Effect of JTP-2942, a novel thyrotropin-releasing hormone analog, on motor deficits after chronic focal cerebral ischemia in rats.
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To investigate the chronic effects of a novel thyrotropin-releasing hormone analog, JTP-2942 (N(alpha)-[(1S, 2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamide monohydrate), on behavioral changes after stroke, the authors examined its effects on motor and neurologic deficits using a middle cerebral artery (MCA) occlusion model in rats. A left MCA was permanently occluded at a proximal site. From 1 week after occlusion, JTP-2942 was intravenously administered once a day for 4 weeks. Sensorimotor performance was evaluated weekly for 10 weeks after the occlusion. The ability of the rat to maintain its body position on an inclined plane and neurologic examination based on hemiparesis and abnormal posture were examined. After all behavioral examinations were completed, the degree of shrinkage of the left hemisphere was measured. The ability of MCA-occluded rats to maintain body position on an inclined plane in the left-headed position was significantly lower than that of sham-operated rats throughout the test period. JTP-2942 gradually improved this deficit dose dependently, and a dose of 0.03 mg/kg of JTP-2942 significantly improved performance to the levels of the sham-operated rats. Neurologic deficits were also observed in MCA-occluded rats. JTP-2942 also significantly improved these deficits dose dependently. On the other hand, CDP-choline (500 mg/kg, administered intravenously), a therapeutic agent for the disturbance of consciousness and hemiparesis after stroke, improved neurologic deficits but did not affect the motor deficits measured using the inclined plane. It is noteworthy that the effects of JTP-2942 on these deficits were observed 4 weeks after cessation of drug administration. Furthermore, there was no difference in the degree of shrinkage of the cerebrum among the MCA-occluded groups. In the present study, long-lasting improving effects of JTP-2942 on the impairment of motor and neurologic functions were observed in rats with MCA occlusion, which continued after cessation of drug administration and which were not attributable to a reduction in ipsilateral cerebral shrinkage. It is considered that the effect of JTP-2942 on functional recovery is attributable to the activation of substitutive functions such as neuronal reconstruction. These pharmacologic properties of JTP-2942 may be of interest for the treatment of patients with motor and neurologic deficits during the chronic or subacute phase of stroke. (+info)
Glucose stimulates and insulin inhibits release of pancreatic TRH in vitro.
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OBJECTIVE: Pancreatic TRH is present in insulin-producing B-cells of the islets of Langerhans. There is fragmentary evidence that it may be involved in glucoregulation. The aim of our present study was to analyze how glucose and insulin affect TRH secretion by the pancreatic islets. DESIGN: Isolated pancreatic islets were incubated with different concentrations of glucose, insulin and glucagon, and TRH release was measured. RESULTS: In the present study, 6 and 12mmol/l d-glucose caused significant TRH release from isolated adult rat pancreatic islets when compared with that in the presence of the same concentrations of biologically ineffective l-glucose. Thirtymmol/l d-glucose was also ineffective, but this was not due to depression of secretion by hyperosmolarity since isosmotic compensation for the high glucose addition did not restore its stimulatory effect. Five micromol/l dibutyryl cyclic 3',5'-adenosine monophosphate (db-cAMP) increased both basal and glucose-stimulated TRH release, but this effect was not seen with 50micromol/l db-cAMP. Stimulation of phosphodiesterase by imidazole resulted in decreased basal but not glucose-stimulated release of TRH. Glucagon (10(-7)mol/l) did not affect either basal or glucose-stimulated release of TRH, while insulin (10(-7) and 10(-6)mol/l) inhibited both. CONCLUSION: Our present data showing that glucose stimulates and insulin inhibits pancreatic TRH release are compatible with the possibility that this substance may play a role in glucoregulation. (+info)
alpha-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression.
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The hypothalamic arcuate nucleus has an essential role in mediating the homeostatic responses of the thyroid axis to fasting by altering the sensitivity of prothyrotropin-releasing hormone (pro-TRH) gene expression in the paraventricular nucleus (PVN) to feedback regulation by thyroid hormone. Because agouti-related protein (AGRP), a leptin-regulated, arcuate nucleus-derived peptide with alpha-MSH antagonist activity, is contained in axon terminals that terminate on TRH neurons in the PVN, we raised the possibility that alpha-MSH may also participate in the mechanism by which leptin influences pro-TRH gene expression. By double-labeling immunocytochemistry, alpha-MSH-IR axon varicosities were juxtaposed to approximately 70% of pro-TRH neurons in the anterior and periventricular parvocellular subdivisions of the PVN and to 34% of pro-TRH neurons in the medial parvocellular subdivision, establishing synaptic contacts both on the cell soma and dendrites. All pro-TRH neurons receiving contacts by alpha-MSH-containing fibers also were innervated by axons containing AGRP. The intracerebroventricular infusion of 300 ng of alpha-MSH every 6 hr for 3 d prevented fasting-induced suppression of pro-TRH in the PVN but had no effect on AGRP mRNA in the arcuate nucleus. alpha-MSH also increased circulating levels of free thyroxine (T4) 2.5-fold over the levels in fasted controls, but free T4 did not reach the levels in fed controls. These data suggest that alpha-MSH has an important role in the activation of pro-TRH gene expression in hypophysiotropic neurons via either a mono- and/or multisynaptic pathway to the PVN, but factors in addition to alpha-MSH also contribute to the mechanism by which leptin administration restores thyroid hormone levels to normal in fasted animals. (+info)
Human thyrotropin-releasing hormone-associated peptide 3 (hTAP-3) in serum.
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Human thyrotropin-releasing hormone (TRH)-associated peptide 3 (hTAP-3), one of the cryptic peptides resulting from the proteolytic processing of preproTRH to produce TRH, was measured in human plasma from normal, hyperthyroid, and hypothyroid subjects. The dilution curve of hTAP-3 immunoreactivity in the serum paralleled the standard curve of the radioimmunoassay. HPLC analysis revealed a single strong immunoreactive peak, which corresponded to the authentic peptide, hTAP-3. The half-life of hTAP-3 in serum was approximately 3.5 min, and the addition of aprotinin and EDTA completely prevented its degradation. In hyperthyroid patients, plasma concentrations of hTAP-3 were significantly higher than those in the control group and hypothyroid patients, but no correlation was found between its level and that of thyroid hormone. These findings indicate the existence of intact hTAP-3 in the human serum and increases in plasma hTAP-3 levels in hyperthyroid patients, suggesting that blood hTAP-3 may be derived from the peripheral organs rather than the hypothalamus. (+info)
Membrane targeting and cytoplasmic sequestration in the spatiotemporal localization of human protein kinase C alpha.
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In order to map the molecular determinants that dictate the subcellular localization of human protein kinase C alpha (hPKCalpha), full-length and deletion mutants of hPKCalpha were tagged with the green fluorescent protein (GFP) and transiently expressed in GH3B6 cells. We found that upon thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate stimulation, hPKCalpha-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKCalpha failed to translocate in single cells despite the presence of TRH receptors, as attested by the TRH-induced rise in intracellular calcium concentration in these cells. TRH-stimulated translocation of hPKCalpha-GFP from the cytoplasm to cell-cell contacts was a biphasic process: a fast (measured in seconds) and transient phase, followed by a slower (approximately 1 hour) and long lasting phase. The latter and the translocation induced by phorbol 12-myristate 13-acetate absolutely required the N-terminal V1 region. In contrast to the full-length hPKCalpha, the N-terminal regulatory domain alone or associated with the V3 hinge region was spontaneously and uniformly localized at the plasma membrane of single and apposed cells. However, treatment with the calcium chelator BAPTA/AM induced a differential cytoplasmic/nuclear redistribution of the regulatory domain, depending on its association with V3, which suggests the existence of a mechanism controlling the cytoplasmic sequestration of inactive hPKCalpha and involving the V3 region. By using other deletion mutants, we were able to map the sequence required for this sequestration to the C2+V3 regions. This work points to the existence of a complex interplay between membrane targeting and cytoplasmic sequestration in the control of the spatiotemporal localization of hPKCalpha. (+info)
Combination pharmacotherapy: a mixture of small doses of naltrexone, fluoxetine, and a thyrotropin-releasing hormone analogue reduces alcohol intake in three strains of alcohol-preferring rats.
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It is common to treat some diseases with more than one medication simultaneously. Since more than one neurotransmitter system is involved in alcohol-seeking behaviour, then a therapeutic approach that targets more than one system should be more effective in reducing alcohol intake than one addressing a single system. To test this hypothesis, we compared the efficacy of low doses of individual drugs reported to reduce voluntary alcohol drinking to the efficacy of a mixture of these agents at the same low doses in reducing alcohol intake in three strains of alcohol-preferring rats (P, HAD, and Fawn-Hooded). After establishment of a stable baseline for alcohol intake in a continuous access paradigm, each rat received separate single i.p. injections of relatively low doses of either naltrexone (2.0 mg/kg), fluoxetine (1.0 mg/kg), the thyrotropin-releasing hormone analogue TA-0910 (0.2 mg/kg), a mixture of all three drugs, or the vehicle at 09:30. Each rat received all treatments, with an inter-injection washout period of at least 3 days. Alcohol and water intakes were measured at 6 and 24 h, and food intake was measured at 24 h, after the injection. Our results show that individual drugs did not significantly affect food, water, or alcohol intake. However, the mixture significantly reduced alcohol intake in all three strains, but had no effect on food intake. Similar results were obtained when the HAD rats received an oral dose of the individual drugs or the mixture. When P rats were given an i.p. injection of the mixture for 10 consecutive days, there was a continued suppressing effect. These findings show that a combination treatment designed to target simultaneously serotonergic, dopaminergic, and opioidergic systems can reduce alcohol intake, even though the doses of the individual drugs in the mixture are relatively low and ineffective when given singly. (+info)
Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1.
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The C-terminal tail of the long splice variant of the rat thyrotropin-releasing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in response to [(3)H]TRH was dependent upon the degree of truncation. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the re-ceptor, which altered both the association and dissociation rates of [(3)H]TRH, the kinetics of ligand binding were unaffected by C-terminal truncation. Equally, the rate of recycling to the plasma membrane of internalized receptors was unaffected by C-terminal truncation. Although the extent of internalization of the full-length receptor was impaired by pre-exposure of cells to TRH, this was not true of C-terminal truncation mutants, which displayed limited steady-state internalization ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length receptor. (+info)
Metabolism of thyrotropin releasing hormone in brain extracts. Isolation and characterization of an imidopeptidase for histidylprolineamide.
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An extract of porcine brain acetone powder incubated with thyrotropin-releasing hormone (TRH; pGlu-His-ProNH2) produces acid TRH (pGlu-His-Pro), histidine, and prolineamide. Fractionation of the brain extract by DEAE-cellulose chromatography produces three protein fractions which metabolize TRH. The activity of these fractions was characterized using TRH with a 3H-label on the histidine or proline as well as [His-3H]His-ProNH2. Fraction I contains pyroglutamate aminopeptidase and Fraction II contains TRH deamidase. Fraction III was found to contain a previously unrecognized enzyme which cleaves His-ProNH2 to histidine and proline. The histidylprolineamide imidopeptidase has been characterized. A competition study using a variety of compounds containing histidine or proline suggests that the best substrates for the imidopeptidase contain a free alpha-amino group on histidine and a blocked carboxyl group on proline, as is found in His-ProNH2. A survey of a variety of polypeptide hormones indicates that many of them inhibit the imidopeptidase activity. A kinetic study of the inhibition of the enzyme by adrenocorticotropic hormone (1-24) shows that the inhibition by polypeptide hormones is noncompetitive. We hypothesize that pituitary hormones may stimulate the production of (cyclo)-His-Pro by inhibiting alternate routes of TRH metabolism. (+info)