Synthetic Thyrotropin-releasing Hormone Research Articles

Effects of Opioid Peptides on Thyrotropin-Releasing Hormone Release from the Rat Caecum in Vitro

Effects of opioid peptides (beta-endorphin, dynorphin (1-13). alpha-neoendorphin, beta-neoendorphin, leucine-enkephalin, methionine-enkephalin) on the release of thyrotropin-releasing hormone (TRH) from the rat caecum were studied in vitro. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium). The amount of TRH release from the rat caecum into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was inhibited significantly in a dose-related manner with the addition of opioid peptides. The inhibitory effects of opioid peptides on ir-TRH release from the rat caecum were blocked with an addition of naloxone. The elution profile of acid-methanol-extracts of rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that opioid peptides inhibit TRH release from the rat caecum in vitro.

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human-beta-thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thyrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.

Ontogenetic development of thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of the mallard embryo

Developmental changes of thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of mallard embryos were studied by means of immunocytochemistry (PAP technique). The primary antibody was generated against synthetic TRH. Immunoreactive neurons were first detected in the hypothalamus of 14-day-old embryos. By day 20, increasing numbers of immunoreactive perikarya were observed in the paraventricular nucleus, anterior preoptic region and supraoptic region. Immunoreactive fiber projections were seen in the median eminence as early as embryonic day 20; they occurred also in some extrahypothalamic regions (lateral septum, accumbens nucleus). The number and staining intensity of the cell bodies increased up to hatching, and continued to increase during the first week after hatching.

Innervation of the Pars intermedia and Control of Alpha-Melanotropin Secretion in the Newt

In this study we have investigated the presence of nerve fibers containing dopamine, thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) in the pars intermedia of the crested newt and we have examined the possible effect of these neurohormones on the release of alpha-melanotropin (alpha-MSH) by neurointermediate lobes in vitro. By means of immunohistochemistry, we observed the presence of tyrosine hydroxylase (TH)-immunoreactive fibers in the pars intermedia of the crested newt. Using a specific antiserum to dopamine, these fibers appeared to be mainly dopaminergic in nature. Unlike anurans, urodele amphibians do not exhibit TRH or NPY-like immunoreactivity in the pars intermedia. A perifusion system technique for newt pituitaries was developed to investigate the effect of dopamine, TRH and NPY on alpha-MSH secretion. Administration of increasing concentrations of dopamine (from 10(-9) to 10(-5)M) induced a dose-related inhibition of alpha-MSH release. This inhibitory effect was mimicked by the dopamine agonist apomorphine (10(-6)M). In contrast, the secretory activity of the newt pars intermedia was not affected by administration of synthetic TRH or NPY (up to 10(-7) and 10(-6)M, respectively). These results indicate that the neurotransmitter dopamine likely plays a pivotal role in the regulation of melanotropin secretion in urodele amphibians.

Effects of Acetylcholine on Thyrotropin-Releasing Hormone Release from the Rat Caecum in Vitro

Effects of acetylcholine on the release of thyrotropin-releasing hormone (TRH) from the rat caecum in vitro were studied. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was enhanced significantly in a dose-related manner with the addition of acetylcholine, but not changed with atropine. The stimulatory effect of acetylcholine on ir-TRH release from the rat caecum was blocked with an addition of atropine. Elution profile of acid-methanol-extracted rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that the cholinergic system stimulates TRH release from the rat caecum in vitro.

The effects of TRH on plasma thyroid hormone levels of rainbow trout ( Salmo gairdneri) and arctic charr ( Salvelinus alpinus)

Plasma levels of l-thyroxine (T 4) and 3,5,3′-triiodo- l-thyronine (T 3) were measured in 2- to 4-year-old rainbow trout ( Salmo gairdneri) and arctic charr ( Salvelinus alpinus) intraperitoneally injected with 0.7% NaCl alone (controls) or 0.7% NaCl containing synthetic thyrotropin-releasing hormone (TRH). Blood was obtained from terminally sampled fish or from serially sampled fish cannulated in the dorsal aorta. In trout, TRH (1 μg/g body wt) significantly raised plasma T 4 to maximal values at 2 hr. A dose of 0.1 μg/g was ineffective in trout but doses below this level elevated plasma T 4 in arctic chart. In charr starved for 2 or 3 weeks the plasma T 4 response to TRH appeared blunted. There was no consistent influence of TRH on plasma T 3 under any condition for either species. It is concluded that TRH influences the salmonid thyroid system at some level to elevate plasma T 4, possibly through thyrotroph stimulation.

Interaction of Thyrotropin-Releasing Hormone (TRH) and serotonin in cardiovascular control

Intravenous injection of the synthetic TRH analog, MK-771, to anesthetised cats raised the blood pressure by a central mechanism, i.e. by activating the outflow of sympathetic stimuli from the CNS to the periphery and raising the plasma concentration of adrenaline and noradrenaline. In contrast, noradrenaline in the CSF was lowered by intravenously injected MK-771. The concentration of 5-HT in the CSF was increased and the pressor action of the peptide was correspondingly enhanced by 5-HT antagonists. These findings suggest that the release of 5-HT into the CSF lowers blood pressure and thus reduces the hypertensive effect of MK-771. Methysergide acts synergistically with MK-771 to combat the sudden fall in pressure induced by acute hemorrhage in the anesthetised cat. Combination of the two drugs enhanced and accelerated recovery from this hypotensive state.

Luteinizing hormone-releasing hormone as a potent stimulator of the thyroidal axis in ranid frogs

Plasma concentrations of T 4, measured by radioimmunoassay, were raised significantly 2 and 4 hr after intravenous injection of synthetic luteinizing hormone-releasing hormone (LHRH) in Rana ridibunda (1 and 10 μg on 2 consecutive days) and in Rana esculenta (10 μg). A dose of 1 μg LHRH was not so effective as 50 μg synthetic thyrotropin-releasing hormone (TRH) when injected in Rana ridibunda in November. However 10 μg LHRH was equipotent to 50 μg TRH. In February somewhat less than half of the Rana temporaria group was responsive to LHRH. There is no clear indication that fluctuating plasma T 3 concentrations were caused by LHRH or TRH. Preinjection levels of T 3 and T 4 were higher during the breeding season (April) in R. esculenta (resp. 35.4 ± 1.4 pg/ml; 744 ± 134 pg/ml; n = 22) compared to the basal concentrations in the very closely related Rana ridibunda (November) (resp. 15.2 ± 1.1; 162 ± 24 pg/ml; n = 28). Four days after removal of the pars distalis plasma T 4 concentrations were significantly decreased in Rana esculenta, whereas T 3 could stay longer in circulation. T 3 and T 4 content of the thyroids was not altered by the short-term hypophysectomy. Injection of 10 μg LHRH had no influence on plasma T 4 nor testosterone concentrations in these frogs, contrary to the sham-ectomized animals in which plasma testosterone remained elevated longer than T 4. The results suggest that the stimulatory effect of intravenous injected LHRH on thyroid (and gonadal) activity in the frog is primarily mediated through the hypophysis. They also point to a possible correlation between the gonadal and thyroidal axis.

Effects of calcium hopantenate on the release of thyrotropin-releasing hormone from the rat adrenal gland in vitro.

The effects of calcium hopantenate (HOPA), a GABA agonist, on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of HOPA to the medium. HOPA's effects on TRH release from the adrenal gland were blocked with the addition of bicuculline, a GABA receptor inhibitor. The elution profile of methanol-extracted rat adrenal gland TRH was identical to that for synthetic TRH. The findings suggest that HOPA inhibits TRH release from the rat adrenal gland, and that its effects are mediated via the GABA receptor.

Effects of thyrotropin-releasing hormone in vitro on thyrotropin and prolactin release from the turtle pituitary

Effects of synthetic thyrotropin-releasing hormone (TRH) on thyrotropin (TSH) and prolactin (PRL) release by hemipituitaries of adult turtles, Chrysemys picta, were studied in an in vitro superfusion system. Significant increases in the rates of secretion of both immunoreactive TSH and PRL occurred at doses between 0.01 and 10 ng/ml TRH. TSH secretion increased acutely by two-, to sixford over nonstimulated secretion levels; responses tended to decline after many hours of continual stimulation, but output remained elevated above baseline in most cases. PRL secretion increased, parallel to TSH secretion during TRH stimulation. No significant difference was found in secretion rates between males and females, and no clear relationship between TRH responsiveness and reproductive stage was evident. These data provide the first direct evidence for the stimulation of TSH secretion by TRH in a reptile and confirm earlier reports that TRH stimulates the release of PRL in the turtle. Although previous in vivo studies indicated that TSH secretion was not affected by TRH in turtles, the present data indicate that the dose sensitivity of the chelonian gland is comparable with that of mammalian and avian pituitaries. Evidence for the role of TRH in endogenous TSH regulation is still lacking in reptiles but the present data provide evidence for functional TRH receptors on the chelonian thyrotrope and, hence, argue against the hypothesis that TSH stimulating activity of TRH evolved relatively recently in association with endothermy.

Dopamine inhibits thyrotropin-releasing hormone release from rat adrenal gland in vitro.

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The rat adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of dopamine and enhanced with the addition of pimozide or domperidone to the medium. Dopamine's effects on ir-TRH release from the adrenal gland were blocked with the addition of pimozide or domperidone. The elution profile of methanol-extracted rat adrenal gland was identical to that of synthetic TRH. The findings suggest that the dopaminergic system inhibits TRH release from the rat adrenal gland.

Interaction of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release in chickens.

The effects of synthetic somatostatin (SRIF) on serum growth hormone (GH) concentrations stimulated by exogenous administration of synthetic thyrotropin-releasing hormone (TRH) and/or human pancreatic GH-releasing factor (hpGRF) were investigated in 4-week-old cockerels. In addition, the additive effects of TRH and hpGRF on serum GH were examined. TRH and hpGRF, when given in combination intravenously, produced an additive effect on serum GH concentration that peaked 10 min after the injection. The somatostatin did not significantly affect basal GH concentrations when given alone, but did significantly decrease the magnitude of the GH response to hpGRF. In contrast, SRIF did not significantly decrease the stimulatory effects of TRH on GH release. These results suggest that TRH and hpGRF are potent GH releasers in vivo and that their stimulating effects on GH release are additive, suggesting different mechanisms for their stimulation. The results obtained from the combination studies suggest that the main site of the stimulatory action of hpGRF is at the pituitary, and that SRIF significantly inhibited the rise in serum GH induced by a synthetic hpGRF, but not that induced by TRH.

Concentrations of thyrotropin-releasing hormone in the brain of ataxic mice

Concentrations of thyrotropin-releasing hormone (TRH) were studied in the brain of the Weaver ataxic mouse, the Purkinje cell degenerative ataxic mouse (pcd-ataxic mouse) and the cytosine arabinoside (ara-C)-induced ataxic mouse. The brain tissue was dissected into 4 parts, e.g., hypothalamus, cerebrum, cerebellum and brain stem. TRH concentrations in each part of the brain were measured by radio-immunoassay. TRH concentrations in the brain of Weaver ataxic mice were significantly higher in the cerebellum and brain stem than in the controls. In pcd-ataxic mice, the TRH concentrations in the brain were significantly higher in the cerebrum and brain stem. In ara-C-induced ataxic mice, the concentrations were significantly higher in the cerebrum, cerebellum and brain stem. TRH levels in the hypothalamus of ataxic mice did not differ from those of controls. The elution profile of methanol-extracted cerebellum of ataxic mice on Sephadex G-10 was identical to that of synthetic TRH. These findings suggest that changes in the TRH concentrations in the brain play a pathophysiological role in ataxic mice.

Ascorbic acid increases the thyrotropin-releasing hormone content of hypothalamic cell cultures

Thyrotropin-releasing hormone (TRH) is one of many COOH-terminal alpha-amidated neuropeptides. Recent work with the intermediate pituitary has indicated that ascorbate is a required cofactor for the COOH-terminal alpha-amidation of alpha-melanotropin. This is consistent with the ascorbate requirement of an enzyme found in pituitary and hypothalamus capable of converting peptides with a COOH-terminal glycine (-X-Gly) to alpha-amidated molecules (-H-NH2). Thus, it has been proposed that COOH-terminal glycine-extended TRH (TRH-Gly) may be the direct precursor to TRH. In the present study, primary hypothalamic cultures supplemented with ascorbate for 7 d contained two- to threefold more TRH immunoactivity (amide-specific) than cultures maintained without ascorbate. A dose-response experiment indicated that 20 microM ascorbate was capable of producing 50% of the maximum observable increase in culture TRH immunoactivity; this concentration is similar to the Km value for ascorbate uptake obtained in adrenal chromaffin and pituitary cells. A stereoisomer of ascorbate, D-isoascorbate, was also capable of producing an increase in TRH immunoactivity, but oxidized ascorbate was not. Recent studies have shown that the amidation enzyme from pituitary is capable of utilizing both L-ascorbate and D-isoascorbate but is incapable of utilizing oxidized ascorbate. The culture extracts were analyzed further by reversed-phase high-performance liquid chromatography; the increased TRH immunoactivity observed in extracts of cultures maintained in ascorbate comigrated with standard synthetic TRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Various types of thyrotropin-releasing hormone receptors in discrete brain regions and the pituitary of the rat.

Receptors for thyrotropin-releasing hormone (TRH) in the rat brain and the pituitary are heterogenous. The receptors were classified into four types according to the dissociation constant (KD). High-affinity receptors (KD less than 3 nM) are present in the pituitary, hypothalamus, amygdala, and limbic forebrain which contains the nucleus accumbens and the septum. Intermediate-affinity receptors (KD, 5-16 nM) are evidently present in the frontal cortex, hippocampus, striatum, thalamus, and the brainstem, but may also be present in other regions. Low-affinity TRH receptors (KD, 50-80 nM) are seen in the limbic forebrain, amygdala, and the hypothalamus. Very-low-affinity receptors (KD, 215 nM) exist in the pituitary. Experiments using DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate), a synthetic TRH analogue with a more potent central activity, indicated the presence of TRH receptors having a high affinity to DN-1417 at least in the limbic forebrain but not in the pituitary. This type of receptor is not labeled by [3H](3-methyl-histidine2)-TRH. Density of the TRH receptor is the highest in the pituitary and next highest in the amygdala.

Mechanisms of release of prolactin from fowl anterior pituitary glands incubated in vitro: effects of calcium and cyclic adenosine monophosphate.

Fowl anterior pituitary glands were bisected and each half was pretreated in either Medium 199 or medium containing EGTA to deplete endogenous calcium (Ca2+) stores, after which they were incubated in Medium 199, or Ca2+-free medium, containing prolactin release-stimulating agents and verapamil, a Ca2+ channel blocker. High K+ concentrations, hypothalamic extract, synthetic thyrotrophin-releasing hormone (TRH) and dibutyryl cyclic AMP (dbcAMP) all stimulated release of prolactin from control (non EGTA-treated) hemianterior pituitary glands. The effects of TRH and dbcAMP were not additive, but the response to submaximal concentrations of TRH was augmented by theophylline, a phosphodiesterase inhibitor. Reduction of Ca2+ availability with EGTA or verapamil reduced basal release of prolactin, prevented the prolactin-stimulating effects of high K+ concentrations and TRH, and markedly attenuated responses to hypothalamic extract and dbcAMP, EGTA being more effective than verapamil. Increasing the Ca2+ concentration of the medium did not augment basal or stimulated release of prolactin. These results suggest that both Ca2+ and cyclic AMP may act as intracellular mediators in the release of prolactin. Both basal and stimulated release of prolactin depend upon the presence of Ca2+. Although influx from the medium may be the major source of Ca2+, endogenous stores of Ca2+, perhaps mobilized by dbcAMP, may be able to maintain some release of prolactin. The prolactin-stimulating effects of TRH may be mediated by cyclic AMP.

Thyrotropin and gonadotropin releasing hormones (TRH and GnRH) do not alter levels of oxytocin and oxytocin does not change the response of luteinizing or follicle stimulating hormones to GnRH in humans.

Four hundred micrograms of synthetic thyrotropin releasing hormone (TRH) were given intravenously to 4 normal men and 4 normal women, and four weeks later, 1000 micrograms of TRH were administered intravenously to 4 of the 8 individuals and oxytocin (OT) was measured in plasma on both occasions. Following injection of either dose of TRH, OT did not change significantly from baseline. Likewise, synthetic gonadotropin releasing hormone (GnRH), 100 micrograms, administered intravenously to 6 normal men did not alter the levels of OT from baseline. Synthetic OT, 300 mU/minute, administered 30 minutes before and for 90 minutes after injection of GnRH, was without effect on the GnRH-induced rise of luteinizing hormone (LH) or follicle stimulating hormone (FSH) in normal men. We conclude that continuous infusion of OT in pharmacologic concentrations does not alter the pituitary release of LH or FSH in response to GnRH in humans. TRH and GnRH given intravenously do not alter basal levels of OT in the plasma of humans, thus a physiologic role for GnRH or TRH in the neuroendocrine control of OT secretion in humans is unlikely.

Effect of DN-1417, a synthetic thyrotropin-releasing hormone analogue, on [3H]GABA binding in the cerebellum of ataxic rats

Effect of DN-1417, a synthetic thyrotropin-releasing hormone analogue, on [3H]GABA binding in the cerebellum of ataxic rats

Effect of DN-1417, a synthetic thyrotropin-releasing hormone analogue, on [ 3H]GABA binding in the cerebellum of ataxic rats

The neonatal administration of cytocine arabinoside (40 mg/kg, s.c.) induced marked ataxia and hypoplastic cerebellum in male Sprague-Dawley rats. Specific [ 3H]GABA binding in the cerebellum was significantly reduced by cytosine arabinoside, whereas [ 3H]GABA binding in the cerebral cortex was not changed. The administration of DN-1417 (1 mg/100 g body wt., i.p.), a synthetic derivative of thyrotropin-releasing hormone (TRH), significantly increased [ 3H]GABA binding in the cerebellum of cytosine arabinoside-induced ataxic rats. These results suggest that TRH may play a role in regulating GABA receptors involved in the function of the rat cerebellum.

Reversal of leukotriene D 4 hypotension by thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) reversed the hypotension produced by leukotriene D 4 (LTD 4) (5 μg/kg, i.v.) in conscious guinea pigs in a dose-dependent fashion at intravenous doses between 0.2 and 2.0 mg/kg. LTD 4 hypotension was also reversed by the synthetic TRH analog MK771 (0.2 or 2 mg/kg, i.v.), suggesting a possible receptor-mediated mechanism. Since LTD 4 has been implicated as a mediator of anaphylaxis, these results provide a basis for further evaluation of TRH and its analogs in the treatment of anaphylactic shock.