Pro-TRH gene expression and precursor peptides in rat brain. Observations by hybridization analysis and immunocytochemistry.
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Animals↗
Biomedical subjects
R M Lechan
Publications and source records attributed to R M Lechan.
Vasoactive intestinal peptide in the anterior pituitary is increased in hypothyroidism.
Vasoactive intestinal peptide (VIP) and PRL have been reported to be colocalized in rat lactotropes. To determine whether induced hypothyroidism, known to reduce pituitary PRL concentration, also reduces pituitary concentration of VIP, rats were treated with antithyroid drugs for 3 weeks. Pituitary PRL concentration in male rats (micrograms/mg protein) was markedly reduced by this treatment (9.4 +/- 1.0 vs. 2.3 +/- 0.4 when extracted at pH 1.1, 17.9 +/- 3.0 vs. 3.4 + 0.4 when extracted at pH 7.4, 21.8 +/- 3.3 vs. 6.7 + 1.3 when extracted at pH 10.0). Contrary to expectation, pituitary VIP concentration was markedly increased in hypothyroidism; in males from 169.5 +/- 20.3 to 834.0 +/- 82.2 pg/mg protein, and in females (whose pituitary PRL had been similarly reduced) from 103.1/I +/- 34.1 to 771.6 +/- 100.9 pg/mg protein. Serum PRL was significantly reduced in hypothyroid males (7.4 +/- 1.6 vs. 28.9 +/- 12.2 ng/ml) whereas in females, serum PRL was not significantly altered (41.4 +/- 11.6 vs. 38.8 +/- 14.3 ng/ml). The effect of hypothyroidism was reversed by administration of T4 in physiological doses. The authenticity of pituitary immunoreactive VIP was further established by demonstrating chromatographic patterns by Sephadex G-50 gel exclusion and reverse phase HPLC separations identical to synthetic VIP. Immunohistochemically reactive VIP cells could not be demonstrated in normal pituitaries, but the marked increase in VIP in hypothyroid animals made it possible to visualize a population of VIP immunoreactive stellate cells which appear to be distinct from hypothyroid lactotropes and thyrotropes.
Animals↗
Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland.
Vasoactive intestinal peptide (VIP) is a secretagogue for pituitary prolactin, but the importance of this peptide in the normal control of prolactin secretion is unclear. Recent studies suggest VIP synthesis within the rat anterior pituitary. We have shown (Endocrinology 124:1077) that the content of rat pituitary VIP increases in hypothyroidism. To confirm in situ pituitary synthesis of VIP and determine whether thyroid hormone effects on pituitary VIP relate to changes in VIP mRNA, Northern and in situ hybridization analyses of VIP mRNA in rat pituitaries were performed. Northern hybridization demonstrated an RNA species from rat pituitary consistent with rat VIP mRNA. Hypothyroidism increased the content of pituitary VIP mRNA, and replacement with 1-thyroxine prevented this increase. In situ hybridization showed multiple, widely-distributed hybridizing cells in pituitaries from hypothyroid animals. A distinct population of VIP-producing pituitary cells exists which may serve to modulate prolactin secretion in a paracrine or autocrine fashion.
Animals↗
Neurons containing a N-terminal sequence of the TRH-prohormone (preproTRH53-74) are present in a unique location of the midbrain periaqueductal gray of the rat.
Using an antiserum (no. 373) raised against a tyrosinated analog of preproTRH53-74 [( Tyr1]preproTRH53-74 or pYT 22), we have demonstrated the presence of a discrete population of immunoreactive neurons in the midbrain periaqueductal gray (PAG). Relative to the distribution of serotonin, somatostatin, peptide histidine isoleucine (PHI), methionine enkephalin, substance P and neurotensin-containing neuronal perikarya in the PAG, neurons containing immunoreactive pYT 22 occupied a unique location in the ventrolateral PAG. In contrast, terminal fields containing these neuroactive substances with the exception of PHI, were seen in abundance in the region of the ventrolateral PAG neurons. These studies indicate that a non-TRH sequence contained within the N-terminal portion of the TRH prohormone are expressed in a distinct group of neurons in the ventrolateral PAG. The location of these neurons in the PAG in a region richly innervated by nerve terminals containing analgesia-mediating substances, suggests a possible role for proTRH-derived peptides in the modulation of nociception.
Animals↗
Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus.
Thyroid hormone administered systemically exerts negative feedback control of biosynthesis of the TRH pro-hormone in the hypothalamic paraventricular nucleus (PVN), the origin of neurons that regulate anterior pituitary TSH secretion, but not in any other group of TRH-synthesizing neurons in the brain. To determine whether this response is mediated by direct effects on PVN neurons, we studied the effect of unilateral stereotaxic implants of L-T3 into the anterior hypothalamus on the concentration of pro-TRH mRNA and pro-TRH in the PVN of hypothyroid rats. Because hypothalamic-pituitary-thyroid function is also regulated by central catecholamines, we also determined the effect of unilateral ablation of ascending catecholaminergic fibers to one side of the PVN by stereotaxic injection of 6-hydroxydopamine or transection of ascending catecholaminergic pathways. T3-implanted hypothyroid animals showed a marked reduction in pro-TRH mRNA and immunoreactive pro-TRH in medial parvocellular neurons of the PVN on the same side as the implant, but not in contralateral PVN neurons or TRH-synthesizing neurons in other hypothalamic regions. In contrast, hypothyroid animals implanted with pellets of hormonally inactive 3,5-diiodo-L-thyronine showed intense symmetric hybridization and immunoreaction product in both wings of the PVN. Despite marked unilateral reduction in the catecholamine innervation to the PVN, no reduction in pro-TRH mRNA or immunoreactive pro-TRH was observed in the PVN on the affected side compared to that on the unaffected side. These studies demonstrate that negative feedback regulation of thyroid hormone occurs directly on TRH neurons and is restricted only to those in the PVN tuberoinfundibular system.
Animals↗
Spinal cord TRH deficiency is associated with incomplete recovery of denervated muscle in the rat.
The raphe-spinal pathway, which contains co-localized serotonin (5-HT), thyrotropin-releasing hormone (TRH), and several TRH-prohormone-derived non-TRH peptides, projects to the ventral horn of the spinal cord. Pharmacologic ablation of this pathway with the 5-HT neurotoxin, 5,7-dihydroxytryptamine, in neonatal rats resulted in deficient recovery of plantar foot muscles, functionally denervated with botulinum toxin type A. Failure of reinnervation was suggested by slower and incomplete recovery of the plantar foot compound muscle action potential amplitude and by a reduced mean diameter of plantar foot muscle fibers in ablated rats. These findings indicate that deprivation of alpha motor neurons from descending raphe-spinal input interferes with their ability to respond to muscle-derived signals for reinnervation.
5,7-Dihydroxytryptamine↗
Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus.
Thyroid hormone is important in the regulation of synthesis and secretion of thyroid-stimulating hormone (TSH) in the anterior pituitary, but its role in the control of hypothalamic thyrotropin-releasing hormone (TRH) is controversial. To determine whether thyroid hormone regulates the function of TRH in the hypothalamic tuberoinfundibular system, a study was made of the effect of hypothyroidism on thyrotropin-releasing hormone messenger RNA (proTRH mRNA) and TRH prohormone in the rat paraventricular nucleus. Extracts of rat hypothalamic paraventricular nucleus were examined by quantitative Northern blot analysis, and coronal sections of rat brain were examined by in situ hybridization histochemistry and immunocytochemistry. A nearly twofold increase in proTRH mRNA was observed in hypothyroid animals; this increase could be obliterated by levothyroxine treatment, suggesting an inverse relation between circulating thyroid hormone and proTRH mRNA. In situ hybridization showed that this response occurred exclusively in medial parvocellular neurons of the paraventricular nucleus. A simultaneous increase in proTRH mRNA and immunoreactive TRH prohormone in this region suggests that hypothyroidism induces both transcription and translation of the TRH prohormone in the paraventricular nucleus.
Animals↗
Effect of spinal cord TRH deficiency on lower motorneuron function in the rat.
Thyrotropin-releasing hormone (TRH), present in high concentrations in the mammalian spinal cord, exerts excitatory effects on the alpha-motorneuron (AMN) via axodendritic contacts. We used the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) to deplete TRH from the ventral horn of the spinal cord of adult rats to determine whether the tripeptide may be trophic to the AMN. The rats were studied blindly and sequentially for 11 weeks. Motor performance remained normal by clinical and electrophysiologic assessments. AMN counts were not reduced in the lumbar cord, and gastrocnemius muscle showed no evidence of denervation in treated rats. We conclude that in the adult rat chronic ventral horn TRH deficiency does not lead to AMN degeneration and is not associated with a significant alteration of AMN function.
5,7-Dihydroxytryptamine↗
Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons. Ultrastructure, adrenergic innervation and putative transmitter action.
The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine-beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvocellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN. These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.
Animals↗
Paraventricular nucleus mediates prolactin secretory responses to restraint stress, ether stress, and 5-hydroxy-L-tryptophan injection in the rat.
The role of the paraventricular nucleus (PVN) in mediating acute stimulatory PRL responses was investigated in conscious male rats. Electrolytic lesions, verified histologically at autopsy, were stereotaxically made in the PVN region, and sham lesions were made in control rats. Blood was obtained through a chronically indwelling catheter in the right atrium. PVN-lesioned (PVL) rats showed significantly lower T3 levels 1 week after surgery (less than 34.4 ng/dl) compared with sham (mean +/- SEM, 91.2 +/- 5.0 ng/dl) and intact (86.8 +/- 2.0 ng/dl) animals, verifying a lesion in the PVN. T3 was restored to normal (95.6 +/- 1.8 ng/dl) by daily sc administration of T4 (10 micrograms/kg BW) for at least 4 days before the day of the experiments. Basal PRL levels in PVL rats did not differ significantly from those in control or sham-lesioned animals. In response to restraint stress, plasma PRL levels of PVL rats did not rise, in contrast to marked elevation in PRL in sham and intact rats [PRL levels (mean +/- SEM; nanograms per ml), basal to peak: PVL, 4.3 +/- 0.3 to 4.5 +/- 0.4; sham, 4.5 +/- 0.5 to 47.0 +/- 4.1; intact, 4.0 +/- 0.3 to 46.3 +/- 4.9]. PVL also resulted in the complete inhibition of PRL secretion induced by 30-min inhalation of ether (basal to peak: PVL, 3.3 +/- 0.3 to 4.5 +/- 0.2; sham, 5.7 +/- 0.8 to 19.9 +/- 0.9; intact, 3.3 +/- 0.4 to 27.9 +/- 4.0). The stimulatory effect on plasma PRL in sham and intact rats by one iv bolus injection of the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP; 10 mg/kg BW) was completely abolished in PVL animals (basal to peak: PVL, 3.7 +/- 0.6 to 5.2 +/- 1.4; sham, 6.7 +/- 0.6 to 36.0 +/- 0.5; intact, 4.1 +/- 1.2 to 33.3 +/- 3.2). In contrast to the marked alteration in PRL regulation, PVL rats exhibited a typical ultradian rhythm of plasma GH secretion during a 6-h observation period and increased release of GH induced by iv injection of 5-HTP [GH (nanograms per ml), basal to peak; PVL, 4.5 +/- 0.6 to 21.0 +/- 4.9; sham, 3.7 +/- 0.3 to 18.4 +/- 4.4; intact, 2.9 +/- 0.1 to 17.8 +/- 3.5]. These findings indicate that PRL responses to stress and to serotonin act through the PVN, the site of origin of several putative PRL-releasing factors.(ABSTRACT TRUNCATED AT 400 WORDS)
5-Hydroxytryptophan↗
Identification and characterization of thyrotropin-releasing hormone precursor peptides in rat brain.
The sequence of rat hypothalamic pro-TRH, deduced by sequencing of cDNA, contains five copies of the TRH progenitor sequence Gln-His-Pro-Gly flanked by paired basic amino acid sequences. The TRH prohormone also contains leader and trailer sequences and four intervening sequences. We have developed two RIAs against synthetic peptides corresponding to sequences within the deduced pro-TRH sequence and have used these assays to identify and partially characterize four pro-TRH-derived peptides distinct from TRH in extracts of rat brain tissue. Two of these peptides contain incompletely processed TRH sequences; the other two peptides are probably derived from the N-terminal leader sequence. The presence of these authentic pro-TRH-derived peptides indicates that pro-TRH may give rise to a family of peptides other than TRH, some of which may be of biological significance.
Amino Acid Sequence↗
Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain in situ hybridization.
We studied the distribution of pro- TRH mRNA in rat brain by in situ hybridization histochemistry using radiolabeled single stranded cRNA probes to confirm the hypothesis that the TRH precursor is distributed beyond regions that contain immunoreactive TRH. All regions of the central nervous system previously recognized to contain TRH showed hybridization. Hypophysiotropic neurons in the medial parvocellular division of the paraventricular nucleus showed more intense hybridization than anterior parvocellular division cells, suggesting regional differences in expression. In addition, regions not previously recognized to contain TRH in neuronal perikarya by immunocytochemistry showed specific hybridization for pro-TRH mRNA. These include cells in the olfactory bulbs, dorsal motor nucleus of the vagus, ventrolateral periaqueductal gray, reticular nucleus of the thalamus, and anterior commissural nucleus. Only a single hybridizing band was observed on Northern blots of RNA extracts of the periaqueductal gray and reticular nucleus, identical to that seen in extracts of the paraventricular nucleus. The appearance of pro-TRH mRNA in neurons not previously recognized to contain TRH but which contain the prohormone suggests that non-TRH peptides within the TRH precursor may be preferentially expressed in certain regions of the brain.
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Immunocytochemical distribution in rat brain of putative peptides derived from thyrotropin-releasing hormone prohormone.
Processing of the TRH prohormone (Pro-TRH), a protein of approximately 26,000 mol wt, could yield 5 copies of TRH, as well as extended forms of TRH and several other non-TRH peptides. To determine whether some of these peptides are formed and transported by axons in the rat brain, we used antiserum to synthetic peptides corresponding to portions of pro-TRH. These included the N-tyrosyl analogs [Tyr0]prepro-TRH-(25-50) (pYE27) and [Tyr1]prepro-TRH-(53-74) (pYT22) contained within the N-terminal flanking region of the prohormone, the N-tyrosyl analog [Tyr0]prepro-TRH-(165-186) (pYS23), expanding the fourth progenitor sequence of TRH in the midportion of the prohormone, and the synthetic peptide pAC12 corresponding to the first 12 amino acids of the C-terminal flanking region or prepro-TRH-(208-219). All antisera showed staining in neuronal perikarya and processes in all regions of the brain previously demonstrated to immunostain for TRH, including dense innervation of the external zone of the median eminence. In addition, these antisera immunostained regions of the brain not previously immunopositive for TRH. Not all regions reactive with antiserum to [Tyr0]prepro-TRH-(25-50) were also recognized by anti-pYT, -pYS, and -pAC. These studies confirm the presence of the deduced non-TRH sequences within the TRH precursor and their formation and transport in vivo in the central nervous system. The presence of immunoreactivity in regions of the brain that do not contain TRH and the variability of immunostaining of the different antisera in some of these regions suggest regional preferential processing of pro-TRH to other peptides that may be biologically active.
Animals↗
Localization of somatostatin mRNA in the gut, pancreas and thyroid gland of the rat using antisense RNA probes for in situ hybridization.
A method for the localization of rat pre-prosomatostatin mRNA by in situ hybridization with 32P- and 3H-labeled antisense RNA probes is reported. Somatostatin mRNA was detected in endocrine cells of the rat gut mucosa, pancreas and thyroid in a distribution identical to immunoreactive somatostatin. In addition, in situ hybridization allowed localization of reactive neurons in the submucous and myenteric plexus of the gut, sites which are variably positive or negative for immunoreactive somatostatin even after colchicine treatment. These studies indicate that in situ hybridization is more sensitive than immunohistochemistry in some instances for demonstration of somatostatin in gene expression.
Animals↗
Neuroendocrinology of pituitary hormone regulation.
Hormone secretion from the pituitary gland is dependent on the intimate association between the hypothalamus and pituitary by vascular and neuronal connections. The anatomic organization of these networks and their specific peptide and/or amide mediators are described in detail. Pertinent factors governing hypothalamic regulation of anterior pituitary secretion are also discussed.
Humans↗
Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice.
Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.
Animals↗
Thyrotropin-releasing hormone precursor: characterization in rat brain.
To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.
Amino Acid Sequence↗
The syndrome of inappropriate antidiuretic hormone secretion associated with induction chemotherapy for squamous cell carcinoma of the head and neck.
Two patients with squamous cell carcinoma of the head and neck are reported in whom the syndrome of inappropriate antidiuretic hormone (SIADH) secretion occurred transiently during the rapid cytolytic phase of tumor destruction after chemotherapy with cis-platinum diamminedichloride and bleomycin. Immunoperoxidase staining for ADH of the original biopsy specimens from both patients was negative. Possible mechanisms for and the implications of the production of SIADH in this setting are discussed.
Antineoplastic Agents↗