Inhibition by testosterone of prolactin and growth hormone release from chicken anterior pituitary glands in vitro

Pituitary glands and hypothalami from broiler fowl were incubated in medium containing testosterone, and prolactin and GH release were determined. Pituitary glands were also preincubated for 20 h in medium containing testosterone, and then in medium containing various secretagogues. Testosterone inhibited the release of prolactin directly from the pituitary gland in a concentration-related manner. The hypothalamus stimulated the release of prolactin, but by a lesser amount in the presence of testosterone. When pituitary glands were preincubated with testosterone, subsequent release of prolactin was inhibited, except with the highest concentration which stimulated prolactin release. Hypothalamic extract (HE) markedly stimulated prolactin release from control pituitary glands although testosterone-primed glands were less responsive. The stimulation of prolactin release by thyrotrophin releasing hormone (TRH) and prostaglandin E2 (PGE2) was also reduced by preincubation of the pituitary glands with testosterone. Priming with testosterone did not affect the release of GH from pituitary glands alone, but reduced the TRH-, HE- and PGE2-stimulated release of GH. These results demonstrate that testosterone directly inhibits prolactin secretion and reduces the sensitivity of pituitary lactotrophs and somatotrophs to provocative stimuli.     

Serotonin and acetylcholine affect the release of prolactin and growth hormone from pituitary glands of domestic fowl in vitro in the presence of hypothalamic tissue

Anterior pituitary glands from broiler fowl were incubated alone or with hypothalamic tissue in medium containing either serotonin or serotoninergic drugs, acetylcholine or cholinergic drugs, and the release of prolactin (Prl) and growth hormone (GH) measured by homologous radioimmunoassays. The neurotransmitters and drugs affected the release of hormones from the pituitary gland only when hypothalamic tissue was also present. Serotonin and its agonist quipazine stimulated the release of Prl and inhibited release of GH in a concentration-related manner. The antagonist methysergide blocked the effects of serotonin and quipazine on Prl. Acetylcholine and its agonist pilocarpine also stimulated release of Prl and inhibited release of GH in a concentration-related manner. Atropine blocked these responses. The results show that serotonin and acetylcholine affect pituitary hormone secretion by acting on the hypothalamus. They may stimulate the secretion of a Prl releasing hormone and somatostatin.     

Hypothalamic control of prolactin and growth hormone secretion in the pituitary gland of the pigeon and the chicken: in vitro studies

Hypothalamic extracts stimulated the release of prolactin and growth hormone from pigeon and chicken pituitary glands incubated in vitro. Release of hormone was proportional to the amount of hypothalamic extract added. Pituitary glands from "lactating" pigeons released more prolactin and their hypothalami contained more prolactin-releasing activity compared with controls. Partial separation of prolactin releasing activity from growth hormone releasing activity in chicken hypothalamic extract was achieved using gel filtration chromatography. Co-incubation studies in vitro with hypothalamic tissue present showed that prolactin release from the pituitary was inhibited and growth hormone release was stimulated when dopamine was added to the medium. The effects of dopamine were blocked by the antagonist pimozide. The possible existence of hypothalamic releasing and inhibiting factors regulating secretion of prolactin and growth hormone is discussed.     

Ontogenetic development of the inhibition of growth hormone release by somatostatin in the rat: in-vivo and in-vitro (perifusion) study

An in-vitro study of GH secretion by rat fetal and neonatal pituitary glands was conducted using a perifusion system. After a 2 h period the GH content of the effluent was constant. Theophylline, thyrotrophin releasing hormone (TRH) and rat stalk median emience extract (SME) were effective stimuli of GH release from the pituitary glands of the 19.5-day-old fetuses. Somatostatin, added to the medium (10 microgram/ml), had no inhibitory effect on GH release (basal or stimulated by either theophylline or SME) before day 4 after birth. After postnatal day 5, somatostatin always inhibited GH secretion. These findings were consistent with the results of experiments in vivo. In rats tested within 4 days of birth, sodium pentobarbitone-stimulated plasma GH levels were not reduced by somatostatin; on day 4 and thereafter somatostatin depressed the response to pentobarbitone injection. These results indicate a postnatal maturation of the regulation of GH release by the hypothalamo-hypophysial system in the rat.     

Growth hormone and prolactin secretion in water-deprived chickens

The deprivation of water for 12 or 24 hr increased the prolactin concentration in the plasma of immature chickens but had no effect on the circulating growth hormone (GH) level. The increase in plasma prolactin level reflected an increase in the basal rate of prolactin release from incubated hemipituitary glands and an increase in the responsiveness of the pituitary gland to hypothalamic releasing factors. The deprivation of water had no effect on basal level of pituitary GH release in vitro but abolished the stimulatory effect of the hypothalamus on in vitro GH secretion.     

Hypothalamic control of prolactin and growth hormone secretion in different vertebrate species.

Pituitaries from different vertebrates representing mammals, birds, reptiles, and amphibians, were incubated in vitro with various hypothalamic extracts (HE). Prolactin and growth hormone (GH) in medium and pituitary were measured by densitometry after polyacrylamide gel electrophoretic separation (PAGE). Rat (Rattus norvegicus), chicken (Gallus domesticus), terrapin (Chrysemys picta), and toad (Xenopus laevis) pituitaries were incubated with homologous HE. Rat HE inhibited prolactin release. In the other species the HE stimulated prolactin release. In all four species GH release was stimulated by HE. The effects on prolactin and GH release were proportional to the dose of HE added. Chicken pituitaries were incubated with chicken HE together with rat HE. The rat HE inhibited the chicken HE-stimulated release of prolactin, as measured by radioimmunoassay. Heterologous incubations were used to test HE for prolactin releasing and inhibiting factors and for GH releasing and inhibiting factors. Chicken pituitaries were incubated with HE from the eel (Anguilla anguilla), the cod (Gadus gadus) and the flounder (Pleuronectes flesus) as well as from the other species listed. Both cod and flounder HE marginally inhibited autonomous chicken prolactin release. HE from these species dose-responsively inhibited chicken HE-stimulated prolactin release. Cod HE also inhibited chicken HE-stimulated GH release. HE from the eel, the terrapin and the toad stimulated chicken prolactin release. Hormone release from terrapin and toad pituitaries incubated with heterologous HE was consistent with hypothalamic control via releasing factors in these species.     

Oestradiol 17 beta modifies fowl pituitary prolactin and growth hormone secretion in vitro

Chicken pituitary glands were incubated in medium containing oestradiol 17 beta (E2), alone or together with single whole hypothalami. E2 stimulated prolactin release from the pituitary and increased the prolactin releasing activity of the hypothalamus, but did not affect growth hormone release. Preincubation of pituitaries with E2 dramatically stimulated subsequent prolactin release. Pituitaries primed with E2 were more responsive to the prolactin-stimulating effects of hypothalamic extract (HE) and thyrotrophin-releasing hormone (TRH) and more sensitive to the prolactin-inhibiting effect of dopamine. E2-primed pituitaries were much less sensitive to the growth hormone releasing activity of TRH and HE. These results show that E2 may regulate pituitary function by direct effects on hormone release by modifying pituitary sensitivity to stimulatory or inhibitory influences and by altering hypothalamic releasing activity.     

Age-related changes in prolactin and growth hormone release from pituitary glands in vitro

The basal release of prolactin from cockerel anterior pituitary glands in vitro declined between 1 and 7 weeks of age, to a level less than that released by pituitary glands from 18 week old (adult) cockerels and hens. Basal growth hormone (GH) release increased between 1 and 7 weeks of age but had declined in adults to a level similar to that released from 4 weeks old cockerels. The responsiveness of the pituitary gland to hypothalamic stimulation, using hypothalami from 8 week old broiler fowl, was also age-related. Prolactin release was considerably higher from pituitaries of 1 week old cockerels compared to the other age groups. Stimulation of GH release by the hypothalamus was higher from pituitaries of both 1 and 7 week old cockerels compared to the other groups of birds. The increase in release of prolactin following incubation with thyrotrophin releasing hormone (TRH) declined between 1 and 7 weeks, but increased slightly in adult birds, whereas the increase in release of GH following TRH was higher from pituitaries of both 1 and 7 week old cockerels. Hypothalamic prolactin (Prl) releasing activity, measured as the ability of the hypothalamus to stimulate hormone release from 8 week old broiler fowl anterior pituitary glands, declined with the age of the donor cockerels. The hypothalami from adult hens secreted significantly more Prl releasing activity than did adult cockerel hypothalami. The secretion of GH releasing activity decreased markedly with the age of the donor bird. These results suggest that maturational patterns of hormone secretion in fowl are partly due to changes in autonomous hormone release, to changing patterns of hypothalamic activity and to differences in pituitary responsiveness to provocative stimuli.     

Mechanism of serotonin effects on prolactin and growth hormone secretion in domestic fowl

Brain serotonin levels were increased in immature chickens by ip injection of pargyline (75 mg/kg) and clorgyline (5 mg/kg) and by L-tryptophan (100 mg/kg) and imipramine (10 mg/kg) treatment. These treatments increased the circulating prolactin level and reduced the concentration of plasma growth hormone (GH). Treatment with para-chlorophenylalanine (PCPA, 100 mg/kg) reduced the brain serotonin content and the level of plasma prolactin. Treatment with these drugs in vivo similarly affected the basal level of prolactin release from pituitary glands in vitro, although it did not affect the basal level of GH release. The in vitro responsiveness of the pituitary gland to hypothalamic stimuli eliciting prolactin secretion was increased by in vivo pargyline and combined tryptophan: imipramine treatment but reduced by PCPA administration. The in vitro GH response to hypothalamic stimulation was reduced after the in vivo injection of pargyline, clorgyline and tryptophan: imipramine. The hypothalami from clorgyline and tryptophan: imipramine treated birds induced a greater stimulation of in vitro prolactin secretion from control pituitary glands than hypothalami from controls birds, whereas the GH releasing activity was reduced. These results suggest that serotonin stimulates prolactin secretion in chickens by increasing pituitary responsiveness to hypothalamic releasing factors and by increasing the prolactin releasing activity of the hypothalamus. Serotonin appears to suppress GH secretion by reducing pituitary sensitivity to releasing factors and by reducing hypothalamic GH releasing activity.     

Role of Ca2+ stores in dopamine- and PACAP-evoked growth hormone release in goldfish

Growth hormone (GH) secretion, evoked by either pituitary adenylate cyclase-activating polypeptide (PACAP) or dopamine (DA), is dependent on both voltage-sensitive calcium channels (VSCC) and cAMP signaling in goldfish. We further characterized the involvement of Ca2+ in evoked release by PACAP and DA, by examining the sensitivity of evoked GH release to perturbations of Ca2+ signaling. Both VSCC and calmodulin/calmodulin-dependent kinase are involved in PACAP signaling as had been shown for DA. In spite of this apparent dependence on VSCC, blockade of TMB-8 but not ryanodine-sensitive intracellular Ca2+ stores inhibited both PACAP- and DA-evoked GH release. Using sarcoplasmic/endoplasmic reticulum Ca-ATPases (SERCA) inhibitors, we found BHQ blocked, whereas thapsigargin (Tg) enhanced stimulated GH release, suggesting that Tg-sensitive SERCA may counteract these cAMP-mobilizing neuroendocrine regulators by sequestering [Ca2+]i. As GH secretion stimulated by two endogenous gonadotropin-releasing hormones is not affected by Tg, it appears that distinct multiple Ca2+ stores mediate the hormone releasing response to different neuroendocrine regulators.     

Thyrotropin-releasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in vitro

We have examined the interaction of thyroid hormone and TRH on GH release from rat pituitary monolayer cultures and perifused rat pituitary fragments. TRH (10(-9) and 10(-8)M) consistently stimulated the release of TSH and PRL, but not GH, in pituitary cell cultures of euthyroid male rats. Basal and TRH-stimulated TSH secretion were significantly increased in cells from thyroidectomized rats cultured in medium supplemented with hypothyroid serum, and a dose-related stimulation of GH release by 10(-9)-10(-8) M TRH was observed. The minimum duration of hypothyroidism required to demonstrate the onset of this GH stimulatory effect of TRH was 4 weeks, a period significantly longer than that required to cause intracellular GH depletion, decreased basal secretion of GH, elevated serum TSH, or increased basal secretion of TSH by cultured cells. In vivo T4 replacement of hypothyroid rats (20 micrograms/kg, ip, daily for 4 days) restored serum TSH, intracellular GH, and basal secretion of GH and TSH to normal levels, but suppressed only slightly the stimulatory effect of TRH on GH release. The GH response to TRH was maintained for up to 10 days of T4 replacement. In vitro addition of T3 (10(-6) M) during the 4-day primary culture period significantly stimulated basal GH release, but did not affect the GH response to TRH. A GH stimulatory effect of TRH was also demonstrated in cultured adenohypophyseal cells from rats rendered hypothyroid by oral administration of methimazole for 6 weeks. TRH stimulated GH secretion in perifused [3H]leucine-prelabeled anterior pituitary fragments from euthyroid rats. A 15-min pulse of 10(-8) M TRH stimulated the release of both immunoprecipitable [3H]rat GH and [3H]rat PRL. The GH release response was markedly enhanced in pituitary fragments from hypothyroid rats, and this enhanced response was significantly suppressed by T4 replacement for 4 days. The PRL response to TRH was enhanced to a lesser extent by thyroidectomy and was not affected by T4 replacement. These data suggest the existence of TRH receptors on somatotrophs which are suppressed by normal amounts of thyroid hormones and may provide an explanation for the TRH-stimulated GH secretion observed clinically in primary hypothyroidism.     

Enhancement by proopiomelanocortin-derived peptides of growth hormone and prolactin secretion by bullfrog pituitary cells.

Corticotrophs in the bullfrog (Rana catesbeiana) are situated mainly in the rostral region of the anterior lobe of the pituitary gland, which receives its blood supply primarily from the portal vessel. On the assumption that the proopiomelanocortin (POMC)-derived peptides released into the pituitary circulation may influence the function of other pituitary cells situated downstream, the effects of three POMC-derived peptides, namely, N-terminal peptide of POMC (NPP), adrenocorticotropic hormone (ACTH), and joining peptide (JP), on the secretion of growth hormone (GH) and prolactin (PRL) by bullfrog dispersed anterior pituitary cells were examined. NPP and ACTH, but not JP, stimulated the release of GH and PRL in a concentration-dependent manner. It was also found that ACTH1-17, but not alpha-melanocyte-stimulating hormone, was effective in enhancing GH and PRL release. A marked difference between the response to NPP and ACTH and the response to thyrotropin-releasing hormone employed as a reference secretagogue in terms of the time required for stimulating the release of GH and PRL was noted. Northern blot analysis of GH and PRL mRNA levels and radioimmunoassay for GH and PRL in the cultured cells revealed that ACTH increases the syntheses of both pituitary hormones as well. The possibility that NPP and ACTH act on neighboring cells to maintain their overall secretory function is discussed.     

Stimulation of growth hormone release by vasoactive intestinal peptide and peptide PHI in rat anterior pituitary reaggregates. Permissive action of a glucocorticoid and inhibition by thyrotropin-releasing hormone

In superfused rat anterior pituitary cell reaggregates, cultured for 5 days in serum-free defined medium, vasoactive intestinal peptide (VIP) concentration-dependently stimulated prolactin (Prl) release but had only a marginal effect on growth hormone (GH) release. When reaggregates were cultured in the presence of 80 nM dexamethasone (Dex) VIP strongly stimulated GH release from a concentration as low as 0.1 nM. VIP did not stimulate LH release. Peptide PHI also stimulated GH release but thyrotropin-releasing hormone (TRH) or angiotensin II did not. In fact, TRH slightly but transiently inhibited basal GH release and strongly inhibited VIP-stimulated GH release. GH-releasing factor (GRF) stimulated GH more potently and with higher intrinsic activity than VIP but GRF did not increase Prl release. The present data indicate that under defined hormonal conditions VIP and PHI are capable of stimulating GH release and that TRH can antagonize this effect by a direct action on the pituitary.     

Annexin 5 inhibits thyrotropin releasing hormone (TRH) stimulated prolactin release in the primary culture of rat anterior pituitary cells

Annexin 5, a novel calcium-phospholipid binding protein, is thought to be involved in hormone secretion by the anterior pituitary gland. Gonadotropin releasing hormone stimulates annexin 5 synthesis, which, in turn, enhances gonadotoropin secretion. On the other hand, annexin 5 was shown to inhibit prolactin release in vitro. To understand the nature of the opposing effects of annexin 5 on these two major pituitary hormones, the present study examines the inhibitory effect of annexin 5 on prolactin release in relation to thyrotropin stimulating hormone (TRH) using primary cultures of anterior pituitary cells of adult female rats. While recombinant rat annexin 5 was found to have little effect on basal prolactin release, it significantly inhibited TRH-stimulated prolactin release. Addition of specific anti-annexin 5 serum to the culture increased basal prolactin release in a concentration dependent manner, and no further increase in prolactin release was observed following application of TRH in the presence of anti-annexin 5. The enhanced basal prolactin release induced by anti-annexin 5 was reversed by the simultaneous administration of indomethacin, an inhibitor of cyclooxygenase. These results demonstrate that endogenous pituitary annexin 5 exerts an inhibitory effect on prolactin release and suggest that this is attained by suppression of eicosanoid synthesis in vitro.     

In vitro effects of thyrotropin-releasing hormone and somatostatin on prolactin and growth hormone release by the pituitary of Poecilia latipinna. I. An electrophoretic study

Pituitary glands were removed from Poecilia latipinna which had been maintained in one-third seawater and were incubated for 18 hr in media of either 300 mosmol/kg (OP300) or 340 mosmol/kg (OP340) osmotic pressure for measurement of both total and newly synthesised prolactin (PRL) and growth hormone (GH) release. Thyrotropin-releasing hormone (TRH) at 100 ng/ml increased release of total and newly synthesised PRL into OP340, but not into OP300, medium. Conversely, 300 ng/ml of somatotropin-release-inhibiting factor (SRIF) inhibited total and newly synthesised PRL release into OP300, but not OP340, medium. At 1000 ng/ml, SRIF inhibited total PRL release into both media, but newly synthesised PRL release was reduced significantly only in OP300 medium. The release of GH was unaffected by 100 ng/ml TRH in OP300 medium, but both total and newly synthesised GH release were enhanced by this dose in OP340 medium. SRIF at 300 ng/ml reduced total GH release into OP300 medium, whereas the release of newly synthesised GH was inhibited in OP340 medium. At 1000 ng/ml, SRIF inhibited total GH release into both media, but release of the newly synthesised hormone was not significantly altered. These results suggest that TRH can stimulate and SRIF inhibit both PRL and GH release by Poecilia pituitaries, but that these effects may be modulated by plasma osmotic pressure.     

TRH is a tonic secretagogue in growth hormone secreting but not in nonfunctioning pituitary tumors

In most patients with growth hormone (GH) secreting pituitary adenomas and clinically nonfunctioning pituitary tumors (NFPT) the intravenous injection of thyrotropin releasing hormone (TRH) augments the secretion of GH and subunits of gonadotropin hormones respectively. Similar hormone responses to TRH have been detected in rat pituitary cell lines and in primary human pituitary tumor cultures in vitro. Nevertheless the TRH effect on tumor hormonal secretion has not been well characterized. In the present study we examined TRH-induced hormone secretion in GH secreting tumors and in NFPT in vitro. Cultured cells secreted betaLH and betaFSH (NFPT) or GH (GH secreting adenomas) up to 14 days in culture. In NFPT TRH (10(-8) mol/l) elicited peak betaLH and betaFSH secretion at 60 to 90 min, with no further increase at 24 h. TRH-stimulated GH secretion peaked at 90-120 min, and decreased after 3 h, but a secondary rise occurred after 24 h of incubation. Chronic daily exposure to TRH followed by an acute TRH challenge resulted in a further increase of GH secretion after one hour. In contrast, acute TRH administration following chronic exposure did not elicit increased P-subunits secretion in NFPT. Coadministration of cycloheximide did not change TRH induced beta-subunits secretion in NFPT. However, when it was administered 24 h prior to TRH, it blocked both basal and TRH induced beta-subunits levels in NFPT. Cycloheximide had no effect on basal or stimulated GH secretion when administered concomitantly or 24 h before TRH. Incubation of cultured GH secreting tumors with cycloheximide during 5 days blocked both basal and TRH stimulated GH secretion, thus indicating dependency on protein synthesis during the chronic, secondary phase. Since the acute secretion was not affected by coadministration of cycloheximide, these early increases in hormone levels apparently reflect the release of stored hormone. In summary, GH secreting adenomas and NFPT differ significantly in their hormonal response to continuous exposure to TRH. The mechanisms underlying the sustained effect of TRH on GH secretion in vitro remain to be investigated. If endogenous TRH exerts a similar continuous effect it may contribute to the disregulated GH secretion in acromegaly.     

Calcium and somatostatin interaction in regulating prolactin and somatotropic hormone release from rat adenohypophysis cells in primary culture

The effect of Ca+2 and somatostatin on prolactin and growth hormone (GH) secretion was studied in the primary cell culture of the rat adenohypophysis. A decrease in Ca+2 concentration of the medium from 1.8 to 0.1 mM inhibited prolactin release and did not influence GH secretion. An elevation of extracellular Ca+2 level up to 5 mM did not affect prolactin secretion and slightly decreased GH release. Somatostatin inhibited prolactin and GH release provided Ca+2 concentration in the medium was normal (1.8 mM) and/or diminished (0.1 mM). An increase in Ca+2 level up to 5 mM blocked the inhibitory effect of somatostatin. The results obtained show Ca+2 and somatostatin to be antagonistic in the prolactin control and GH release from adenohypophyseal cells.     

Selectivity of melatonin pituitary inhibition for luteinizing hormone-releasing hormone

The pineal indole melatonin suppresses the neonatal rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to LH-releasing hormone (LHRH), as shown in previous studies from this laboratory. We show in this study that the melatonin inhibition is a selective effect and is not due to general inhibition of pituitary function. The effects of the indole on the responses to thyrotropin-releasing hormone (TRH) and somatostatin (SRIF) and on basal pituitary hormone secretion were examined with cells in culture. Neonatal rat anterior pituitary cells dissociated with collagenase and hyaluronidase were cultured overnight and distributed to 35-mm dishes at the time of use. For examination of melatonin effects on the response to releasing hormones, the cells were incubated for 3 h in control medium or medium containing LHRH (10-9-10-6 M), TRH (10-10-10-6 M), or SRIF (10-9-10-6 M), either alone or in the presence of melatonin (10-8 or 10-6 M). For examination of basal hormone secretion, the cells were incubated for 1.5, 3, 6, 15, or 24 h in either medium alone or medium containing melatonin (10-6 M). Medium and cell lysate concentrations of LH, FSH, thyroid-stimulating hormone (TSh), prolactin (PRL) and growth hormone (GH) were determined by double antibody RIA. As previously, melatonin (10-8 M) significantly suppressed LH and FSH release by all concentrations of LHRH. This concentration of the indole produced maximal suppression of both LH and FSH responses to LHRH. By contrast, melatonin at a 100-fold greater concentration (10-6 M) had no effect on TRH stimulation of TSH or PRL release or on SRIF inhibition of GH release. Similarly, melatonin had no effect on basal release of TSH, PRL, or GH at the times examined. These findings show that melatonin inhibition of the gonadotroph response to LHRH is a selective effect.     

Thyroid-stimulating hormone and prolactin responses to thyrotropin-releasing hormone during infusion of epinephrine and propranolol in man.

Male volunteers were administered 100 microgram thyrotropin-releasing hormone (TRH) intravenously during control (saline) and drug (epinephrine-propranolol) infusions. There were no differences in the thyroid-stimulating hormone (TSH) or prolactin responses to TRH during the epinephrine-propranolol infusion periods. There were no significant differences in growth hormone (GH) responses to epinephrine-propranolol infusions. Epinephrine-propranolol had no detectable effect on basal TSH, prolactin and GH concentrations. We conclude that the alpha-adrenergic system does not play any role at the pituitary level in modulating the effect of TRH-stimulated TSH or prolactin secretion in male volunteers.     

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro.The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.