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A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.
A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.
Pituitaries from hatchling turtles (Pseudemys scripta elegans) were incubated with different doses of L-thyroxine (T4: 1-500 ng/ml) or 3,5,3'-triiodothyronine (T3: 1-50 ngiml) for various times (1-48 hr) to assess their effects on basal thyrotropin (TSH) and growth hormone (GH) secretion and responsiveness to thyrotropin-releasing hormone (TRH: 10 ngiml). Pretreatment with a pharmacological dose of T4 (500 ngiml) for 1 hr inhibited the TSH response to TRH, and physiological doses were effective after slightly longer times. The TRH effect was reduced by exposure to 50 ngiml T, for 1 hr, and abolished after 4 hr. Pretreatment with 50 ngiml T, or T, for 6 hr did not affect basal TSH secretion, but the attenuation of the TRH response persisted for at least 20 hr after removal of thyroid hormones. Longer exposure (16 hr) to 50 ngiml T4 or T3 reduced both TSH basal secretion and the response to TRH. A low dose (1 ngiml) of T4 or T, for the same amount of time had no effect on basal TSH secretion and only slightly reduced the TRH response; T3 was more potent than T4. Basal secretion and TRH responses of glands pretreated with 5 ng/ml T4 were not different from controls after 4 and 6 hr, but were significantly reduced after 24 hr of exposure; 15 ng/ml T4 reduced the TRH response after 6 hr, but basal secretion was not affected until 24 hr. Treatment with 2 ng/ml T, for 48 hr reduced the response to TRH but did not affect the baseline. Basal and TRH-stimulated GH secretion was also inhibited by thyroid hormones, but the onset of these effects were delayed (16 to 24 hr after exposure) and required a relatively high dose (50 ng/ml).These data are consistent with an earlier report from this laboratory (Denver and Licht: J . Exp. Zool. [in press], '88) that in vivo treatment with thyroid hormone or a goitrogen alters in vitro pituitary hormone secretion in turtles and provide evidence for a direct action of thyroid hormones at the level of the pituitary.The mammalian hypothalamo-hypophyseal-thyroid axis has long been considered a classic example of a negative-feedback loop, with thyroid hormones (TH) determining the set-point for pituitary thyrotropin (TSH) secretion. It has been established that the primary site of TH feedback regulation of pituitary TSH secretion in mammals is at the level of the thyrotrope (see Morley , '8l>, although compelling evidence for an important role of TH in the regulation of hypothalamic TRH secretion was recently presented (Segerson et al., '87). The nature of the thyroidal feedback in nonmammalian vertebrates is poorly understood. In particular, the paucity of data describing changes in the functional status of the pituitary or the hypothalamus in relation to thyroidal state prohibits evaluation of hypotheses regarding the site(s) of action of TH in relation to the secretion of TSH or any other hormones.Previous work from this laboratory demonstrated that turtle pituitaries respond in vitro to the tripeptide amide, thyrotropin-releasing hormone (TRH) by releasing TSH, prolactin (PRL), ...
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