Increase of thyrotropin response to thyrotropin-releasing hormone (TRH) and TRH release in rats during pregnancy

Regulation of thyrotropin (TSH) release by thyrotropin releasing hormone (TRH) in the anterior pituitary gland (AP) of pregnant rats was studied. The pregnant (day 7, 14, and 21) and diestrous rats were decapitated. AP was divided into 2 halves, and then incubated with Locke's solution at 37 degrees C for 30 min following a preincubation. After replacing with media, APs were incubated with Locke's solution containing 0, or 10 nM TRH for 30 min. Both basal and TRH-stimulated media were collected at the end of incubation. Medial basal hypothalamus (MBH) was incubated with Locke's medium at 37 degrees C for 30 min. Concentrations of TSH in medium and plasma samples as well as the cyclic 3':5' adenosine monophosphate (cAMP) content in APs and the levels of TRH in MBH medium were measured by radioimmunoassay. The levels of plasma TSH were higher in pregnant rats of day 21 than in diestrous rats. The spontaneous release of TSH in vitro was unaltered by pregnancy. TRH increased the release of TSH by AP, which was higher in pregnant than in diestrous rats. Maternal serum concentration of total T3 was decreased during the pregnancy. The basal release of hypothalamic TRH in vitro was greater in late pregnant rats than in diestrous rats. After TRH stimulation, the increase of the content of pituitary cAMP was greater in late pregnant rats than in diestrus animals. These results suggest that the greater secretion of TSH in pregnant rats is in part due to an increase of spontaneous release of TRH by MBH and a decrease of plasma thyroid hormones. Moreover, the higher level of plasma TSH in rats during late pregnancy is associated with the greater response of pituitary cAMP and TSH to TRH.     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Vasopressin rapidly stimulates phosphatidic acid-phosphatidylinositol turnover in rat anterior pituitary cells

In cultured rat anterior pituitary cells, the agonist [Asu1,6, Arg8]vasopressin (AVP-A) increased by 1.5-fold 32Pi incorporation into phosphatidic acid (PA), as early as 15 s after its addition. Increased phosphatidylinositol (PI) labeling became significant 4 min after AVP-A addition. Dose-response measurements with AVP-A showed ED50 values of 76 and 62 nM for PA and PI labeling, respectively. Peptide corticotropin-releasing factor (CRF) (0.1 microM) did not affect the stimulatory effect of AVP-A on PA and PI labeling. These data suggest that stimulation of PI metabolism in corticotrophs may be one of the early events involved in the stimulation of ACTH release induced by vasopressin.     

Release of thyroid stimulating hormone from chick anterior pituitary glands by thyrotropin releasing hormone (TRH)

Chicks two and ten days-of-age respond to a wide range of thyrotropin releasing hormone (TRH) dosages as measured by thyroid uptake of ³²P. The duration of hormone and ³²P action is important. Excellent responses were obtained with the injection of 1.0 μCi³²P at one hour and TRH either at one or four hours before autopsy in both two-day and ten-day-old birds. The ³²P uptake in the thyroid glands was increased by doses of hormone which ranged from 40 nanograms to 125,000 nanograms and was bimodal. Analysis of the data when calculated using log¹⁰ of dose was best accomplished by the use of 5th-degree polynomial equations. It is suggested that the bimodal response is a result of a dual action of TRH. First, TRH initiates the release of stored TSH from the anterior pituitary; and second, TRH stimulates the secretion of newly synthesized TSH by the anterior pituitary.     

Central nervous system action of TRH to stimulate gastric emptying in rats

The effects of intracisternal injection of TRH on gastric emptying of a liquid meal was investigated in 24 h fasted rats using the phenol red method. Intracisternal injection of TRH, RX 77368, or [N-Val2]-TRH, an analog devoid of TSH-releasing activity, 5 min prior to a meal, stimulated gastric emptying measured 20 min later. TRH action was dose dependent (1-100 ng), and rapid in onset. The calculated time for emptying half of the meal was decreased from 16 +/- 3 min (control group) to 4 +/- 1 min (TRH 30 ng). The stable analog, RX 77368, unlike TRH, stimulated gastric emptying when the meal was given 60 min after peptide injection. Intravenous injection of atropine (2.5 micrograms) inhibited and that of carbachol (1 microgram) stimulated gastric emptying whereas i.v. injection of TRH (0.1-1 microgram) had no effect. Vagotomy but not adrenalectomy reversed the increase in gastric emptying induced by intracisternal TRH. Atropine blocked the stimulatory effect of TRH and carbachol. These results demonstrate that TRH acts within the brain to stimulate gastric emptying through vagus-dependent and cholinergic pathways whereas alterations of adrenal and pituitary-thyroid secretion do not play an important role.     

Hypophysial portal vascular infusion of TRH in the rat: an ultrasturctural and radioimmunoassay study.

The hypophysial portal vessels and anterior pituitary gland of adult male Wistar rats were exposed surgically. A hypophysial portal vessel was cannulated and infused for one minute with saline or thyrotrophin (TRH). Anterior pituitary glands were collected at 1,5,15,30 or 60 minutes after cessation of infusion, for light and electron microscopic examination. Before and immediately after cannulation of a portal vessel, a 1-ml sample of blood was collected at 1,5,15,30, or 60 minutes, from the femoral vein for radioimmunoassay (RIA) of growth hormone. Thyrotrophs from anterior pituitary glands of rats infused with TRH displayed emiocytic activity at all time-periods studied. Rough endoplasmic reticular (RER) cisternae were dilated at 15 minutes following infusion and remained dilated at 30 and 60 minutes. TRH was observed to stimulate emiocytic activity in most pituitary cell-types. Extensive dilations of RER cisternae were also observed in mammotrophs and gonadotrophs, but were not observed in somatotrophs or adrenocorticotrophs. The demonstration that thyrotrophs, mammotrophs, somatotrophs, and gonadotrophs respond to TRH suggests that some common features may be shared by these cells. Preliminary analysis of the RIA data show that TRH was potent in elevating radioimmunoassayable growth hormone levels. Significant increases (p less than 0.02) in plasma GH levels were present at the earlier time periods studied (1,5, and 15 minutes) following the infusion of TRH, but no at 30 or 60 minutes. These findings provide additional support for the non-specific action of TRH upon hte various adenohypophysial cell types, and demonstrate that TRH stimulates these cells by a direct action on the adenohypophysis.     

Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

The effect of thyrotrophin-releasing hormone (TRH, 10(-7) M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone-releasing hormone (LH-RH, 10(-7) M). Actinomycin D (2 X 10(-5) M) and cycloheximide (10(-4) M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).     

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.     

Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect.

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of growth hormone release in dwarf and normal chickens by thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF)

The effect of thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF) on growth hormone (GH) release was studied in both dwarf and normal Rhode Island Red chickens with a similar genotype except for a sex-linked dw gene. Both TRH (10 micrograms/kg) and hpGRF (20 micrograms/kg) injections stimulated plasma GH release within 15 min in young and adult chickens. The increase in GH release was higher in young cockerels than that in adult chickens. The age-related decline in the response to TRH stimulation was observed in both strains, while hpGRF was a still potent GH-releaser in adult chickens. The maximal and long acting response was observed in young dwarf chickens, suggesting differences in GH pools releasable by TRH and GRF in the anterior pituitary gland. The pituitary gland was stimulated directly by perifusion with hpGRF (1 microgram/ml and 10 micrograms/ml) or TRH (1 microgram/ml). Repeated perifusion of GRF at 40 min intervals blunted further increase in GH release, but successive perifusion with TRH stimulated GH release. The results suggest the possibility that desensitization to the effects of hpGRF occurs in vitro and that the extent of response depends on the number of receptors for hpGRF or TRH and/or the amount of GH stored in the pituitary gland.     

Thyroid hormone modulation of TRH precursor levels in rat hypothalamus, pituitary, thyroid and blood

In the present study we have examined the in vivo effects of thyroid hormones and TRH on tissue and blood levels of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Using specific radioimmunoassays (RIAs), we measured TRH immunoreactivity (TRH-IR) and TRH-Gly-IR concentrations in blood, hypothalamus, anterior and posterior pituitary, and thyroid in euthyroid, hypothyroid and thyroxine (T4)-treated 250 g male Sprague-Dawley rats. TRH-Gly-IR and TRH-IR were detected in all of these tissues. Highly significant positive correlations between whole blood TRH-Gly-IR levels and the corresponding serum TSH values (p less than 0.01), whole blood TRH-IR versus serum TSH (p less than 0.01) and whole blood TRH-Gly-IR versus whole blood TRH-IR (p less than 0.01) are consistent with cosecretion of TRH and TRH precursor peptides into the circulation. Euthyroid rats injected with TRH IP (1 microgram/100 g b.wt.) and hypothyroid rats had 4-fold higher whole blood TRH-Gly-IR levels compared to euthyroid controls (p less than 0.0005). Injection of TRH into euthyroid rats significantly increased the TRH-Gly-IR concentration in the hypothalamus, anterior and posterior pituitary and thyroid. The increase in blood TRH-Gly-IR following intravenous TRH may be due, in part, to partial saturation of TRH-degrading enzymes in blood and cell membranes. The ratio of TRH-Gly to TRH was significantly increased in the anterior pituitary by hypothyroidism and TRH injection, suggesting that thyroid hormones and TRH regulate the alpha-amidation of TRH-Gly to form TRH in this tissue. TRH-Gly levels of pooled pituitary and thyroid extracts quantitated by a combination of TRH-Gly RIA and high performance liquid chromatography (HPLC) revealed several-fold increases following incubation at 60 degrees C. Heating at this temperature may block the alpha-amidation activity in extra-hypothalamic tissues but not the "trypsin-like" enzymes which cleave prepro-TRH into TRH-Gly-immunoreactive peptides.     

Detection of thyrotropin-releasing hormone (TRH) mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary.

To investigate the presence of TRH mRNA in the human anterior pituitary tissue, total RNA from human normal and tumoral anterior pituitary, hypothalamus (positive control) and muscle tissues (negative control) was reverse transcribed (RT) to the first strand of cDNA. RT products were then amplified by polymerase chain reaction (PCR) using a set of three exon-specific primers (two external 5' and 3' primers and one internal 3' primer) for a target sequence of the TRH gene including an intronic sequence of about 650 base pairs (bp). Southern analysis of the RT-PCR products specifically hybridizing with a 45-mer TRH probe showed two bands of the predicted sizes (399 and 351 bp) far more intense in hypothalamus than in normal and tumoral anterior pituitary tissue. The 399 and 351 bp RT-PCR products contained the BglII enzyme restriction site included in the TRH cDNA sequences spanned by the primers and the two respective digested fragments which were, as predicted, 337 and 289 bp long, hybridized with the TRH probe. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target TRH sequences in the human normal and tumoral anterior pituitary tissue as well as in the hypothalamus. Our data imply TRH gene expression in the human anterior pituitary.     

Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures

Intact anterior pituitary tissue and primary anterior pituitary cultures were stained with 1:30,000 anti-TRH and 1:10,000 anti-GnRH using the peroxidase antiperoxidase immunocytochemical technique. Stains applied to serial ultrathin sections of intact pituitaries showed that TRH immunoreactivity could be localized in secretory granules of thyrotropes, gonadotropes and corticotropes whereas GnRH immunoreactivity was found only in gonadotropes and corticotropes. Long-term primary pituitary cultures were studied to remove the anterior pituitary cells from hypothalamic influences. In these cell populations both TRH and GnRH immunoreactivity persisted. In addition, quantification of the stained cells at the light microscopic level demonstrated that the volume fraction of TRH and GnRH immunoreactive cells remained constant up to 3 weeks of culture. Studies of serial ultrathin sections through cells from these cultures showed TRH or GnRH localized in secretory granules of cells that contained LH and ACTH, but not TSH. Both liquid and solid phase immunoabsorption specificity controls were used to validate the immunocytochemical stains. These studies suggest that the pituitary TRH and GnRH immunoreactivities may not be completely of hypothalamic origin, but may also be endogenous to a subpopulation of unique multihormonal pituitary cells.     

Multifactorial Modulation of TRH Metabolism

1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through medianeminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary.2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell.3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure.4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times.TRH mRNA are thus controlled by transsynaptic and hormonal influences.5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglu-tamyl peptidase II (PPII).6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity.7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.     

Ca2(+)-independent secretory mechanism of thyrotropin-releasing hormone (TRH) involves protein kinase C in rat pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates biphasic prolactin (PRL) secretion from rat pituitary GH3 cells. The pretreatment of cells with EGTA (100 microM) plus arachidonic acid (15 microM), a condition which decreased TRH-responsive intracellular Ca2+ pools, eliminated the activity of TRH on burst PRL secretion (2 min) but did not alter that on sustained PRL secretion (30 min). However, the treatment of cells with EGTA, arachidonic acid and H-7 (300 microM), a potent inhibitor of protein kinase C (PKC), almost completely suppressed the activity of TRH for sustained PRL secretion. In cells down-modulated for PKC, TRH abolished this Ca2(+)-independent sustained PRL secretion. These results suggest that TRH acts through a separate, Ca2(+)-independent secretory mechanism, besides by modulating the Ca2(+)-dependent mechanism and that PKC is involved in this Ca2(+)-independent secretory pathway.     

Effect of active and passive immunization with TRH on plasma TSH response to propylthiouracil.

The role of thyrotropin-releasing hormone (TRH) in the secretion of TSH from the anterior pituitary was investigated in rats by active and passive immunization with TRH. The plasma TSH response to propylthiouracil (PTU) in TRH-bovine serum albumin (BSA)-immunized rats was significantly lower than that of BSA-immunized or non-immunized rats. Similarly, the increased plasma TSH level following PTU treatment was significantly suppressed after iv injection of antiserum to TRH. However, the decline in plasma TSH levels was not complete. The results of the present study indicate, at least in part, the physiological significance of endogenous TRH in the regulation of pituitary TSH secretion.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Central inhibitory action of TRH on prolactin secretion in the rat

Intravenous (iv) injection of FK33-824 [( D-Ala2, MePhe4, Met-(O)5-ol]-enkephalin, 8 and 16 nmole/100 g body wt), a potent Met5-enkephalin analog, and domperidone (1.2, 2.4, and 24 nmole/100 g body wt), a dopamine antagonist, resulted in a dose-related increase in plasma prolactin (PRL) levels in urethane-anesthetized male rats. PRL release induced by FK33-824 (16 nmole/100 g body wt, iv) was inhibited by intraventricular (icv) injection of TRH (0.6 nmole/rat). DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate, 0.6 nmole/rat, icv), a TRH analog, also blunted PRL release induced by FK33-824. PRL release induced by a smaller dose of domperidone (1.2 nmole/100 g body wt, iv) was blunted by TRH and DN-1417, whereas both peptides failed to suppress elevated PRL levels induced by larger doses of domperidone. These results suggest that TRH not only stimulates PRL secretion by acting directly at the pituitary, but has an inhibitory action on PRL release through activation of the central dopaminergic mechanism.     

Effect of caerulein on pituitary response to TRH in humans

The effect of caerulein (100 ng/kg/h X 1 h) on basal as well as on thyrotropin-releasing hormone (TRH)-stimulated prolactin and thyroid-stimulating hormone (TSH) secretion was studied in healthy male volunteers. The peptide did not change the basal levels of prolactin and TSH. However, during the infusion of caerulein, prolactin response to TRH was significantly increased whereas the TSH response was decreased. These data, showing an action of caerulein (a frog peptide which mimics the biological actions of cholecystokinin) on prolactin and TSH release, suggest that cholecystokinin may be involved in the physiological control of human pituitary secretion.     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.