Effect Of TRH Research Articles

Regulation of thyrotropin (TSH) bioactivity by TSH-releasing hormone and thyroid hormone.

The regulation of TSH biological activity by thyroid hormone and TRH was studied by comparison of pituitary and in vitro secreted TSH from normal and thyroidectomized rats that were alternatively treated with TRH either in vivo or in vitro. Normal and thyroidectomized (3 weeks postthyroidectomy), rats were injected with saline or TRH (100 micrograms) three times over 24 h. Pituitaries were incubated in vitro for 6 h, and six groups of samples from both pituitary and secreted TSH were analyzed: normal (n = 6), thyroidectomized (n = 6), normal and thyroidectomized groups treated with TRH in vitro (n = 2 each) with 10(-8) M TRH added to the incubation medium, and normal and thyroidectomized groups TRH treated in vivo, their incubation medium also supplemented with 10(-8) M TRH (n = 4 each). The biological activity of TSH in pituitary extracts and media was analyzed in terms of the ability to stimulate adenylate cyclase in human thyroid membranes. Thyroidectomy significantly decreased pituitary TSH bioactivity (70%) compared to normal, with no effect on secreted TSH in the medium. TRH, both in vivo and in vitro, when compared to the corresponding untreated groups, produced a significant increase in bioactive TSH in media from both normal (TRH in vivo, 131%; TRH in vitro, 139%) and thyroidectomized samples after TRH in vivo (158%). The TRH effect in the pituitary showed a significant increase in TSH bioactivity from normal samples treated with TRH in vivo (137%), whereas in thyroidectomized pituitary samples with TRH in vitro, TSH bioactivity was decreased (69%). These results indicate that thyroid hormone deficiency and TRH differentially regulate TSH bioactivity. Thyroid hormone deficiency induced a decrease in pituitary TSH bioactivity and favored the effect of TRH on secretion of more bioactive forms. TRH not only induced the formation of more bioactive forms but also stimulated their secretion into the medium.

Thyrotrophin-releasing hormone does not accumulate glycosylated thyrotrophin, but changes heterogeneous forms of thyrotrophin within the rat anterior pituitary gland.

We have investigated the effect of TRH on the accumulation of glycosylated TSH in the rat anterior pituitary gland. Hemipituitaries from adult male rats were incubated in medium containing [3H]glucosamine in the presence of TRH. [3H]Glucosamine-labelled TSH in media and pituitaries was measured by immunoprecipitation and characterized by isoelectric focusing after affinity chromatography. Incorporation of [3H]glucosamine into immunoprecipitable TSH in the media and pituitaries increased progressively with the period of incubation. Although the release of [3H]glucosamine-labelled and unlabelled TSH into media was stimulated by the addition of TRH in a time- and dose-dependent manner, TRH administration did not alter the amounts of labelled or unlabelled TSH in the anterior pituitary lobes. The anterior pituitaries were found, by isoelectric focusing analysis, to be composed of four major component peaks of [3H]glucosamine-labelled TSH. Administration of TRH caused profound changes in the radioactivity of these components and evoked new radioactive peaks, resulting in the appearance of six components in total. The present data provide evidence that TRH significantly changes the heterogeneity of glycosylated TSH in the anterior pituitary without altering the amount of the glycosylated form.

Regulation of Thyroxine 5′-Deiodinase by Thyroid Hormones and Activators of Protein Kinase C in GH4C1Cells*

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.

The phorbol ester TPA induces hormone release and electrical activity in clonal rat pituitary cells.

The phorbol ester TPA activates the protein kinase C in a similar way as 1,2-diacylglycerol. The effect of TPA on prolactin (PRL) secretion and electrical properties of rat pituitary cells in culture (GH4C1 cells) were compared with the effects of thyroliberin (TRH) on the corresponding parameters. The rate of hormone release was measured using a parafusion system optimized to give high time resolution. Samples for PRL measurements were taken every 4 s. The TRH evoked a biphasic PRL release, with a transient peak after about 30 s followed by a lower but sustained enhancement of the secretion. The TPA mimicked the late phase of the secretory response to TRH. The TPA analogue, 4 alpha-PDD, had no effect on the PRL release. The TRH also evoked biphasic membrane potential changes in the GH4C1 cells; the late phase consisting of membrane depolarisation associated with increased input resistance and enhanced firing of Ca2+ dependent action potentials. The TPA mimicked to a great extent these late phase effects of TRH, whereas the inactive analogue 4 alpha-PDD was ineffective. Continuous exposure to TPA masked the late phase of the electrophysiological response to TRH, suggesting that TPA and TRH share common mechanisms in their action on GH4C1 cells. We suggest that TRH enhances the electrical activity in these cells due to protein phosphorylation induced by diacylglycerol activation of protein kinase C, which in turn suppresses the membrane permeability to K+.

ERRATUM

Abstract. The pharmacokinetics and the endocrine profile of a new low molecular somatostatin derivative, SMS 201–995, were investigated in a group of 35 normal subjects. Clearance studies (n = 6) for this peptide showed a prolonged half-life in plasma, 113 min, following single sc injections of 50 or 100 μg. Arginine stimulation tests (n = 6) were conducted immediately and 180 min after sc injection of 50 μg of SMS 201–995. The stimulatory effect of arginine on GH and insulin was counteracted by the peptide at the P < 0.001 and P < 0.02 significance level, respectively. Delayed arginine stimulation revealed a persistent blockade of the GH release (P < 0.02), whereas a recovery of the insulin response was observed. Plasma glucagon increments following a standard protein meal (n = 10) were significantly (P < 0.001) inhibited by previous sc injection of 50 μg of SMS 201-995. Pretreatment with 50 and 100 μg of SMS 201-995 sc (n = 9) inhibited (P < 0.001) the stimulatory effect of TRH (200 μg iv) on TSH without modifying basal levels. The injection of 100 μg/h during sleep completely abolished the nocturnal GH peak in 4 volunteers. No rebound rise after decline of the suppressive action on GH was recorded in any of the trials. Safety chemistries and blood coagulation studies remained normal and no side-effects or untoward reactions were recorded throughout the investigation. With its high potency, slow plasma clearance, and long action SMS 201–995 may represent a valuable tool in the long-term management of states of inappropriate GH secretion.

Endocrine profile of a long-acting somatostatin derivative SMS 201-995. Study in normal volunteers following subcutaneous administration.

The pharmacokinetics and the endocrine profile of a new low molecular somatostatin derivative, SMS 201-995, were investigated in a group of 35 normal subjects. Clearance studies (n = 6) for this peptide showed a prolonged half-life in plasma, 113 min, following single sc injections of 50 or 100 micrograms. Arginine stimulation tests (n = 6) were conducted immediately and 180 min after sc injection of 50 micrograms of SMS 201-995. The stimulatory effect of arginine on GH and insulin was counteracted by the peptide at the P less than 0.001 and P less than 0.02 significance level, respectively. Delayed arginine stimulation revealed a persistent blockade of the GH release (P less than 0.02), whereas a recovery of the insulin response was observed. Plasma glucagon increments following a standard protein meal (n = 10) were significantly (P less than 0.001) inhibited by previous sc injection of 50 micrograms of SMS 202-995. Pretreatment with 50 and 100 micrograms of SMS 202-995 sc (n = 9) inhibited (P less than 0.001) the stimulatory effect of TRH (200 micrograms iv) on TSH without modifying basal levels. The injection of 100 micrograms/h during sleep completely abolished the nocturnal GH peak in 4 volunteers. No rebound rise after decline of the suppressive action on GH was recorded in any of the trials. Safety chemistries and blood coagulation studies remained normal and no side-effects or untoward reactions were recorded throughout the investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioventilator effects of TRH in anaesthetized rats: Role of the brain stem

Cardioventilstor responses were studied in anaesthetized rats after injections of TRH into either the lateral (i.c.v. lat) or the fourth (i.c.v. IV) cerebral ventricles. TRH induced a more rapid hypertensive effect i.c.v. IV than i.c.v. lat. Blocking of the cerebral aqueduct abolished the hypertensive and tachypnoeic effects of TRH i.c.v. lat but not those of TRH i.c.v. IV. It is concluded that TRH increased blood pressure and ventilation rate via brain stem structures close to the fourth ventricle.

P-348 - Effect of TRH, 5-HTP and 5-methoxy-N,N-dimethyltryptamine on spinal reflexes in 5,6-dihydroxytryptamine-treated rats

P-348 - Effect of TRH, 5-HTP and 5-methoxy-N,N-dimethyltryptamine on spinal reflexes in 5,6-dihydroxytryptamine-treated rats

P-56 - Central regulation of gastric functions by neuropeptides: Effects of TRH and bombesin

P-56 - Central regulation of gastric functions by neuropeptides: Effects of TRH and bombesin

Effect of TRH on catecholamine levels in brainstem of rat with severe cortical damage

Effect of TRH on catecholamine levels in brainstem of rat with severe cortical damage

Comparison of the TRH effect and descending slow E.P.S.P. in the motoneuron of the rat spinal cord

Comparison of the TRH effect and descending slow E.P.S.P. in the motoneuron of the rat spinal cord

The effects of subchronic TRH and DN 1417 on tryptophan hydroxylation in rat raphe slices; role of biopterin in serotonin biosynthesis

The effects of subchronic TRH and DN 1417 on tryptophan hydroxylation in rat raphe slices; role of biopterin in serotonin biosynthesis

Effects of thyrotropin-releasing hormone on phosphoinositides and cytoplasmic free calcium in thyrotropic pituitary cells.

We have previously demonstrated differences in several cellular responses to TRH in mouse thyrotropic pituitary (TtT) cells and in rat mammotropic pituitary (GH3) cells. In this report, we further explore the mechanism of TRH action in TtT cells by measuring its effects on phosphoinositides and on cytoplasmic free Ca2+ concentration [( Ca2+]i). We demonstrate that TRH stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phospholipase C and elevates [Ca2+]i. Furthermore, we present evidence that hydrolysis of PtdIns(4,5)P2 is not secondary to the elevation of [Ca2+]i. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 57% of control and stimulated an increase in inositoltriphosphate, the unique product of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2, to a peak of 280% of control. In control cells, resting [Ca2+]i was 106 +/- (SE) 27 nM, and TRH stimulated a rapid elevation to 700 +/- 210 nM. In experiments performed to determine whether PtdIns(4,5)P2 hydrolysis induced by TRH may have been caused by the elevation of [Ca2+]i, the following results were obtained: the effect of TRH to decrease the level of PtdIns(4,5)P2 was not reproduced by the calcium ionophore A23187 or by membrane depolarization with 50 mM K+; the calcium antagonist TMB-8 did not inhibit the TRH-induced decrease in PtdIns(4,5)P2; and, most importantly, inhibition by EGTA of the elevation of [Ca2+]i did not inhibit the TRH-induced decrease in PtdIns(4,5)P2. We suggest that phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to yield inositoltriphosphate may be the initial event in TRH action in TtT cells, as in GH3 cells, that leads to elevation of [Ca2+]i and to TSH secretion.

Dorsal medullary oxytocin, vasopressin, oxytocin antagonist, and TRH effects on gastric acid secretion and heart rate

Injections of oxytocin and TRH (11 picomoles), centered on the dorsal motor nucleus of the vagus, substantially increased gastric acid secretion. Additionally, oxytocin, but not TRH, simultaneously produced a consistent reduction in heart rate. Vasopressin injected into the same locus, at doses of 11 and 110 picomoles, had no effect on either function. Both the gastric and cardiac effects of oxytocin were eliminated by the central injections of oxytocin antagonist dEt 2Tyr(Et)Orn 8Vasotocin (ETOV; 6 picomoles) or peripheral administration of atropine (300 μg/kg, IP). Application of oxytocin or TRH to the area postrema, at double the dosage (22 picomoles) yielded no consistent effects on either gastric secretion or heart rate. These findings indicate that oxytocin in the dorsal motor nucleus of the vagus may act as a regulator of vagally-mediated gastric and cardiovascular functions while TRH effects, in this medullary area, seem limited to the regulation of gastric function.

Cells of neural crest origin as possible models to investigate thyrotropin releasing hormone action in the central nervous system

Since TRH has been linked to trophic events in the motor neuron and is being used for symptomatic treatment of motor neuron disorders, we have examined seven neural crest cell lines for TRH-receptors (TRH-Rs) and for the enzymes choline acetyltransferase (ChAT) and creatine kinase (CK), with the intent to identify one that can be used for studies of TRH-R mediated cellular events in the CNS. Three of the seven (neuroblastomas) were ChAT-positive but were without detectable TRH-Rs and showed no major alterations in ChAT/CK-activities following acute (24 hr) treatment with 0.01–10 μM TRH. In contrast, high affinity TRH receptor sites were detected in a melanoma (RPMI 3460) cell line. Linking cellular events with these receptors could lead to the use of this established cell line as a tool for the biochemical investigation of the pathways and mechanisms of TRH action in the CNS. Clinically, we have demonstrated that high-dose TRH produces rapid, temporary improvement of motor neuron function, namely increased strength and decreased spasticity, in patients with amyotrophic lateral sclerosis (ALS) (Engel, Siddique and Nicoloff, 1983). Known effects of TRH on animal spinal motor neurons include a) a rapid, neurotransmitter-like excitatory action (Ono and Fukuda, 1982) similar to that seen in ALS patients and b) a longer-term nutritive/trophic effect on cultured motor neurons that results in increased production of such important cellular enzymes as choline acetyltransferase (ChAT) and creatine kinase (CK) (Schmidt-Achert, Askanas and Engel, 1984). The mechanism(s)/pathway(s) of these TRH actions remains unknown but may be based on the binding of the hormone to cellular receptors of some type e.g. the membrane-bound TRH-receptor (TRH-R) that is present in brain and spinal cord (Hawkins and Engel, 1985). As an alternative to motor neurons that are difficult to culture in sufficient quantities for detailed biochemical analyses, we have screened seven cell lines of neural crest origin with the intent to identify one responsive to TRH and containing ChAT and/or CK that can serve as an established cell line for the further investigation of receptor-mediated trophic effects of TRH in the CNS. We have assayed for TRH-receptors and for ChAT and CK activities in six neuroblastomas (N18TG2, NG108-15, LA-N-1, LA-N-2, LA-N-5, SK-N-MC) and a Syrian hamster melanoma (RPMI 3460).

Differential motor and blood pressure effects of intrathecal oxytocin and TRH in the rat

While previous reports have immunocytochemically localized oxytocin and TRH in the spinal cord, the functional significance of these peptides is unclear. The present paper examined this issue and tested the effects of these peptides upon intrathecal administration. We found both peptides produced lasting motor and blood pressure changes. Oxytocin elicited prolonged jerks of the hindlimbs and the tail, while TRH produced an increase of hindlimb muscle tone and tail tremor. TRH in larger doses (5, 10 μg) also caused tail erections and whipping. The motor effects of both peptides were dose-dependent. Intrathecal oxytocin (0.75 or 1.5 IU) caused a transient drop in blood pressure followed by a rise, in 4 out of 7 rats. The other 3 only showed a hypertensive effect. In contrast, intrathecal TRH produced a rise in blood pressure in all the animals tested. These findings suggest that both oxytocin and TRH may play a role in the regulation of motor and automatic functioning at the spinal level.

Brain cortex phosphatidylserine inhibits phosphatidylinositol turnover in rat anterior pituitary glands.

The in vitro effect of bovine brain cortex phosphatidylserine on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine of rat anterior pituitary glands was studied. Phosphatidylserine (0.1 to 66.6 microM) decreased the incorporation of 32Pi into phosphatidylinositol, but not phosphatidylcholine or phosphatidylethanolamine, in a concentration-related manner. The inhibitory effect of phosphatidylinositol was similar to that of dopamine in the same experimental conditions. The combined effects of submaximal concentrations of dopamine and phosphatidylserine elicited an apparently additive inhibitory effect on phosphatidylinositol synthesis. The inhibitory effect of phosphatidylserine was completely reversed by haloperidol and sulpiride and only partially by pimozide, antidopaminergic agents which per se do not affect phosphatidylinositol synthesis. The stimulatory effect of TRH to increase 32Pi incorporation into phosphatidylinositol was decreased by phosphatidylserine. These observations suggest that the decrease in prolactin release in the presence of phosphatidylserine may be evoked through a dopaminergic mechanism.

Effect of TRH analogs on neurologic recovery after experimental spinal trauma.

Effects of two stable TRH analogs were compared after experimental spinal trauma in cats. CG3509, which has a substitution at the amino-terminus of the peptide, improved neurologic recovery; MK771, which has modifications at both ends of the tripeptide, was ineffective. These findings indicate that TRH analogs may be of use in spinal cord injury, and suggest that the C-terminal amino acid may be critical for the beneficial effects of TRH or TRH-analogs in CNS injury.

Intracerebroventricular injection of a TRH analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-prolinamide, induces gastric lesions and gastric acid stimulation in rats.

The effects of TRH and its biologically stable analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide (DN-1417), on gastric mucosa and acid secretion were examined in rats. Intracerebroventricular (ICV) injection of DN-1417 (0.1-10 micrograms) caused a dose-dependent gastric lesion in the corpus and antrum 6 h after administration. The gastric lesions produced by 1 microgram of DN-1417 were more severe than those produced by ICV TRH (10 micrograms), intravenous DN-1417 (200 micrograms) and stress. Although the lesion-generating effect of TRH (10 micrograms) tended to be reduced 6 h after the injection, that of DN-1417 (1 microgram) was sustained during 6 h. Atropine (0.1 and 1 mg/kg s.c.) inhibited DN-1417-induced gastric lesions in a dose-related manner while sulpiride (10 and 30 mg/kg s.c.), haloperidol (1 mg/kg i.p.), phentolamine (1 and 5 mg/kg s.c.) and yohimbine (5 mg/kg s.c.) did not prevent the lesions. ICV DN-1417 also stimulated basal gastric acid secretion and the effect was stronger and longer-lasting than that of TRH. Atropine (0.1 mg/kg s.c.) stopped DN-1417-stimulated gastric acid secretion. In conclusion, the possibility that TRH may be involved in the CNS modulation of gastric mucosal integrity deserves further attention. The enhanced potency of action of DN-1417 over TRH could make ICV injection of this peptide a useful tool for inducing centrally-mediated gastric lesions in rats.

Growth hormone response to thyrotropin-releasing hormone in the urethane-anesthetized rat: effect of thyroid status.

To evaluate the role of thyroid hormones in regulation of the GH-stimulatory effects of TRH and human pancreatic GH-releasing factor (hpGRF-40), we studied the plasma GH responses to these secretagogues under conditions of thyroid hormone deprivation and replacement in the urethane-anesthetized rat. In euthyroid control rats, TRH (1 microgram/kg) elicited a small transient rise in plasma GH, which peaked at 2-5 min and returned to basal by 20 min. In chronically hypothyroid rats (10 weeks after thyroidectomy), intrapituitary GH was markedly depleted to less than 0.1% of normal, and TRH was completely ineffective in eliciting a plasma GH response to TRH. In chronically hypothyroid rats given T4 (20 micrograms/kg daily) for 4 days, intrapituitary GH was partially repleted, and the GH response to TRH was markedly enhanced compared to that in euthyroid rats. The extent to which the GH response to TRH was enhanced by chronic hypothyroidism, followed by short term T4 treatment, depended on the duration of T4 administration. The plasma GH response was greatest after 1-3 days of T4 treatment; treatment for 7 days, on the other hand, suppressed the GH response to below that of euthyroid rats. The minimal duration of hypothyroidism which, in combination with short term (2 days) T4 treatment, enhanced the plasma GH response to TRH, was 6 weeks, with 8 weeks or longer being optimal. The effect of TRH on plasma GH was dose dependent in thyroidectomized rats given T4 for 2 days; the lowest maximally stimulatory dose was 10 times less than that in the euthyroid rat (1 vs. 10 micrograms/kg). The GH-stimulatory effect of TRH in thyroidectomized rats given T4 for 2 days was abolished by the simultaneous administration of SRIF (40 micrograms/kg). That the failure of TRH to stimulate GH release in the chronically hypothyroid rat may have been the consequence of a depletion of intrapituitary GH available for release was suggested by the finding that in parallel studies, hpGRF-40, a more potent stimulator of GH release in the euthyroid rat, was also without effect. In contrast to the GH response to TRH, the GH response to hpGRF-40 was only partially restored by T4 treatment of chronically hypothyroid rats for 2 days. We conclude that chronic thyroid hormone deficiency selectively sensitizes the somatotroph to TRH. Short term thyroid hormone replacement is needed to replete intrapituitary GH and allow the expression of this enhanced GH secretory response to TRH. More prolonged treatment with T4, on the other hand, appears to desensitize the somatotroph to TRH.