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.     

Effects of dopamine on the release of thyrotropin-releasing hormone from the rat retina in vitro.

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in the medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of dopamine, but not with pimozide. The inhibitory effects of dopamine on TRH release from the rat retina were blocked with an addition of pimozide to the medium. The elution profile of methanol-extracted rat retina on sephadex G-10 was identical to that of synthetic TRH. From these findings it is concluded that the dopaminergic system inhibits TRH release from the rat retina in vitro.     

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.     

Effect of thyrotropin-releasing hormone on rat myometrium

The effect of TRH in vitro was observed on electromyograms and isometric tension changes in the uterine horn isolated from the rat. TRH induced transient prolongation of the duration of spike bursts in the electromyogram and an increased tension in contraction of diestrous uterine horns. No distinct response to TRH was elicited in preparations from rats during other estrous stages. TRH produced a contraction associated with a burst of spike potentials in the quiescent horn from the estrogen-primed ovariectomized rat. Priming with progesterone was not a prerequisite for responsiveness to TRH. In a medium with a high Ca concentration, diestrous uteri were quiescent but a transient contraction associated with a burst of spike potentials was induced by TRH. In a Ca-free medium, TRH failed to elicit any response in the diestrous uterus but acetylcholine induced a contraction without associated spike potentials. It appears that TRH stimulates Ca-influx into the uterine muscle in which responsiveness is dependent on estrogen priming.     

Effects of subconvulsive and repeated electroconvulsive shock on thyrotropin-releasing hormone in rat brain

Male Sprague-Dawley rats were given a single electroconvulsive shock (ECS) on alternate days and sacrificed 48 hrs after 1, 3, or 5 seizures. The content of TRH in hippocampus, pyriform cortex and amygdala was increased 2.5-fold, 5.4-fold and 4.3-fold respectively, 48 hrs. after 3 alternate-day electroconvulsive shocks (ECS) and remained unchanged after 2 additional shocks. Pyriform cortex exhibited a significant intermediate increase (1.7-fold) after only 1 ECS. In a second study, rats were sacrificed 48 hrs after a series of 5 alternate-day ECS vs. subconvulsive shocks (SCS). SCS had no significant effect in these same regions, but was seen to alter TRH in striatum. These results provide an interesting parallel to several aspects of clinical electroconvulsive treatment (ECT) of depression. Together with other findings, these data suggest also, that endogenous TRH may play a role in the modulation of convulsive seizures.     

Effect of thyrotropin-releasing hormone on dog thyroid in vitro

The in vitro action of thyrotropin-releasing hormone (TRH) on the cyclic AMP level and iodine metabolism in dog thyroid, has been studied. TRH inhibited cyclic AMP accumulation and subsequent secretion in slices stimulated by thyrotropic hormone (TSH), prostaglandin E1, cholera toxin and to a lesser extent forskolin. The effect of TRH was suppressed in a medium deprived of calcium or in the presence of isobutylmethylxanthine. TRH also stimulated iodide binding to proteins, but not cyclic GMP accumulation. Although all these characteristics of TRH action on dog thyroid fit those of prostaglandin F1 alpha in this tissue, TRH effects were not relieved by indomethacine. The possibility of a TRH action through other known inhibitors of the cyclic AMP system in dog thyroid such as: acetylcholine, alpha-adrenergic agents, adenosine, iodide were checked and ruled out. The possible involvement of other neurotransmitters, such as ATP or vasoactive intestinal peptide were studied but could not be substantiated. Our data suggest the existence of a direct negative action of TRH on the thyroid itself besides its stimulatory role at the pituitary level. The great variability of the TRH effect was overcome by pretreatment of the dog by pyridostigmine, an acetylcholinesterase inhibitor.     

Stimulation of growth hormone release by thyrotropin-releasing hormone in elderly subjects

The effect of thyrotropin-releasing hormone (TRH) on the release of growth hormone (GH) was investigated in 16 elderly male subjects aged 74-88 years. Intravenous injection of 200 micrograms TRH induced a clear-cut GH rise (greater than or equal to 10 ng/ml) in 7 of 16 subjects. TRH administration did not raise plasma GH in 10 adult subjects aged 36-58 years. The results suggest disorders in neurobiochemical mechanisms regulating hypothalamopituitary function in elderly men.     

Effects of rat serum on gonadotrophin release in vitro

Gonadotrophin release from a monolayer of cultured pituitary cells from the rat was increased by the addition of fresh rat serum or rat serum albumin to the culture medium. Similar effects were obtained when horse, chicken, lamb or porcine serum was used. Fetal bovine serum or bovine serum albumin showed a stimulatory effect on FSH and LH release but, unlike the case with rat serum, a dose-response release could not be demonstrated. In addition, fresh rat serum caused the accumulation of secretory granules in the gonadotrophs. The addition of LHRH to the culture medium also stimulated gonadotrophin release in a dose-response dependent manner. The dependence of the gonadotrophs on LHRH was markedly increased by the addition of 50% fresh rat serum to the culture medium. These results indicate that fresh rat serum contains some unknown factor(s) which 1) stimulates gonadotrophin release, 2) causes an accumulation of secretory granules, and 3) is important for maintaining secretory capacity of gonadotrophs.     

Effects of opioid peptides on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of opioid peptides on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. beta-Endorphin (0.3 - 30 nM), dynorphin (0.3 - 30 nM) and FK 33-824 (1 - 10 microM) suppressed basal I-CRF release in a dose-dependent fashion. At 2.2 nM concentrations of these peptides, mean percent inhibition was 56% for beta-endorphin; less than 5% for alpha-endorphin; 44% for dynorphin; 23% for leucine-enkephalin; 6% for methionine-enkephalin; less than 5% for FK 33-824; and less than 5% for D-ala2, D-leu5-enkephalin. The inhibitory effects of beta-endorphin and enkephalins were completely blocked by naloxone, but those of dynorphin were only partially blocked. These results suggest that opioid peptides act through opioid receptors and inhibit I-CRF release from the hypothalamus under our conditions. Therefore, endogenious opioid peptides may have a physiological role in the CRF-releasing mechanism of the hypothalamus.     

Effects of choline augmentation on acetylcholine synthesis and release in rat atrial minces

The uptake and acetylation of [3H]-choline, as well as the calcium-dependent release of a newly synthesized [3H]-ACh, was studied in a new rat atrial mince preparation. The hemicholinium-3-sensitive uptake and acetylation of [3H]-choline increased as [3H]-choline concentrations were elevated to 100 microM in atrial minces. In contrast, hemicholinium-3-sensitive [3H]-choline uptake was saturated with 15 microM [3H]-choline in brain synaptosomes. The increased atrial [3H]-ACh synthesized in the presence of [3H]-choline augmentation was releasable by 50 mM K+-depolarization in a 1 mM cobalt-sensitive manner. These results suggest that atrial parasympathetic activity may be more sensitive to circulating choline concentrations than brain cholinergic neurons are.     

Brain thyrotropin-releasing hormone matures independently of the hypothalamus in the rat

To evaluate the relationship of the extrahypothalamic brain thyrotropin-releasing hormone (TRH) to its hypothalamic counterpart, we studied the maturation of hypothalamic and extrahypothalamic TRH in the rat. The absolute increase of TRH in the whole brain and the extrahypothalamus reached adult levels at 7 days of age, whereas the hypothalamic TRH concentrations did not differ from the adult levels at 23 days. Moreover, the TRH concentrations at 7 days were greater than the adult levels in the striatum, hippocampus, pons-medulla and cerebellum, and similar to the adult levels in the midbrain and cortex. These data indicate the developmental divergency of hypothalamic and extrahypothalamic TRH, implying that the maturation of extrahypothalamic TRH is independent of the hypothalamus. The present study suggests that extrahypothalamic TRH may play a neurophysiological role in the central nervous system at an early infantile age, at which hypothalamic TRH is not ripe for its endocrinological action.     

Effects of thyroidectomy and thyroxine replacement on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The effects of thyroidectomy and thyroxine (T4) replacement on the release of luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) in ovariectomized (Ovx) rats were studied. Immediately after ovariectomy, rats were thyroidectomized (Tx) or sham-Tx. The Ovx-Tx rats were injected subcutaneously with either saline or T4 (2 micrograms/100 g body weight) daily for 30 days before sacrifice. Sham-Tx rats were treated with saline only. Twenty hours after the last injection, the blood sample was obtained by decapitation. The excised anterior pituitary gland (AP) was bisected and incubated in vitro with or without 0.1, 0.5, 2.5, and 50 ng GnRH at 37 degrees C for 4 h. The mediobasal hypothalamus (MBH) was bisected and incubated with or without the AP of Ovx donor rat in vitro. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. LH in the serum of Tx rats was higher than that in the serum of sham-Tx and Tx-T4 rats. Thyroidectomy resulted in an increase of LH release by Ovx rat AP, stimulated with or without 0.1 and 50 ng GnRH, as well as in an increase of immunoreactive GnRH release from MBH of Ovx rats in vitro. After a 4-hour incubation with donor APs, the LH in the medium containing MBH obtained from Tx rats was significantly higher than that obtained from sham-Tx and Tx-T4 rats. LH concentrations, in both sera and media, as well as GnRH concentration in the media of euthyroid and T4-replaced Tx groups were nonsignificantly different. These results suggest that T4 is inhibitory to the basal and GnRH-stimulated LH release as well as to the release of GnRH in the absence of ovarian hormones.     

Effect of electroconvulsive shock on the content of thyrotropin-releasing hormone in rat brain

Five grand-mal seizures were electrically induced in rats on alternate days. Forty-eight hours following the last seizure, TRH was quantitated in extracts of anterior cortex, hippocampus, striatum, thalamus plus midbrain, and hypothalamus. When compared to sham treated controls, TRH was found to be elevated 5-fold in the hippocampus and 2-fold in the striatum with no changes observed in the remaining regions. Since the time chosen for analysis excludes acute post-ictal effects, these results draw attention to a prolonged alteration of TRH levels in specific brain regions in an animal model of electroconvulsive treatment.     

Effects of epinephrine and oxytocin on the release of prostaglandin F from the rat uterus in vitro

Isolated uteri from rats with regular 4-day cycles were incubated in Krebs-Ringer bicarbonate buffer and the release of PGF into the medium was measured by radioimmunoassay after extraction of the incubation medium with ethyl acetate at pH 3.0-3.5. PGF was produced from endogenous precursors and accumulated in equal amounts in the medium during two successive 60 min periods on each day of cycle, but the magnitude of the production varied significantly during the cycle, being greatest in estrus. Oxytocin in doses up to 500 mU/ml had no effect on PGF accumulation in the incubation period at any stage of the cycle, while epinephrine (10(-3)) greatly stimulated PGF release from the estrous uterus but had no effect on PGF release from the diestrous uterus. Phentolamine, an alpha-blocking agent, had no effect on the epinephrine-induced release of PGF, while propranolol, a beta-blocking agent, not only prevented in increase in PGF production induced by epinephrine but also reduced the basal release of PGF by the estrous uterus. Since oxytocin contracts and epinephrine relaxes the nonpregnant rat uterus both in vivo and in vitro, it is unlikely that the effects of these two compounds on uterine contractility are mediated by the release of PGF2alpha.     

Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain.

This report describes the purification of a rat brain thyrotropin-releasing hormone (TRH) deamidating enzyme to apparent homogeneity. Criteria for purity include sodium dodecyl sulfate and disc gel electrophoresis, as well as isoelectric focusing (pI = 4.5). Enzyme purification was facilitated by development of a rapid and sensitive continuous assay using the substrate L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide, which, upon hydrolysis of the naphthylamide, results in the appearance of the fluorescent product, beta-naphthylamine (beta NA). With this substrate the homogeneous enzyme had a specific activity of 14.5 mumol of beta NA min-1 mg-1. The only peptide product formed was shown to be L-pyroglutamyl-Nim-benzylhistidyl-L-proline. Hydrolysis of [L-prolyl-2,3-3H]TRH was shown to yield L-pyro-glutamyl-L-histidyl-L-proline as the only radiolabeled product. Characterization of the brain deamidase by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis indicated that the enzyme consists of a single polypeptide chain having molecular weights of 70,000 and 73,500, respectively. Rat brain TRH deamidase has an apparent Km of 34 micron, and a pH optimum between 7 and 8 using L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide as a substrate. With this substrate, TRH was shown to be a competitive inhibitor with an apparent Ki of 120 +/- 20 micron.