Addition Of Thyrotropin-releasing Hormone Research Articles

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action.

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Thyrotropin-releasing hormone reduces the plasma levels of beta-endorphin-like immunoreactivity in rats.

Effects of thyrotropin-releasing hormone (TRH) on the plasma beta-endorphin-like immunoreactivity (beta-Ep-LI) levels in rats have been studied. TRH (10 mg/kg) were injected i.v., and the animals were serially decapitated. The plasma beta-Ep-LI levels were measured by radioimmunoassay. Effects of TRH on beta-Ep-LI release from the anterior pituitary were also investigated by means of an in vitro experiment. The beta-Ep-LI content in the hypothalamus, pituitary gland or adrenal gland did not change significantly after TRH injection. The plasma beta-Ep-LI levels decreased significantly in a dose-related manner with a nadir at 40 min after the injection. In the hypophysectomized rats, the plasma beta-Ep-LI levels were lower than that of the control, and did not change significantly after TRH injection. beta-Ep-LI release from anterior pituitary in vitro significantly decreased after the addition of TRH to medium. These findings suggest that TRH may act on the pituitary gland to reduce plasma beta-Ep-LI levels in rats.

β-Arrestin Mediates Desensitization and Internalization but Does Not Affect Dephosphorylation of the Thyrotropin-releasing Hormone Receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.

A role of thyrotropin-releasing hormone in insulin secretion by isolated rat pancreatic islets

Insulin-secreting pancreatic beta cells also express thyrotropin-releasing hormone (TRH). Although the physiological role of TRH in this localization is unclear, its participation in glucoregulation has been implied. To test this hypothesis, we blocked the last step of post-translational maturation of the TRH molecule by disulfiram, which is an active inhibitor of peptide alpha-amidation (PAM) within pancreatic islet cells. The treatment of male rats with 200 mg/kg/day of disulfiram during a 5-day period resulted in a low PAM activity, a high insulin content and its basal secretion from pancreatic islets, and the inability to release insulin in response to glucose (16.7 mM) or hypo-osmotic (30%) challenge in vitro. The addition of TRH (1 nM) to the medium during incubation restored the insulin content and both basal and glucose stimulated insulin secretions to control levels. TRH plays an important role in the mechanism of insulin secretion and its response to glucose stimulation.

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Is there a role for antenatal TRH therapy for the prevention of neonatal lung disease?

Unfortunately, surfactant therapy is not routinely available to infants in some parts of the world because of its cost. It is the hypothesis of this article that in situations where surfactant is not available, there may be a role for antenatal thyrotropin-releasing hormone (TRH) plus glucocorticoid therapy. Data from randomized clinical trials, which compared therapy with antenatal glucocorticoid plus TRH to that with glucocorticoid alone were extracted and subjected to meta-analysis. The trials that incorporated surfactant therapy were analyzed separately from those in which surfactant was not used. In addition, because surfactant therapy was only available to some patients in the Australian ACTOBAT trial, each group analysis was performed with and without the ACTOBAT data. A characteristic of the earlier presurfactant trials is that few were designed for "intention to treat" analysis. In most of these studies, it was decided a priori to include babies who delivered within a specified time period after hormone therapy. The addition of TRH did not decrease respiratory distress syndrome in those trials in which surfactant therapy was used. In the presurfactant trials, respiratory distress syndrome was significantly decreased when "intention to treat" data were examined, as well as in those infants who delivered between 1 and 10 days after maternal therapy. There was also a significant decrease in oxygen dependency at 28 days after birth, and in oxygen dependency or death at this time, in those infants who delivered 1 to 10 days after treatment. Antenatal TRH had no significant effect of on neonatal complications such as air leak, intraventricular hemmorhage, patent ductus arteriosus, retinopathy of prematurity, or necrotizing enterocolitis. However, TRH did produce transient suppression of the pituitary thyroid axis. There were also a variety of transient complications in the mothers, including nausea, vomiting or flushing, light-headed feeling, and increased blood pressure. The authors conclude that the implementation of appropriate antenatal glucocorticoid treatment is the first priority. Once this has been established, the data presented here suggest that addition of antenatal TRH should be considered in those situations where surfactant is not available.

Visualization of Agonist-induced Association and Trafficking of Green Fluorescent Protein-tagged Forms of Both β-Arrestin-1 and the Thyrotropin-releasing Hormone Receptor-1

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.

Modulation of human erg K+ channel gating by activation of a G protein-coupled receptor and protein kinase C.

1. Modulation of the human ether-à-go-go-related gene (HERG) K+ channel was studied in two-electrode voltage-clamped Xenopus oocytes co-expressing the channel protein and the thyrotropin-releasing hormone (TRH) receptor. 2. Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization-induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH. 3. The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+. 4. The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with beta-phorbol 12-myristate, 13-acetate (PMA). The TRH-induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA-dependent regulation of the channels. 5. It is concluded that a PKC-dependent pathway links G protein-coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C-activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.

Prenatal hormones alter antioxidant enzymes and lung histology in rats with congenital diaphragmatic hernia.

Prenatal administration of dexamethasone (Dex) and thyrotropin-releasing hormone (TRH) synergistically enhances lung maturity, but TRH suppresses the antioxidant enzyme activity. Prenatal hormonal therapy improves alveolar surfactant content and lung compliance in rats with congenital diaphragmatic hernia (CDH). In full term neonatal rats with CDH we studied the effects of prenatal Dex or Dex+TRH on antioxidant enzyme activity at birth, on survival, and on lung morphometry after 4 h of ventilation with 100% O2. CDH was induced by administration of 2,4-dichlorophenyl-p-nitro-phenylether (Nitrofen) on gestational day 10. Dex+TRH-treated CDH rats had lower activity of glutathione reductase after birth than did sham-treated CDH pups. Dex-treated and sham-treated pups had similar antioxidant enzyme activity. Hormonal treatment did not change survival during ventilation. The average airspace volume increased in Dex-treated CDH pups after ventilation, with a small synergistic effect after addition of TRH. On the basis of our findings, we speculate that prenatal administration of Dex is the best choice to improve lung maturity and airspace volume in CDH patients.

Dynamics of Prolactin Secretion from Diethylstilbestrol-Induced Rat Prolactinoma Tissue In Vitro

Experiments were performed to determine whether PRL secretion in the rat diethylstilbestrol (DES)-induced prolactinoma model is affected by the addition of thyrotropin-releasing hormone (TRH) and/or immunoneutralization of intrapituitary vasoactive intestinal polypeptide (VIP) in vitro. Male Fischer 344 rats were implanted with either a 10 mg DES or placebo pellet 30 days prior to obtaining the anterior pituitary glands for perifusion. The anterior pituitaries were quartered and used in three different perifusion experiments. In Experiment I, placebo-treated tissue channels were perifused for 2 baseline hr followed consecutively by a 30-min exposure to 1:100 nonimmune rabbit serum (NRS), a 30-min wash, and a final 30-min exposure to 10(-5) M TRH. Additional placebo channels were run as above except 1:100 VIP antiserum (AVIP) was substituted for NRS and AVIP was added to the TRH. In Experiment II, the same perifusion protocol was used as in Experiment I, except DES-induced tumor tissue was used instead of placebo tissue. Results from Experiment I and II reveal that AVIP significantly decreased PRL secretory rate in both DES and placebo groups. In the tumor group, both TRH alone and in the presence of AVIP significantly increased the PRL secretory rate. In Experiment III DES-induced tumor tissue channels were perifused with a similar protocol, except the concentrations of NRS and AVIP were increased to 1:10. Both NRS and AVIP significantly decreased PRL secretory rate; however, AVIP had a significantly greater effect than NRS. In this experiment, 1:10 AVIP overcame the stimulatory effect of TRH. In conclusion, AVIP decreases and TRH increases, even in the presence of AVIP, PRL release in DES-induced prolactinoma tissue in vitro. Increasing the AVIP concentration 10-fold diminished the PRL-releasing action of TRH in the tumor tissue. These data suggested that PRL secretion is not autonomous in these prolactinomas and can be affected by exogenous TRH and partial immunoneutralization of endogenous VIP.

Cyclosporine Effects on in vitro Responsiveness of Anterior Pituitary Hormone Release to Dopamine and Thyrotropin‐Releasing Hormone in Young Female Rats

Endocrine side effects of the immunosuppressive drug cyclosporine (CyA) include changes in anterior pituitary hormone secretion. The aim of the present study was to examine the effects of CyA on the responsiveness of in situ and ectopic anterior pituitary prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) release response to dopamine (DA) and thyrotropin-releasing hormone (TRH) treatment in young female rats, and to evaluate the possible PRL participation in these effects. Thirty day old rats were rendered hyperprolactinemic by transplanting an anterior pituitary gland of a littermate donor, under the kidney capsule, and were then injected with CyA or vehicle for 2 or 8 days. Sham-operated rats were used as controls and treated in the same way. CyA treatment prevented the increase in plasma PRL levels which occurred in controls after pituitary grafting. In vitro basal PRL release of in situ pituitaries from either sham-operated and/or pituitary-grafted animals was decreased by CyA treatment at any point studied. Basal in vitro secretion of GH was only decreased in the in situ pituitaries from grafted animals after 2 days of CyA therapy. The presence of an ectopic pituitary lead to an increase in the in vitro basal LH secretion from in situ pituitaries as compared to those from sham-operated rats. Basal LH release rates were not changed by CyA treatment, although the LH release in vitro did increase in the in situ pituitaries from sham-operated animals treated with the drug for 2 days. DA addition to the incubation media decreased the in vitro release of PRL, GH and LH from the in situ pituitaries of sham-operated and pituitary-grafted animals treated with vehicle. In CyA treated animals, DA decreased in vitro PRL release from the in situ pituitaries of animals, independently of the presence or absence of an ectopic pituitary. Reductions of the in vitro GH and LH release release after DA treatment were higher in the in situ pituitaries from grafted animals on day 8 of CyA or vehicle treatment. TRH increased the in vitro release of the three hormones with differential effects related to the length of the treatment with CyA and/or the presence of an ectopic pituitary. In vitro release of PRL and GH by ectopic pituitaries was inhibited by previous treatment with CyA and this effect was decreased proportional to the duration of the treatment with the drug, while LH secretion was not modified. Addition of DA to the incubation media resulted in a marked reduction of in vitro PRL and GH release, but only at day 8 of vehicle treatment on GH release did DA addition to media further decrease the release of both hormones from ectopic pituitaries from animals treated for 2 or 8 days with the drug, whereas LH secretion was not modified. TRH addition to the incubation media of ectopic pituitaries surprisingly reduced PRL and GH secretion on day 8 of CyA treatment or after surgery. The results of these studies suggest that CyA can act directly at the hypophyseal level modifying pituitary responsiveness to external stimuli. CyA seems to exert its main effects on lactotroph activity while its effects on somatotrophs and gonadotrophs are less.

Australian collaborative trial of antenatal thyrotropin-releasing hormone (ACTOBAT) for prevention of neonatal respiratory disease

The addition of thyrotropin-releasing hormone (TRH) to antenatal glucocorticoid treatment of women at risk of preterm delivery has been reported to lower the risk of respiratory distress syndrome (RDS) in the infant. We have assessed the efficacy of 200 μg TRH in a multicentre randomised trial. 1234 women at 24 weeks to 31 weeks 6 days of gestation with a singleton or twin pregnancy and at risk of preterm delivery were randomly allocated to groups receiving 200 μg TRH or placebo intravenously every 12 h up to a maximum of four doses. Randomisation was stratified by duration of gestation and centre. All women received glucocorticoids. Clinical outcome is known for 1231 women and their 1397 infants. The frequencies of the main prespecified study outcomes RDS (relative risk 1·17 [95% Cl 1·00-1·36], p=0·05) and need for ventilation (1·15 [1·01-1·31], p=0·04) were higher in TRH-group infants than in control infants. The excess risk in the TRH group was greater in infants who were born more than 10 days after treatment. Multivariate analysis adjusting for duration of gestation at randomisation, time from randomisation to delivery, parity, history of perinatal death, and infant's sex did not affect the risk estimates. TRH administration was associated with increased risks of maternal nausea, vomiting, lightheadedness, and a rise in blood pressure to 140/90 mm Hg or higher. Antenatal TRH given with glucocorticoids to women at high risk of preterm delivery is associated with maternal and perinatal risks and cannot be recommended for widespread clinical use.

Okadaic acid and calyculin A enhance the effect of thyrotropin-releasing hormone on GH 3 rat anterior pituitary cells excitability

Thyrotropin-releasing hormone (TRH) causes a transient hyperpolarization followed by several minutes of increased action potential frequency in patch-perforated current-clamped GH 3 cells. Treatment of cells for 5 min with either 2 or 100 nM of the protein phosphatase inhibitor okadaic acid does not affect electrical activity of the cells, but potentiates the enhancement of action potential frequency elicited by a subsequent addition of TRH. Alternatively, 100 nM (but not 2 nM) of okadaic acid added during the second phase of TRH action, further increases the frequency of firing above that produced by the hormone. Similar effects to those of 2 nM okadaic acid are observed with 20 nM calyculin A. These data suggest that a protein phosphatase plays a major role in regulating the delayed effects of TRH on cell excitability in GH 3 cells.

Thyrotropin-releasing hormone-induced cytosolic calcium transients: Characterisation of store refilling in bovine anterior pituitary cells

The intracellular calcium ion concentration ([Ca 2+] i) in individual bovine anterior pituitary cells was measured using fura-2 and ratiometric imaging. Addition of thyrotropin-releasing hormone (TRH) in the presence of external calcium ion ([Ca 2+] e; 1 mM) caused a rapid transient increase in [Ca 2+] i falling to a plateau which remained above pre-stimulation levels in the continued presence of TRH. Decreasing [Ca 2+] e to 0.1 μM decreased [Ca 2+] i. At 0.1 μM [Ca 2+] e, the first TRH addition caused the rapid transient rise in [Ca 2+] i but no plateau phase and a second addition of TRH did not cause a second transient rise. However, the second application of TRH in 0.1 μM [Ca 2+] e caused a rise in [Ca 2+] i if it was preceded by transient exposure of the cells to 2 mM [Ca 2+] e. The presence of nitrendipine, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ), or TRH during the re-exposure to external calcium blocked this recovery of subsequent responses to TRH in the presence of only 0.1 μM [Ca 2+] e. We conclude that refilling of the calcium stores depleted by TRH occurred only after the removal of agonist, used a tBHQ-sensitive uptake mechanism, and was mainly sustained by voltage-gated calcium entry into the cells.

Neuropeptides can interfere with the recording properties of voltammetric carbon fibre electrodes

The effects of neuropeptides on the recording properties of carbon fibre micro-electrodes used with differential pulse voltammetry were examined both in vitro and in vivo. The in vitro voltammetric signal recorded in a solution containing 3,4-dihydroxyphenylacetic acid (10 −4 M) was attenuated after the addition of thyrotropin releasing hormone, neuromedin N and neurotensin (10 −5 M). The administration of neurotensin (1 or 3 μg/μl) into the nucleus accumbens adjacent to the carbon fibre electrode produced a decrease in the extracellular 3,4-dihydroxyphenylacetic acid peak. However, evidence was obtained that peptides alter the recording properties of the microelectrodes when present at sufficient concentrations and such an effect may explain the result obtained in vivo. In contrast, neurotensin administered into the ventral tegmental area increased the 3,4-dihydroxyphenylacetic acid peak in the nucleus accumbens as previously reported. Thus, attenuation of the voltammetric signal was caused by an effect of the peptide on the recording properties of the electrode rather than real diminution of the free oxidisable amine around the electrode. Caution should be taken when investigating the effects of peptides on extracellular amines or their metabolites using in vivo voltammetry with carbon fibre electrodes if the peptides are administered in high concentrations close to the electrode.

Transient Dopamine Withdrawal Differentially Potentiates Thyrotropin Releasing Hormone-Induced Release of Prolactin of Various Ages

Transient removal of dopamine (DA) potentiates the prolactin (PRL) releasing action of thyrotropin releasing hormone (TRH) in cultured lactotrophs. We have determined if this potentiating action on PRL release is related to the intracellular processing of PRL by studying this phenomenon in cells in which PRL was pulse-labeled at varying times. Anterior pituitaries from ovariectomized estradiol-treated rats were dispersed and cultured on plastic coverslips. Cells were incubated for 24 h in media containing 500 nM DA. Cells were exposed to three consecutive 20-min incubation periods. During the first period all cells were maintained in 500 nM DA, during the second period half of the cells were removed from DA inhibition and during the third period DA inhibition was restored and the cells challenged with 100 nM TRH. In experiments involving labeling of PRL, cells were incubated in leucine-free media containing 100 microCi/ml [3H] leucine for 1 h prior to treatment, for 1 h starting 5 h prior to treatment or for 16 h starting 24 h prior to treatment. Total PRL (by radioimmunoassay) and radiolabeled PRL (by liquid scintillation after electrophoretic isolation) were determined both in media and cells. The withdrawal of DA alone resulted in a 2-fold stimulation of total PRL release. The efficacy of TRH to release total PRL was also increased 2- to 4-fold after transient DA withdrawal. Whereas release of 1-hour-old labeled PRL was not affected by DA withdrawal, 5-hour-old and 8- to 24-hour-old [3H] PRLs were stimulated 2.6- and 2-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of thyrotropin-releasing hormone and potassium depolarization on phosphoinositide metabolism and cytoplasmic calcium in bovine pituitary cells

Addition of thyrotropin-releasing hormone (TRH) (10 nM to 10 μM) to bovine anterior pituitary cells labelled with [ 3H]inositol decreased the radioactivity in inositol-containing lipids and increased it in inositol phosphates. TRH also increased the cytoplasmic calcium concentration biphysically. At TRH concentrations below 10 nM, the increase was sustained and sensitive to inhibitors of calcium influx through voltage-gated channels, whereas concentrations over 10 nM elicited in addition a rapid transient increase in calcium, which was relatively insensitive to such inhibition. Incubation of the cells in medium containing 25 mM KCl increased the cytoplasmic calcium concentration by stimulating influx through voltage-gated channels, and markedly enhanced the initial transient increase of calcium seen at TRH concentrations above 10 nM. It did not affect the generation of Ins P 3 and it also enhanced the calcium response to ionomycin. It is suggested that stimulation of calcium entry through voltage-gated channels can increase the amount of calcium available for mobilisation by TRH.

Dopamine Withdrawal and Addition of Thyrotropin-Releasing Hormone Stimulate Membrane Translocation of Protein Kinase-C and Phosphorylation of an Endogenous 80K Substrate in Enriched Lactotrophs*

The biochemical mechanisms underlying the direct stimulatory action of dopamine (DA) withdrawal on PRL release and on the potentiation of TRH stimulation are not known. These actions can be mimicked by pretreatment of lactotrophs with the protein kinase-C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate. Previous studies have shown that administration of TRH or withdrawal of DA stimulates polyphosphoinositide breakdown, although to different degrees. We have tested whether the acute withdrawal of DA activates PKC and determined if the prior removal of DA modifies the activation of PKC by TRH. Primary cultures of dispersed anterior pituitaries from estradiol-treated rats consisting of approximately 80% lactotrophs were maintained overnight in 500 nM DA. Activation of PKC was assayed immunochemically as translocation of PKC to a membrane fraction and by in situ phosphorylation of an acid-soluble heat-stable 80K substrate. Acute withdrawal of DA induced translocation of immunoreactive PKC to the membrane fraction (25-250%) and enhanced phosphorylation (40-100%) of an 80K protein. These effects were detected within 5-15 sec of DA withdrawal and were prolonged (10-30 min). TRH induced a rapid and transient activation of both parameters. The duration and magnitude of the action of TRH were increased by prior removal of DA. These results are consistent with a role for PKC activation in transduction of the stimulation of PRL release by the withdrawal of DA. The longer lasting activation of PKC may explain at least in part the potentiation of the PRL-releasing action of TRH by the withdrawal of DA.

Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells.

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.

Effect of thyrotropin-releasing hormone on development of the pituitary-thyroid system in fetal rats in organ culture.

Six groups of thyroid glands from 18-day fetal rats were explanted to organ culture for 2 days. In one group, thyroid was cultured alone and in the remaining five groups thyroid was cocultured with pituitaries from fetuses ranging in age from 17 to 21 days. In each of the groups, half of the cultures had thyrotropin-releasing hormone (TRH) added to the medium. Histometric parameters of the thyroid follicle such as diameter and cell height were used as indicators of development of the thyroid gland. When 18-day thyroid was cultured alone, addition of TRH did not accelerate development. When either one 18-day or two 17-day pituitaries were cocultured with thyroid, a significant increase in diameter and cell height was seen. Addition of TRH to the medium induced little or no further change. When the thyroid was cultured with 19- to 21-day pituitaries, a marked increase in thyroid development was observed; and the addition of TRH caused further acceleration in thyroid development. These results suggest that, in organ culture, 17- to 18-day pituitary glands can release some thyrotropin (TSH) with or without additional TRH. Older pituitaries (19- to 21-day) apparently can release an amount of TSH in the presence of TRH that is greater than their own spontaneous TSH secretion.