Neuropharmacological evaluation of RX 77368--a stabilised analogue of thyrotropin-releasing hormone (TRH)

L-Pyroglutamyl-L-histidyl-L-2,3-dimethylprolineamide (Pyr-His-Dmp . NH2; RX 77368) a stabilised analogue of thyrotropin-releasing hormone (TRH) has been examined for neuropharmacological effects in animal tests. The compound was more potent than either TRH or clinically established drugs in four animal tests of antidepressant potential (reserpine reversal, clonidine antagonism, tremorine reversal and learned immobility). RX 77368 also antagonised barbiturate sleeping time. Given by itself to rats the peptide produced arousal as characterised by EEG and EMG measurements and delayed the onset of sleep. The arousal induced was not accompanied by increases in locomotor activity. The profile of pharmacological activity for RX 77368 did not correspond to the profiles of tricyclic antidepressants, psychic-stimulants or analeptic drugs. The possible clinical uses for such a molecule are discussed.     

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Phosphatidic acid and phospholipase D both stimulate phosphoinositide turnover in cultured human keratinocytes

Phosphatidic acid (PA) induced a rapid dose-dependent increase in production of inositol phosphates in cultured adult human keratinocytes, peaking at 30 s. Natural and dioleoyl PA were equally effective, while other phospholipid classes had no effect. Lipid A was also active. Lyso-PA also induced inositol phosphate production, but contamination of the PA preparation by lyso-PA could not account for the effect of PA. The effect of PA could not be reproduced by treatment of cells with calcium ionophore. PA-induced inositol phosphate production could be inhibited (> 50%) by pre-treatment of cells with either pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate, suggesting the involvement of a GTP-binding protein and a protein kinase C-mediated negative feedback mechanism. PA also stimulated release of arachidonic acid from keratinocytes. Treatment of cells with exogenous phospholipase D similarly induced inositol phosphate production in the keratinocytes. Since PA may be formed by receptor-mediated activation of phospholipase D, or by phosphorylation of diacylglycerol, the results suggest that PA may play a significant role in signalling mechanisms of human keratinocytes.     

促甲状腺激素释放激素的分布及生理作用

促甲状腺激素释放激素(TRH)广泛分布于中枢神经系统和某些外周器官中,它除了有促进垂体前叶释放TSH和PRL等内分泌作用外,作为神经递质或神经调质,对中枢神经系统还可产生广泛的生理效应。     

Extrahypothalamic thyrotropin releasing hormone (TRH) -- its distribution and its functions.

Thyrotropin releasing hormone (TRH) is distributed throughout the extrahypothalamic nervous system and spinal cord, in the retina, in the pancreas and gastrointestinal tract, in the placenta, in amniotic fluid, in the adrenals and in frog skin. TRH has been shown to have a variety of effects in the central nervous system, both on isolated neurones and in a number of in vivo situations. TRH interacts with endogenous and exogenous opiates and it has been suggested that endogenous TRH may mediate part of the opiate withdrawal syndrome. The presence of TRH in the retina suggests the possibility that TRH plays a role in the visual process. TRH appears to be integrally related to central thermoregulatory mechanisms. The role of TRH in psychiatric disorders is at present controversial. Recent studies suggest a role for TRH as a modulator of gastrointestinal and pancreatic function. The gastrointestinal actions of TRH include inhibition of gastric acid secretion and alterations in gastic motility. The high concentrations of TRH in the neonatal pancreas suggest a role for TRH in the early development of the pancreas. One of the metabolites of TRH histidyl-proline diketopiperazone, appears to have a number of extrahypothalamic actions and this suggests the need for further exploration of the affects of this compound both on the central nervous system and the gastrointestinal tract. The multiple extrahypothalamic actions of TRH have led to the concept that it is an ubiquitous neurotransmitter that has been co-opted by the pituitary as a releasing factor.     

Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain

Characteristics of TRH-receptors were studied in the rat central nervous system (CNS). Ion species, pH and temperature importantly influenced TRH-receptor binding. Subcellular distribution of TRH-receptor binding revealed that synaptic membranes had the greatest percentage of total sites. Scatchard analysis suggested that the rat CNS had two distinct TRH binding sites with apparent dissociation constants (Kd) of 5 X 10(09) M and 13 X 10(-8) M. Receptor activity is sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for the binding of [3H]TRH. Thiol reagents reduced the binding activity of the receptor, suggesting that an intrachain disulfide bond may form an important constituent of the binding site for TRH. The TRH-receptor in the rat brain was successfully solubilized with non-ionic detergent Triton X-100. On gel chromatography with Sepharose 6B column, the solubilized TRH-receptor molecule eluted at the fraction corresponding to an apparent molecular weight of 300,000 daltons, with Stokes' radius of 5.8 nm. The regional distribution of TRH-receptor binding was examined to clarify the site of TRH action. The highest level of binding was in the hypothalamus, cerebral cortex and hippocampus, indicating that TRH affects the CNS function mainly through the limbic system, cerebral cortex and hypothalamus. Moreover, tricyclic anti-depressants and Li+ decreased the binding of [3H]TRH. These findings suggest that endogenous TRH and TRH receptor may play the role of a neurotransmission modulator in the brain to control emotional and mental functions.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Influence of prostaglandins and thyrotropin releasing hormone (TRH) on hormone secretion and growth in wether lambs.

A series of experiments were conducted in ewes and whether (castrate male) lambs to evaluate the influence of prostaglandins on secretion of anabolic hormones and to determine if repeated injections of prostaglandin (PG) F2alpha would chronically influence the secretion of these hormones and perhaps growth rate as well. A single intravenous injection of PGA1 and PGB1 (100 microgram/kg) exerted no significant (P greater than .10) influence on plasma concentrations of prolactin (PRL), growth hormone (GH) or thyrotropin (TSH) in ewes. PGA1, but not PGB1, stimulated an increase in the plasma concentration of insulin. Infusion of PGF2alpha for 5.5 hr into ewes resulted in increased (P less than .05) plasma concentrations of both GH and ARL while TSH and insulin were not significantly influenced. Prostaglandin F2alpha, when injected subcutaneously into wether lambs (10 mg twice weekly) stimulated (P less than .05) plasma GH concentrations after the first injection, but not after 3 weeks of treatment. Changes in plasma PRL or TSH were not observed consistently in the lambs treated chronically with PGF2alpha or TRH. Prostaglandin F2alpha, in the present studies, and PGE1 in previously reported studies (1-3), has been demonstrated to be stimulatory to the secretion of PRL and GH. In contrast, PGA1 and PGB1, which lack an 11-hydroxyl group, failed to influence the secretion of either PRL or GH. It would, therefore, appear that the 11-hydroxyl group is a structural requirement for prostaglandins to influence the secretion of these two hormones in sheep. Treatment with thyrotropin releasing hormone (TRH), alone or in combination with PGF 2alpha, significantly (P less than .05) increased growth rate (average daily gains) while PGF2alpha did not, despite the fact that both compounds exerted similar effects on plasma GH.     

Thyrotropin releasing hormine (TRH): distribution in the brain, blood and urine of the rat

Measurement of thyrotropin releasing hormone (TRH) in the rat by a radioimmunoassay capable of detecting 6 pg is described. TRH was found in high concentration in the hypothalamus, especially in the stalk median eminence (SME). Small but significant concentrations were also detected hroughout the extrahypothalmic brain. Quantitatively, these levels are substantial, and suggest that this tripeptide may have an extrathyroidal brain function. TRH was measurable in the blood only in low concentrations, but large amounts were excreted in the urine (18.4ng/day).     

The effects of thyrotropin releasing hormone (TRH) on the gastrointestinal tract.

Thyrotropin-releasing hormone (TRH) immunoreactivity is distributed throughout the gastrointestinal tract and the pancreas. We have studied the effect of TRH on several gastrointestinal functions in intact, unanesthetized dogs. Intravenous TRH stimulated gastric action potentials (p<0.01) and transiently inhibited tetragastrin-stimulated gastric acid secretion (p<0.05). TRH had no effect on basal or secretin-stimulated pancreatic exocrine secretion. TRH did not alter water absorption in dogs with Thiry-Vella loops constructed from proximal jejunum.     

Thyrotropin-releasing hormone (TRH) acts centrally to stimulate gastric contractility in rats

Changes in gastric contractility induced by intracisternal (ic) injection of thyrotropin-releasing hormone (TRH) or a stable TRH analog, RX77368 [p-Glu-His-(3,3'-dimethyl)-Pro NH2] were investigated in 24 h fasted-conscious rats. Gastric contractility was monitored using chronically implanted extraluminal force transducers sutured to the corpus. Response elicited by a standard meal was used as a physiologic standard. Intracisternal injection of TRH (1 microgram) or RX77368 (100 ng), unlike saline, stimulated high amplitude gastric contractions. The stimulation of gastric contractions induced by ic RX77368 was dose dependent (3-100 ng), rapid in onset, long lasting and not mimicked by the intravenous route of administration. Atropine (0.1 mg/kg) partially antagonized and vagotomy totally blocked the RX77368 (100 ng, ic)-induced stimulation of gastric contractility. These results demonstrated that TRH or RX77368 acts within the brain to elicit potent contractions of the stomach; TRH action appears vagally mediated probably through cholinergic mechanism.     

Thyroid hormone response to thyrotrophin releasing hormone (TRH) in the sex-linked dwarf chicken

The effect of an injection of thyrotrophin releasing hormone (TRH) on plasma levels of thyroid hormones was studied in dwarf and normal Rhode Island Red chickens with similar genotypes other than for the sex-linked dwarf gene dw. The sex-linked dwarf chickens had different plasma iodothyronine levels from control normal chickens: high thyroxine (T4), low triiodothyronine (T3) and similar reverse T3 (rT3) levels. The injection of TRH (10 micrograms/kg) in 5-day- and 5-week-old normal chickens increased the plasma T4 within 30 min without a significant increase in T3, whereas the injection of TRH in 11-and 26-week-old normal chickens increased plasma T3 60 min later. In dwarfs the response of T4 to TRH was the same as that in normals but no increased T3 response was observed. The plasma level of rT3 was not influenced by the TRH injection in either strain. These results suggest that although in the sex-linked dwarfs thyroidal response to exogenous TRH is similar to that of normals, the dwarf gene dw inhibits the conversion of T4 to T3 in peripheral tissues without any inhibitory effect on rT3 production.     

Lack of effect of thyrotropin releasing hormone (TRH) in circulatory shock

Thyrotropin releasing hormone (TRH) has been reported to reverse hypotension induced by a variety of agents and thus it has been suggested to be of therapeutic value in circulatory shock. We have investigated TRH (2 mg/kg bolus plus 2 mg/kg/hr infusion) in both hemorrhagic (cats) and traumatic shock (rats). TRH induced a pressor effect of 23 +/- 8 mm Hg (p less than 0.05) in cats and 19 +/- 3 mm Hg (p less than 0.01) in rats during hypotension. However, this transient (10-15 min) response did not result in any sustained improvement in the cardiovascular status of the animals in either shock model when compared to the vehicle. In addition, TRH did not attenuate any of the biochemical indices of the severity of the shock state (i.e., plasma amino-nitrogen concentrations, or plasma cathepsin D and MDF activities) nor did it improve survival time in traumatic shock (2.8 +/- 0.4 vs. 2.0 +/- 0.2 hours). Furthermore, TRH resulted in a significant blunting of the maximum post-reinfusion superior mesenteric artery flow and enhanced beta-glucuronidase release from liver lysosomal preparations in vitro. These potentially detrimental effects in conjunction with the lack of any overt protective effect under the conditions existing in these two shock models, do not provide evidence that TRH is beneficial as a therapeutic agent in circulatory shock.     

Potential anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues

The anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues (DN-1417: gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; MK-771: L-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) were examined in behavioral despair rats, an animal model of depression. TRH, DN-1417, MK-771, amitriptyline and diazepam were injected three times after the first forced swimming. One hr after the last injection, a 5-min swimming test was performed. Experimental animals were placed in a Hall's type open-field apparatus immediately before and after the 5-min test, and their locomotor activities were determined. No significant difference was noted in the locomotor activity immediately before the 5-min test among any group. In the 5-min swimming test, TRH, DN-1417 and MK-771 caused a dose-dependent decrease in immobility, showing an anti-depressive effect similar to amitriptyline. Diazepam showed no difference compared with the control group. After the swimming test, locomotor activity remarkably decreased in the control rats, while decreased locomotor activity was partially prevented in the TRH, DN-1417, MK-771 and amitriptyline treated rats which exhibited active movement not only during the swimming period but also after it. In terms of the minimum effective dose, TRH and DN-1417 seemed to be of similar potency, while MK-771 was 40-fold stronger than TRH. An examination of a possible correlation between the cross-reactivity of TRH analogues in a radioreceptor assay and the effects of the analogues on despair rats suggested that the structure-binding relationship was proportional to the structure-activity relationship.     

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.     

Increase in muscarinic receptors in rat intestine by thyrotropin releasing hormone (TRH)

The effect of Thyrotropin Releasing Hormone (TRH) on the contractile activity elicited by acetylcholine and electric stimulation in the rat ileus terminalis was investigated. TRH did not show any intrinsic contractile activity but, after a 30 minute latency period, the peptide caused a shift to the left of the dose-response curve for both acetylcholine and electric stimulation. The binding of 3H-quinuclidinylbenzilate (3H-QNB) assayed on ileum slices disclosed that the addition of TRH increased the number of muscarinic cholinergic receptors without changes in affinity when incubation was performed at pH 7.8, but no effect TRH was demonstrated at pH 7.4. Therefore, in spite of its neural and direct actions on intestine motor activity, TRH may affect the acetylcholine induced contraction by increasing the number of muscarinic receptors at a specific pH.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.