence and absence of growth hormone releasing factor (GRF). Following collagenase dispersion and culture (preincubation, 48 hr) cells were exposed (incubation, 2 hr) to test agents, except for thyroid hormones which were added during the preincubation, and incubation period. Growth hormone release was increased (P95%) determined by the trypan blue exclusion method. The cells were aliquoted into 24 well plates (Falcon, Lincoln Park, NJ) at a concentration of 2XlO 5 cells/ml and maintained for 48 hr in a humidified incubator (37C) under 5% CO2 and air. Following the 48 hr preincubation period, media was aspirated and attached cells were washed with serum free M199. Cultured cells were then washed with fresh M199 and then exposed (2 hr) to M199 containing the test agents except for T3 and T4. Thyroid hormones were added during both the preincubation and incubation period, or, in a subsequent study, T3 ( 1 . 3 ) < lO-SM) was added during the preincubation or incubation period or both. Following the 2 hr incubation period, the media were collected, centrifuged and the supernatant solutions were retained for GH assay. Materials: Human pancreatic GRF (hpGRF 1-44 NH2), employed as the source of GRF, and clonidine were kindly donated by respectively Dr. T. Mowles (Hoffman La Roche) and Boehringer, prostaglandins El, E2, F2~, T3, T4, TRH, epinephrine, norepinephrine, dopamine, isoproterenol, and phenylephrine were obtained from SIGMA (St. Louis, M O ) . Cell culture medium was purchased from Gibco (Grand Island, NY). G r o w t h H o r m o n e RIA: Samples were assayed for chicken GH by the homologous chicken radioimmunoassay described previously in this laboratory
CHICKEN GH RELEASE IN VITRO
37
(23). All samples within experiments were assayed in a single assay and the coefficient of variation was less than 8%. Statistical Analysis: All data were analyzed by ANOVA and treatment means were partitioned by Fisher Least Squares difference with GRF, TRH, prostaglandins, catecholamine, T3 or I"4 as main effects. A probability of P
