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.     

The prolonged increase in thyrotropin-releasing hormone in rat limbic forebrain regions following electroconvulsive shock

We have previously demonstrated substantial increases in thyrotropin-releasing hormone (TRH) in specific regions of rat forebrain two days after single or repeated alternate-day electroconvulsive shock (ECS). To determine longer term effects of ECS-induced seizures on forebrain TRH content, we extended the time of the post-ECS observations to 6 and 12 days following 1 (ECS x 1) or 3 (ECS x 3) alternate-day ECS. Previous observations at 2 days post-ECS were confirmed except that hippocampal content of TRH was higher after ECS x 1. In pyriform cortex TRH remained elevated for 6 days after ECS x 1 and 3, and for 12 days after ECS x 3. In hippocampus TRH was elevated for 6 days after ECS x 1 and tended to remain elevated beyond 2 days after ECS x 3. In anterior cortex the increase persisted 6 days after ECS x 1 and 12 days after ECS x 3. These data show that convulsive seizures can induce sustained elevations of TRH beyond 48 h. This finding may be especially important in pyriform cortex and hippocampus where TRH may function as an endogenous anti-epileptic. Our data are also consistent with a possible role for TRH in affective regulation in the hippocampus, amygdala, pyriform and other cortical regions. Moreover, the present results further advance the analogy of the time-course of the TRH changes in rat to the course of the antidepressant response to electroconvulsive treatment in humans.     

The effects of hemorrhagic shock on thyrotropin-releasing hormone and its receptors in discrete regions of rat brain

The effects of hemorrhagic shock on thyrotropin-releasing hormone (TRH) levels and its receptors were studied in different regions of the rat brain. Rats were bled for 30 min from the left femoral artery, and their mean arterial pressure was kept at 40 mmHg for the following hour. The rats were killed by decapitation. Rat brains were immediately removed and dissected into 7 regions. Hemorrhagic shock decreased TRH significantly in the frontal cortex, septum, hippocampus, and hindbrain but TRH was not changed in the striatum, hypothalamus, and midbrain. Hemorrhagic shock significantly decreased TRH receptor binding in the septum and hindbrain. Scatchard analysis of saturation isotherms of specific TRH binding showed that the decreased specific TRH binding in the hindbrain resulted not from an increase of the dissociation constant (Kd), but from a decrease in the maximum number of binding sites (Bmax). In the septum, the decrease in specific binding was due both to a decrease in Bmax and an increase in Kd. The findings indicate that TRH plays a role in the physiological response to hemorrhagic shock.     

Effects of chronic electroconvulsive shock on the expression of beta-adrenergic receptors in rat brain: immunological study

The purpose of the present study is to determine the effect of chronic electroconvulsive shock (ECS) on the expression of beta-adrenergic receptors in rat brain by Western blot using mAb beta CO2, a monoclonal antibody against beta-adrenergic receptors. Rats in ECS treated groups received maximal ECS (70 mA, 0.5 second, 60 Hz) through ear-clip electrodes for 12 consecutive days. The experiment was carried out in 14 discrete regions of brain. Chronic ECS reduced the expression of beta-adrenergic receptors in frontal cortex, temporal cortex, parietooccipital cortex, hippocampus and limbic forebrain, but not in other areas of brain. The regional specificity and the magnitude of the reduction of receptor expression are well correlated with those of the reduction of receptor ligand binding, which was determined using [3H]dihydroalprenolol. To the best of our knowledge, this is the first report to demonstrate that chronic ECS decreases the expression of receptor protein in specific regions of rat brain.     

Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain

Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of [3H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system.     

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.     

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 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.     

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.     

autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor

The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_max) but not the binding affinity (K_D) of the receptor. The effect was reversible and depended on the duration of treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis.     

Intraventricular 6-hydroxydopamine increases thyrotropin-releasing hormone (TRH) content in regions of rat brain

Rats were given intraventricular (ivt) injections of various doses (50-400 micrograms, hydrobromide salt) of 6-hydroxydopamine (6-OHDA) and killed 1, 3 or 6 days later. Brains were removed, dissected into 11 regions, and the thyrotropin-releasing hormone (TRH) content of each region was measured by radioimmunoassay. 6-OHDA (400 micrograms) caused significant elevations in the TRH content of 6 regions: olfactory bulb, anterior cortex, brainstem, posterior cortex, hippocampus, and amygdala-piriform cortex. The magnitude of these increases ranged from 59% in olfactory bulb to 497% in hippocampus and was, in all cases, greatest at 3 days. These results suggest that the TRH content of certain brain regions may be regulated by catecholamine neurotransmitters.     

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.     

The effect of electroconvulsive shock on brain tubulin during development and aging

Effect of electroconvulsive shock on rat brain tubulin content was studied during maturation and aging. The results show that electroconvulsive shock had no effect on soluble tubulin in different brain structures of young animals (22 days) while the same treatment produced a marked decline in adult (95 days) and aged (490-511 days) animals. The same treatment produced inhibition of 3H-leucine incorporation into tubulin and decrease of 3H-colchicine binding in the proteins of synaptosomes isolated from the centricephalic structures of all the ages examined. Tubulin biosynthesis by free polysomes was not diminished to the extent which could explain the decrease of tubulin level found in the soluble or synaptosomal fraction. Thus, our results suggest that changes in soluble tubulin content in response to electroconvulsive shock could be a reflection of changes in equilibrium: tubulin dimers--microtubules--membrane-bound tubulin.     

The effect of repeated electroconvulsive shock treatment and chronic lithium feeding on the release of norepinephrine from rat cortical vesicular preparations

The effect of repeated electroconvulsive shock (ECS) treatment and chronic LiCl feeding on calcium-dependent, K+-evoked release of [3H] norepinephrine from rat cortical vesicular preparation was studied. There was no significant effect of either acute or repeated ECS treatment on [3H]norepinephrine release in cortical vesicles obtained from animals treated for either 1 or 10 days. Release of norepinephrine was examined over a range of CaCl2 concentrations. Clonidine effectively inhibited release of [3H]norepinephrine in cortical vesicles obtained from control and ECS-treated animals. K+-evoked release of [3H]norepinephrine at low (0.2 mM) and high (1.0 mM) CaCl2 concentrations was significantly increased in cortical vesicles obtained from LiCl-treated animals. Clonidine effectively inhibited release of [3H]norepinephrine in cortical vesicles obtained from both control and LiCl-fed animals. These results suggest a possible common mechanism of action of antidepressant drug therapy on presynaptic release of norepinephrine from nerve terminals.     

An analogue of thyrotropin-releasing hormone, DN1417, decreases naloxone binding in the rat brain

We explored whether thyrotropin-releasing hormone may affect opioid receptors in the rat brain. Adult male rats were intraperitoneally injected twice a day with varying doses of DN1417, a potent analogue of thyrotropin-releasing hormone, for 2 days, and opioid receptors of the brain (hypothalamus, striatum, hippocampus, midbrain, ponsmedulla, and cortex) homogenates were determined using [3H]naloxone. Intraperitoneal injection of DN1417 in a dose of 0.3 mg/100 g body wt resulted in a significant reduction in naloxone binding of the striatum as compared with the saline-injected group, whereas naloxone binding of other brain regions was not affected by DN1417. DN1417 produced a dose-dependent decrease in naloxone binding of the striatum. The affinity constant of naloxone binding was similar between the saline- and DN1417-injected groups. In vitro addition of DN1417 did not interfere with the brain naloxone binding. The distribution of 3H-labeled DN1417 injected peritoneally did not differ among the brain regions. The present data imply that the opioid antagonistic action of thyrotropin-releasing hormone may be due, at least in part, to the significant decrease in the striatal opioid binding in the rat brain.     

Effect of repeated stimulation by thyrotropin-releasing hormone (TRH) on thyrotropin and prolactin secretion in perfused euthyroid and hypothyroid rat pituitary fragments

The previously reported refractoriness of pituitary response to thyrotropin-releasing hormone (TRH) stimuli was investigated here in an in vitro perfusion system using pituitary tissue from euthyroid and hypothyroid rats. Thyroid-stimulating hormone (TSH) and prolactin (PRL) responses to TRH (28 pmol) were significantly greater in hypothyroid tissue compared with euthyroid. Hypothyroid tissue showed a reduction in response to two consecutive stimuli in both TSH and PRL, however the TSH decline in response was more marked than PRL. Euthyroid tissue showed no significant decline in response to TRH. An increase in the dose of TRH (112 pmol), administered to euthyroid tissue, resulted in increased TSH and PRL response, but no decline in response to sequential stimuli was observed. Three consecutive stimuli by TRH (28 pmol) of hypothyroid tissue resulted in a consistent decline in TSH response. The decline in PRL response only reached statistical significance by the third stimulation. Euthyroid and hypothyroid pituitary tissue was subjected to sequential depolarising stimulation with KCl (50 mumol). Euthyroid tissue showed no decline in response in either TSH or PRL. In hypothyroid tissue only, the decline in TSH response reached statistical significance. This decline in TSH response was significantly smaller than the decline in response observed in hypothyroid tissue stimulated with TRH. Refractoriness of hypothyroid pituitary tissue to repeated TRH stimuli is reported here. Our data suggest that the decline in hormonal response cannot be explained solely on the basis of tissue depletion.