Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

Effect of TRH analog (DN-1417) on tyrosine hydroxylase activity in mesocortical, mesolimbic and nigrostriatal dopaminergic neurons of rat brain

Tyrosine hydroxylase (TH) was assayed in eight regions of rat brain following repeated treatment with a TRH analog, DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide). Repeated DN-1417 treatment (20 mg/kg/day, IP) for 7 days increased TH activity in the ventral tegmental area and decreased in the prefrontal cortex polar, medial and lateral fields and olfactory tubercles. No significant change in TH activity was found in the nucleus accumbens, striatum and substantia nigra. Kinetic analysis showed that the increased TH activity in the ventral tegmental area was due to an increase in Vmax, but not a change in the apparent Km of TH for a cofactor, 6-methyl-tetrahydropteridine. When TH was assayed at a suboptimal pH and in the presence of a subsaturating cofactor, the striatal TH was activated significantly after DN-1417. In the prefrontal cortex medial field, nucleus accumbens and olfactory tubercles, TH activity assayed under the suboptimal condition was not modified by DN-1417 treatment. These results suggest an intimate involvement of central dopaminergic systems in the actions of DN-1417.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.     

Characterization of adenosine receptor-mediated generation of cyclic AMP in slices of rat cerebral cortex with chronic epileptic activity

Cyclic AMP accumulations elicited by adenosine analogues 2-chloroadenosine (2-CADO),R-N ⁶-phenylisopropyladenosine (R-PIA), andN ⁶-cyclohexyladenosine (CHA) were investigated in cortical slices of chronic iron-induced epileptic rats. Cyclic AMP accumulation was elicited 9-to 18-fold by 2-CADO and it was elicited 5-to 7-fold by eitherR-PIA or CHA; 2-CADO was more potent thanR-PIA or CHA in eliciting cyclic AMP accumulation. The adenosine analogues elicited cyclic AMP accumulation in a dose-dependent manner, and the elicitation was inhibited by the adenosine antagonist 8-phenyltheophylline. The 2-CADO-elicited accumulation of cyclic AMP was greatly increased in the cortical region on the primary epileptic side, while theR-PIA-or CHA-elicited accumulation did not change in any cortical region. The deviation detected only in the 2-CADO-elicited accumulation of cyclic AMP may be due to the difference in relative potency for adenosine receptors of the adenosine analogues. The results suggest that adenosine receptormediated generation of cyclic AMP is altered in the primary region of iron-induced epileptic cortex, in which heterogeneous alterations in different adenosine receptor subtypes may occur in the epileptic process.     

Effect of monoamine receptor agonists and antagonists on cyclic AMP accumulation in human cerebral cortex slices.

In human cerebral cortex slices noradrenaline, isoproterenol (a beta-adrenergic agonist), dopamine, apomorphine (a dopaminergic agonist), and serotonin stimulated cyclic AMP formation: noradrenaline greater than or equal to isoproterenol greater than dopamine = apomorphine = serotonin. Clonidine (and alpha-adrenergic agonist) was ineffective in stimulating cyclic AMP formation in temporal cortex slices. The stimulatory effect of noradrenaline and isoproterenol was blocked by propranolol (a beta-adrenergic blocker) but not by phentolamine (an alpha-adrenergic blocker). Pimozide (a selective dopaminergic antagonist) inhibited the increase of cyclic AMP formation induced by dopamine or apomorphine but not that induced by noradrenaline, isoproterenol, or serotonin. Neither propranolol or phentolamine had any effect on dopamine- or serotonin-stimulated cyclic AMP formation. Chlorpromazine blocked the increase of cyclic AMP formation induced by noradrenaline, dopamine or serotonin, while cyproheptadine, a putative central serotonergic antagonist, was ineffective. These observations suggest that there may be at least two monoamine-sensitive adenylate cyclases in human cerebral cortex which have the characteristics of a beta-adrenergic and a dopaminergic receptor, respectively, and also possibly a serotonergic receptor.     

Thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of the domestic mallard

Summary The distribution of immunoreactive thyrotropin-releasing hormone (TRH) in the central nervous system of the domestic mallard was studied by means of the peroxidase-antiperoxidase technique. After colchicine pretreatment, the highest number of TRH-immunoreactive perikarya was found in the parvocellular subdivision of the paraventricular nucleus and in the preoptic region; a smaller number of immunostained perikarya was observed in the lateral hypothalamic area and in the posterior medial hypothalamic nucleus. TRH-immunoreactive nerve fibers were detected throughout the hypothalamus, forming a dense network in the periventricular area, paraventricular nucleus, preoptic-suprachiasmatic region, and baso-lateral hypothalamic area. TRH-containing nerve fibers and terminals occurred in the organon vasculosum of the lamina terminalis and in the external zone of the median eminence in juxtaposition with hypophyseal portal vessels. Scattered fibers were also seen in the internal zone of the median eminence and in the rostral portion of the neural lobe. Numerous TRH-immunoreactive fibers were detected in extra-hypothalamic brain regions: the highest number of immunoreactive nerve fibers was found in the lateral septum, nucleus accumbens, olfactory tubercle, and parolfactory lobe. Moderate numbers of fibers were located in the basal forebrain, dorsomedial thalamic nuclei, hippocampus, interpeduncular nucleus, and the central gray of the mesencephalon. The present findings suggest that TRH may be involved in hypophysiotropic regulatory mechanisms and, in addition, may also act as neuromodulator or neurotransmitter in other regions of the avian brain.     

Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect.

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-adrenergic interaction with stimulators of cyclic AMP concentrations in canine thyroid slices.

Thyroid stimulating hormone (TSH) increased cyclic AMP levels approximately 10–20 fold in canine thyroid slices after 30 min incubation. Thereafter the cyclic AMP level declined reaching about 50% of the maximal by 90 min even in the presence of 10 mM theophylline. When phentolamine, an α-adrenergic blocker, was added with TSH to the incubation medium, the decline of cyclic AMP levels that followed the peak was markedly diminished. The maximal effect of phentolamine was observed at a concentration of 10^?6M. A similar decline of the cyclic AMP levels after the peak was observed when the tissues was stimulated by prostaglandin E₁ or cholera toxin and the decline was again prevented by phentolamine. Phentolamine alone had no significant effect on the basal cyclic AMP levels. Phenylephrine, an α-adrenergic agonist, diminished the rise of cyclic AMP levels induced by TSH.Norephinephrine, a physiologic adrenergic stimulator, caused a marked inhibition of the elevation of cyclic AMP levels induced by prostaglandin E₁ or cholera toxin as was the case by TSH (Life Sciences 21, 607, 1977). The norepinephrine effect was abolished by phentolamine, but not by propranolol, a β-adrenergic blocker.These results indicate that α-adrenergic actions may be involved in the counter-regulation of cyclic AMP levels in canine thyroid glands.     

Thyrotropin-releasing hormone and cyclic AMP activate distinctive pathways of protein phosphorylation in GH pituitary cells

The studies reported here were undertaken to clarify the cellular mechanism of the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), in clonal, hormone-responsive GH pituitary cells and to assess the possibility of a role for cyclic AMP as a mediator of TRH action. We investigated patterns of protein phosphorylation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of high speed supernatant and pellet fractions from untreated and treated GH cells. Brief treatment of cells with agents which elevate or mimic cellular cyclic AMP (8-bromo cyclic AMP, dibutyryl cyclic AMP, vasoactive intestinal polypeptide or cholera toxin) stimulated the phosphorylation of five supernatant peptides (41, 45, 47, 72, and 82 kilodaltons) and one pellet peptide (135 kilodaltons) and decreased the phosphorylation of one supernatant peptide (55 kilodaltons). In contrast, TRH promoted the phosphorylation of four different supernatant peptides (two 59, 65, and 80 kilodaltons). In addition, TRH also stimulated the phosphorylation of cyclic AMP-responsive 41-, 45-, and 82-kilodalton supernatant peptides and 135-kilodalton pellet protein and decreased the phosphorylation of 55-kilodalton supernatant peptide. Altered labeling of 47- and 72-kilodalton supernatant peptides, however, was not observed with TRH. Time course studies, as well as the overlapping biological action of TRH and vasoactive intestinal polypeptide, lead us to conclude that these peptide hormones utilize distinct, parallel pathways which converge at some late step. Furthermore, the results indicate that effects of TRH are mediated by a cyclic AMP-independent pathway.     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.     

Thyrotropin-releasing hormone (TRH) precursor processing. Characterization of mature TRH and non-TRH peptides synthesized by transfected mammalian cells

Prepro-thyrotropin-releasing hormone (TRH) contains five TRH progenitor sequences and at least six other potential peptides (Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G., and Goodman, R. H. (1986a) Science 231, 159-161). Previous studies using radioimmunoassays developed against discrete regions of prepro-TRH have demonstrated that several of the potential peptides are present in rat brain and pancreas (Wu, P., Lechan, R. M., and Jackson, I. M. D. (1987) Endocrinology 121, 108-115; Wu, P. and Jackson, I. M. D. (1988a) Brain Res. 456, 22-28; Wu, P., and Jackson, I. M. D. (1988b) Regul. Pept. 22, 347-360). However, the low level of peptides present in intact tissues has made isolation of the peptides difficult. CA77 cells, a medullary thyroid carcinoma cell line, also express prepro-TRH and display processing similar to that found in tissues. However, peptide content in this tumor cell line is enhanced only 3-fold compared with normal tissues (Sevarino, K. A., Wu, P., Jackson, I. M. D., Roos, B. A., Mandel, G., and Goodman, R. H. (1988) J. Biol. Chem. 263, 620-623). To achieve higher levels of expression for facilitating peptide sequencing studies and to see if alternate processing of prepro-TRH could be detected in different cell types, we transfected into 3T3, GH4, AtT20, and RIN 5F cells a cDNA vector under control of the cytomegalovirus immediate-early promoter. 3T3 and GH4 cells failed to process prepro-TRH beyond cleavage of the signal sequence. Both AtT20 and RIN 5F cells efficiently cleaved the precursor at dibasic sites to generate mature TRH and the non-TRH peptides previously identified in vivo. Peptide content was up to 30 times greater than in hypothalamic extracts and 10 times greater than in CA77 cells. Secretion experiments with transfected AtT20 cells demonstrated that both mature TRH and the non-TRH peptides were secreted via a regulated secretory pathway similar to that utilized by endogenously synthesized peptides. We isolated several of the non-TRH peptides synthesized by transfected AtT20 cells and characterized these peptides by sequential Edman degradation. These studies identified the signal sequence cleavage site and determined that the non-TRH peptides are generated by cleavage at the dibasic sites flanking the five TRH progenitor sequences. Further, we determined that processing occurs at the Arg51-Arg52 site located in the amino-terminal portion of the precursor, the only dibasic site not flanking a TRH progenitor sequence.     

Thyrotropin-releasing hormone (TRH) is not thyrotropic but somatotropic in fed and starved adult chickens.

Adult fed and starved Warren chickens, 2 yr of age, and approaching the end of the second laying year, were injected iv with 1 of the following products: 10 micrograms of thyrotropin releasing hormone (TRH); 100 micrograms of bovine thyrotropin (bTSH); 100 micrograms of ovine growth hormone (oGH); saline. The influence on plasma concentrations of thyroxine (T4), triiodothyronine (T3) or chicken GH (cGH) were followed. Prior to injection, it was clear from the control values that starvation for 3 d decreased plasma levels of T3 and increased cGH, whereas 7 d of fasting increased T4 and cGH. The plasma levels of cGH were elevated greater than 10-fold at 15 min following the TRH challenge in food-deprived chickens compared to a less than 4-fold increase in normal fed hens. This increase was followed by a rise in T3 after 1 h, which was also more pronounced in the starved animals, whereas T4 decreased or remained unaffected. Increases in T4 can, however, be obtained with 100 micrograms TSH in normal fed (2-fold) or starved animals (greater than 3-fold). Following injection of 100 micrograms oGH, a significant increase in T3 levels was observed which in fed animals was already present at 30 min, but the higher levels persisted for 1 and 2 h in fed and starved hens. At the same time, a decrease in T4 was observed in both groups of GH-treated chickens. It is concluded that TRH at the dose used is not thyrotropic but has a somatotropic effect and is responsible for the peripheral conversion of T4 into T3.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Effects of thyrotropin releasing hormone and its analogue, DN 1417, on biopterin concentration and tryptophan hydroxylation in rat raphe slices

The effects of subchronic administration of thyrotropin releasing hormone (TRH) and its analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN 1417), on serotonin biosynthesis in situ were investigated in tissue slices of the midbrain raphe of rats. TRH or DN 1417 (10 mg/kg per day intraperitoneally) were administered to male Wistar rats for ten days. At twenty four hr after the last injection, tissue slices of the midbrain raphe were prepared and the rate of serotonin biosynthesis was estimated by measuring formation of 5-hydroxytryptophan (5-HTP) from tryptophan during inhibition of aromatic L-amino acid decarboxylase using high-performance liquid chromatography with fluorescence detection. Total biopterin content was determined by a specific radioimmunoassay. 5-HTP formation was decreased 22% and 29%, and total biopterin content 69% and 72%, in TRH- and DN 1417-treated rats, respectively. However, tryptophan concentration in raphe slices did not change. In contrast, the Vmax of tryptophan hydroxylase in the homogenate of the raphe nucleus in the presence of a saturating concentration of (6R)-L-erythro-tetrahydrobiopterin, the naturally occurring pterin cofactor, was significantly increased after repeated administration of TRH or DN 1417. These results indicate that reduction of in situ serotonin biosynthesis in tissue slices from the rats treated with TRH or DN 1417 subchronically contray to the increase in in vitro tryptophan hydroxylase may result from the decrease of the biopterin cofactor, and that changes in concentrations of the biopterin cofactor may play a regulatory role in serotonin biosynthesis in vivo under certain conditions.