Thyrotropin-releasing Hormone Immunoreactivity Research Articles

TRH and NPY Interact to Regulate Dynamic Changes in Energy Balance in the Male Zebra Finch.

In contrast to mammals, birds have a higher basal metabolic rate and undertake wide range of energy-demanding activities. As a consequence, food deprivation for birds, even for a short period, poses major energy challenge. The energy-regulating hypothalamic homeostatic mechanisms, although extensively studied in mammals, are far from clear in the case of birds. We focus on the interplay between neuropeptide Y (NPY) and thyrotropin-releasing hormone (TRH), 2 of the most important hypothalamic signaling agents, in modulating the energy balance in a bird model, the zebra finch, Taeniopygia guttata. TRH neurons were confined to a few nuclei in the preoptic area and hypothalamus, and fibers widely distributed. The majority of TRH neurons in the hypothalamic paraventricular nucleus (PVN) whose axons terminate in median eminence were contacted by NPY-containing axons. Compared to fed animals, fasting significantly reduced body weight, PVN pro-TRH messenger RNA (mRNA) and TRH immunoreactivity, but increased NPY mRNA and NPY immunoreactivity in the infundibular nucleus (IN, avian homologue of mammalian arcuate nucleus) and PVN. Refeeding for a short duration restored PVN pro-TRH and IN NPY mRNA, and PVN NPY innervation to fed levels. Compared to control tissues, treatment of the hypothalamic superfused slices with NPY or an NPY-Y1 receptor agonist significantly reduced TRH immunoreactivity, a response blocked by treatment with a Y1-receptor antagonist. We describe a detailed neuroanatomical map of TRH-equipped elements, identify new TRH-producing neuronal groups in the avian brain, and demonstrate rapid restoration of the fasting-induced suppression of PVN TRH following refeeding. We further show that NPY via Y1 receptors may regulate PVN TRH neurons to control energy balance in T. guttata.

Receptors for thyrotropin-releasing hormone, thyroid-stimulating hormone, and thyroid hormones in the macaque uterus

Thyroid gland dysfunction is associated with menstrual cycle disturbances, infertility, and increased risk of miscarriage, but the mechanisms are poorly understood. However, little is known about the regulation of these receptors in the uterus. The aim of this study was to determine the effects of long-term treatment with steroid hormones on the expression, distribution, and regulation of the receptors for thyrotropin-releasing hormone (TRHR) and thyroid-stimulating hormone (TSHR), thyroid hormone receptor α1/α2 (THRα1/α2), and THRβ1 in the uterus of surgically menopausal monkeys. Eighty-eight cynomolgus macaques were ovariectomized and treated orally with conjugated equine estrogens (CEE; n = 20), a combination of CEE and medroxyprogesterone acetate (MPA; n = 20), or tibolone (n = 28) for 2 years. The control group (OvxC; n = 20) received no treatment. Immunohistochemistry was used to evaluate the protein expression and distribution of the receptors in luminal epithelium, glands, stroma, and myometrium of the uterus. Immunostaining of TRHR, TSHR, and THRs was detected in all uterine compartments. Epithelial immunostaining of TRHR was down-regulated in the CEE + MPA group, whereas in stroma, both TRHR and TSHR were increased by CEE + MPA treatment as compared with OvxC. TRHR immunoreactivity was up-regulated, but THRα and THRβ were down-regulated, in the myometrium of the CEE and CEE + MPA groups. The thyroid-stimulating hormone level was higher in the CEE and tibolone groups as compared with OvxC, but the level of free thyroxin did not differ between groups. All receptors involved in thyroid hormone function are expressed in monkey uterus, and they are all regulated by long-term steroid hormone treatment. These findings suggest that there is a possibility of direct actions of thyroid hormones, thyroid-stimulating hormone and thyrotropin-releasing hormone on uterine function.

Beta-casomorphin inhibits thyrotropin secretion in rats.

The effects of beta-casomorphin on thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) secretion in rats were studied. beta-casomorphin (300 micrograms/kg) was injected iv, and the rats were serially decapitated. TRH, TSH and thyroid hormone were measured by radioimmunoassay. The hypothalamic immunoreactive TRH (ir-TRH) contents increased significantly after beta-casomorphin injection, whereas its plasma concentrations tended to decrease, but not significantly. The plasma TSH levels decreased significantly in a dose-related manner with a nadir at 40 min. after the injection. The thyroid hormone levels showed no change. The plasma ir-TRH and TSH responses to cold were inhibited by beta-casomorphin, but the plasma TSH response to TRH was not. Naloxone partially blocked the inhibitory effect of beta-casomorphin on plasma TSH levels. In the haloperidol-pretreated group, the inhibitory effect of beta-casomorphin on plasma TSH levels was prevented, but not in the L-DOPA, para-chlorophenylalanine- or 5-hydroxytryptophan-pretreated group. These drugs alone did not affect plasma TSH levels in terms of the dose used. The findings suggest that beta-casomorphin acts at the hypothalamus to inhibit TRH release, and its effect are modified by a dopaminergic system.

Effects of growth hormone-releasing hormone and corticotropin-releasing hormone on the release of thyrotropin-releasing hormone from the rat hypothalamus in vitro.

Effects of growth hormone-releasing hormone (GRH) and corticotropin-releasing hormone (CRH) on the release of immunoreactive thyrotropin-releasing hormone (ir-TRH) from the rat hypothalamus in vitro were studied. The rat hypothalamus was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) for 20 min. The amount of ir-TRH release into the medium was measured by radioimmunoassay. The ir-TRH release from the rat hypothalamus was inhibited significantly in a dose-related manner with the addition of GRH or CRH. These findings suggest that GRH and CRH inhibit ir-TRH release from the rat hypothalamus in vitro.

Litorin (bombesin family) inhibits thyrotropin secretion in rats.

The effects of the peripheral administration of litorin on thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) secretion were studied in rats. Litorin (400 micrograms/kg) was injected iv, and the rats were serially decapitated. TRH, TSH and thyroid hormone were measured by radioimmunoassay. The hypothalamic immunoreactive TRH (ir-TRH) content increased significantly after litorin injection, whereas its plasma concentration tended to decrease, but not significantly. The plasma TSH levels decreased significantly in a dose-related manner with a nadir at 20 min. after the injection. The plasma thyroid hormone levels showed no changes. The plasma ir-TRH and TSH responses to cold were inhibited by litorin, but the plasma TSH response to TRH was not affected. In the pimozide- or para-chlorophenylalanine-pretreated group, the inhibitory effect of litorin on TSH levels was prevented, but not in the L-DOPA- or 5-hydroxytryptophan-pretreated group. These drugs alone did not affect plasma TSH levels in terms of the dose used. The inactivation of TRH immunoreactivity by hypothalamus or plasma in vitro after litorin injection did not differ from that of the saline-treated group. These findings suggest that litorin acts on the hypothalamus to inhibit TRH release, and that its effects are modified by amines of the central nervous system.

Physalaemin inhibits thyrotropin secretion in rats.

The effects of physalaemin on thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) secretion were studied in rats. Physalaemin (150 micrograms/kg) was injected iv, and the rats were serially decapitated. TRH, TSH and thyroid hormone were measured by radioimmunoassay. The hypothalamic immunoreactive TRH (ir-TRH) contents increased significantly after physalaemin injection, whereas its plasma concentrations tended to decrease, but not significantly. The plasma TSH levels decreased significantly in a dose-related manner with a nadir at 30 min. The plasma ir-TRH and TSH responses to cold were inhibited by physalaemin, but the plasma TSH response to TRH was not affected. The plasma thyroid hormone levels did not change significantly. In the pimozide- or para-chlorophenylalanine-pretreated group, the inhibitory effect of physalaemin on TSH levels was prevented, but not in the L-DOPA- or 5-hydroxytryptophan-pretreated group. These drugs alone did not affect plasma TSH levels in terms of the dose used. The inactivation of TRH immunoreactivity by the hypothalamus or plasma in vitro after physalaemin injection did not differ from that of the control. These findings suggest that physalaemin acts on the hypothalamus to inhibit TRH release, and its effects are at least partially modified by amines of the central nervous system.

Expression of thyrotropin-releasing hormone by human melanoma and nevi.

Thyrotropin-releasing hormone (TRH) is a tripeptide hormone produced by the hypothalamus in response to hypothyroidism. RNA transcripts for the TRH prohormone have recently been described in melanoma cell lines. To expand these findings, we have examined cultured melanoma cells and melanocytes, human melanoma tumors, and nevi for the expression of TRH. Five melanoma cell lines were analyzed by reverse transcription-PCR/Southern blotting for preproTRH message. The same melanoma lines and two melanocyte lines were examined by immunocytochemistry for TRH protein expression and for growth response to exogenous TRH. Immunohistochemistry was used to test for TRH protein in sections of 19 melanomas, 33 dysplastic nevi, and 27 benign nevi. TRH message and protein were detected in all melanoma cell lines examined. Melanocytes were also found to express TRH protein. Four of the five melanoma cell lines but neither melanocyte line responded with a increase in proliferation to low concentrations of exogenous TRH. TRH immunoreactivity was observed in 12 of 19 melanomas (63%), 23 of 33 (69.7%) dysplastic nevi, and 14 of 27 (51.9%) benign nevi. Expression in dysplastic nevi was significantly greater than in benign nevi. Upon separate analysis of nevi from melanoma patients, the difference between dysplastic and benign nevi was even more significant. However, in healthy individuals, no difference between dysplastic and benign nevi was observed. Furthermore, dysplastic nevi from melanoma patients had a significantly higher percentage of TRH-positive cells when compared with healthy individuals. TRH is commonly expressed by melanomas and dysplastic nevi and may function as a melanoma autocrine growth factor. The presence of TRH in dysplastic nevi may be predictive for the development of melanoma. Our findings have significant clinical and biological implications for future research into the early stages of melanoma initiation and progression.

Distribution of thyrotropin-releasing hormone immunoreactivity in the brain of the dogfish Scyliorhinus canicula.

To improve knowledge of the peptidergic systems of elasmobranch brains, the distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons and fibers was studied in the brain of the small-spotted dogfish (Scyliorhinus canicula L.). In the olfactory bulbs, small granule neurons richly innervated the olfactory glomeruli. In the telencephalic hemispheres, small TRHir neurons were observed in the superficial dorsal pallium, whereas TRHir fibers were widely distributed in pallial and subpallial regions. In the preoptic region, TRHir neurons formed a caudal ventrolateral group in the preoptic nucleus. In the hypothalamus, the most conspicuous TRHir populations were associated with the lateral hypothalamic recess, but small TRHir populations were found in the posterior tubercle and ventral wall of the posterior recess. The preoptic region and hypothalamus exhibited rich innervation by TRHir fibers. TRHir fibers were observed coursing to the neurohypophysis and the neuroepithelium of the saccus vasculosus, but not to the neurohemal region of the median eminence. Some stellate-like TRHir cells were observed in a few cell cords of the neurointermediate lobe of the hypophysis. The thalamus, pretectum, and midbrain lacked TRHir neurons. Further TRHir neuronal populations were observed in the central gray and superior raphe nucleus of the isthmus, and a few TRHir cells were located in the nucleus of the trigeminal descending tract at the level of the rostral spinal cord. In the brainstem, the central gray, interpeduncular nucleus, secondary visceral region of the isthmus, rhombencephalic raphe, inferior olive, vagal lobe, and Cajal's commissural nucleus were all richly TRHir-innervated. Comparison of the distribution of TRHir neurons observed in the dogfish brain with that observed in teleosts and tetrapods reveals strong resemblance but also interesting differences, indicating the presence of both a conserved basic vertebrate pattern and a number of derived characters.

Electroconvulsive seizures modulate levels of thyrotropin releasing hormone and related peptides in rat hypothalamus, cingulate and lateral cerebellum

We have studied the neuroanatomic extent of electroconvulsive (ECS)-responsive prepro-TRH and TRH-related gene expression and its possible interaction with forced swimming. Young adult male Wistar rats were treated in a 2×2 Latin square protocol of swimming, no swimming, three daily ECS or sham ECS. Sixteen different brain regions were dissected and immunoreactivity measured for TRH ( pGlu–His–Pro–NH 2); TRH–Gly, a TRH precursor; Ps4, a prepro-TRH-derived TRH-enhancing decapeptide, and EEP ( pGlu–Glu–Pro–NH 2). ECS, in addition to elevating TRH–immunoreactivity (TRH–IR), TRH–Gly–IR, Ps4–IR and EEP–IR levels in the limbic regions, as we have previously reported, also significantly increased Ps4–IR levels in hypothalamus, posterior cingulate and lateral cerebellum, and increased TRH–Gly–IR levels in hypothalamus. Interestingly, the combination of ECS and swimming significantly reduced the levels of TRH–Gly–IR in the anterior cingulate compared to the sham ECS-no swim group. The combined use of high-pressure liquid chromatography and the EEP radioimmunoassay (RIA) revealed that pGlu–Tyr–Pro–NH 2 and/or pGlu–Phe–Pro–NH 2 occur in amygdala, anterior cingulate, frontal cortex, entorhinal cortex, lateral cerebellum and striatum and make a substantial contribution to the EEP–IR and TRH–IR. We conclude that ECS can alter the expression and secretion of TRH-related peptides in the hypothalamus, cingulate and lateral cerebellum. Such effects have not previously been reported in these limbic and extra-limbic regions which are increasingly implicated in the autonomic, behavioral and volitional changes which accompany severe depression and its treatment.

Thyrotropin-releasing hormone immunoreactivity in the brain and the pituitary during Bufo arenarum development

The ontogeny of the thyrotropin releasing hormone (TRH) neuronal system was evaluated by immunocytochemistry in Bufo arenarum. The first appearance of TRH immunoreactive fibers was at early premetamorphosis. These fibers were found in small numbers and weakly stained in the median eminence and pars nervosa. With the advance of larval development, TRH-like material stained intensely and tended to aggregate in the median eminence, pars nervosa and pars intermedia. At climax stages immunoreactive fibers and perikarya (weakly stained) were also identified in the preoptic area. In adult specimens TRH perikarya and neuronal fibers were found in the preoptic and infundibular nuclei of the hypothalamus and in the amygdala, septum and diagonal band of Broca of the telencephalon. In addition, TRH neuronal fibers and endings were found in the preoptic-hypophyseal tract, the external zone of the median eminence, the pars nervosa and pars intermedia. Fibers were absent in the pars distalis. This study represents the first immunocytochemical demonstration of TRH in Bufo species, and serves as a basis for clarification of the neuroendocrine regulation of metamorphosis.

Development of thyrotropin-releasing hormone immunoreactivity in the brain of the brown troutSalmo trutta fario

The development and adult distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons in the brain of the brown trout, Salmo trutta fario, was studied with the streptavidin-biotin immunohistochemical method. Study of embryos, alevin, and juveniles revealed groups of TRHir neurons in the mesencephalon and rhombencephalon that have not been noted previously in adult teleosts. The earliest TRHir cells observed were those of the trigeminal motor nucleus, which expressed this substance only in embryos and alevins. In the forebrain, early-arising TRH populations were observed in the supra- and postcommissural region of the ventral telencephalic area, the anterior parvocellular preoptic nucleus, the organon vasculosum laminae terminalis, the magnocellular preoptic nucleus, the suprachiasmatic nucleus, and the posterior tuberal nucleus. TRHir cells of the olfactory bulb, abundant in the adult, appeared later. A small TRHir neuronal population was transiently observed in the habenula of alevins and juveniles. The laminar nucleus of the mesencephalon contained a small population of TRH cells in alevins and juveniles. In the isthmus, TRH was observed in cells of the interpeduncular nucleus, the nucleus isthmi, the dorsolateral tegmental nucleus, the superior reticular nucleus, and the central gray, although perikarya were TRHir only in alevin and/or juvenile stages. Some vagal motoneurons were TRHir from the late embryo stage onward. TRHir fibers were abundant in several forebrain regions of alevins and juveniles, including the medial region of the dorsal telencephalic area, the ventral telencephalic area and commissural region, the preoptic neuropil, the posterior tubercle, the anterior tuberal nucleus, and the posterior hypothalamic lobe. TRHir fibers invaded the neurohypophysis in early alevins, and their number increased subsequently to adulthood. The parvocellular superficial pretectal nucleus and the optic tectum received a rich TRHir innervation from juvenile stages onward. The interpeduncular nucleus and the secondary gustatory nucleus contained many TRHir fibers. In the rhombencephalon, TRHir fibers were scarce, except in the ventrolateral regions and the inferior olive. The distribution of TRHir fibers suggests that they were mainly related to hypophysiotropic and visceral centers, although the presence of TRH in centers related to the visual system indicates that TRH also plays other roles in the brain. We discuss the possibility that the strong expression of TRH in the embryonic trigeminal motoneurons plays a role in head morphogenesis.

Cloning of Thyrotropin-Releasing Hormone Precursor and Receptor in Rat Thymus, Adrenal Gland, and Testis

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Cloning of thyrotropin-releasing hormone precursor and receptor in rat thymus, adrenal gland, and testis.

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Implications of Gastric Topical Bioactive Peptides in Ammonia-induced Acute Gastric Mucosal Lesions in Rats

Background: Ammonia, one of the pathogenic factors in Helicobacter pylori-induced mucosal injury, induces acute mucosal lesions in the rat glandular stomach. Methods: The effect of ammonia administered intragastrically on gastric peptides was investigated in urethane-anesthetized rats. Results: Gastric mucosal lesions were observed 5 min after 0.3% ammonia (4 ml/kg, intragastrically). Immunoreactive endothelin-1 (ET-1) and immunoreactive thyrotropin-releasing hormone (TRH) concentrations in the gastric wall decreased significantly 2 min and 5 min after ammonia, respectively. A significant increase in gastric juice immunoreactive ET-1 and TRH levels was reciprocally observed. The severity of gastric mucosal injury and changes in gastric immunoreactive ET-1 and TRH concentrations were shown to be concentration-dependent 30 min after ammonia. Atropine (5 mg/kg, intraperitoneally, -20 min) prevented ammonia-induced injury accompanied by a block of changes in gastric immunorective ET-1 and TRH concentrations. BQ-485 (ETA receptor antagonist; 2 mg/kg, subcutaneously) also abolished ammonia-induced lesions and gastric immunoreactive TRH changes. Conclusions: These findings suggested that gastric ET-1 and TRH play a role in ammonia-induced gastric mucosal injury mediated via a muscarine and an ETA receptor.

Thyrotropin-releasing hormone immunoreactivity in rat adrenal tissue is localized in mast cells.

Pro-thyrotropin-releasing hormone (pro-TRH) has been shown to be present throughout the central nervous system and in several peripheral tissues. In adrenals, TRH immunoreactivity has been reported but not characterized. We show here that two rat pro-TRH-derived peptides, TRH and prepro-TRH[160-169] (Ps4), were detected in extracts of rat adrenal glands by enzyme immunoassay. Endogenous TRH and Ps4 were purified by gel exclusion chromatography and reverse-phase HPLC. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards. By using the indirect immunofluorescence technique, TRH-immunoreactive cell bodies were found rather widely scattered outside the adrenal, in the brown adipose tissue in which the gland is embedded. These immunofluorescent cells have the typical appearance of mast cells and are metachromatic after histological staining with acidic Toluidine Blue. Our findings suggest that pro-TRH-derived peptides exist in rat mast cells.

Neurotransmission by neurons that use serotonin, noradrenaline, glutamate, glycine, and gamma-aminobutyric acid in the normal and injured spinal cord.

The science of neurotransmission in the normal and injured spinal cord has grown. This is a review of neurotransmission using serotonin, noradrenaline, glutamate, glycine, and gamma-aminobutyric acid. The literature on spinal cord neurotransmission and changes that occur with trauma are reviewed. Serotonergic and noradrenergic bulbospinal tracts influence interneurons and motor neurons via postsynaptic inhibition. Colocalization of serotonin and thyrotropin-releasing hormone occur in bulbospinal tracts, and reduction in uptake and thyrotropin-releasing hormone immunoreactivity quantitates the degree of injury in chronic spinal cord injury (SCI). Glutamate functions as an excitatory transmitter of some dorsal root afferent neurons and interneurons modulating nociceptive and motor neurons via at least five different receptors. Reactive synaptogenesis occurs after SCI, leading to an increase in the number of excitatory glutamatergic synapses below the level of SCI. gamma-Aminobutyric acid is an inhibitory transmitter of spinal interneurons that functions both pre- and postsynaptically. After SCI, a reduction occurs in the number of inhibitory synapses related to gamma-aminobutyric acid. Glycine is an inhibitory neurotransmitter that functions postsynaptically and also modulates the N-methyl-D-aspartate receptor. After SCI, a reduction in glycine adds to the loss of local inhibition below the SCI.

Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis.

Endogenous TRH-like products were analyzed in rat testis using a TRH enzyme immunoassay coupled to molecular sieve filtration and HPLC identification. Of the three immunoreactive peptides detected, the two major forms exhibited the same chromatographic properties as synthetic TRH and pGlu-Phe-Pro-NH2. These peptides accounted respectively for 33% and 54% of the total TRH immunoreactivity in the testis. In rat serum, HPLC analysis showed the presence of only one immunoreactive peak with a retention time similar to that of authentic TRH. Ethylene dimethanesulfonate treatment of adult rat was used to assess the effect of Leydig cell destruction on the TRH immunoreactivity content. The concentration of the three TRH immunoreactive peptides fell gradually after treatment and reached a minimum at day 21, where a marked decrease (98%) was observed. At day 41, the regeneration of Leydig cells was achieved, as shown by histochemistry and measurements of serum testosterone and testicular weight. However, no restoration of the TRH immunoreactive content was achieved, the three TRH-related peptides being only detectable in trace amount. On the other hand, no significant change in hypothalamic levels of TRH was observed at any treatment time, indicating that hypothalamic TRH biosynthesis was not influenced by testosterone. By using the indirect immunofluorescence technique, TRH immunoreactive cells were found in the interstitial space of the testis. These results suggest that Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis. A paracrine, or autocrine role for these products is suggested.

Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis

Endogenous TRH-like products were analyzed in rat testis using a TRH enzyme immunoassay coupled to molecular sieve filtration and HPLC identification. Of the three immunoreactive peptides detected, the two major forms exhibited the same chromatographic properties as synthetic TRH and pGlu-Phe-Pro-NH2. These peptides accounted respectively for 33% and 54% of the total TRH immunoreactivity in the testis. In rat serum, HPLC analysis showed the presence of only one immunoreactive peak with a retention time similar to that of authentic TRH. Ethylene dimethanesulfonate treatment of adult rat was used to assess the effect of Leydig cell destruction on the TRH immunoreactivity content. The concentration of the three TRH immunoreactive peptides fell gradually after treatment and reached a minimum at day 21, where a marked decrease (98%) was observed. At day 41, the regeneration of Leydig cells was achieved, as shown by histochemistry and measurements of serum testosterone and testicular weight. However, no restoration of the TRH immunoreactive content was achieved, the three TRH-related peptides being only detectable in trace amount. On the other hand, no significant change in hypothalamic levels of TRH was observed at any treatment time, indicating that hypothalamic TRH biosynthesis was not influenced by testosterone. By using the indirect immunofluorescence technique, TRH immunoreactive cells were found in the interstitial space of the testis. These results suggest that Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis. A paracrine, or autocrine role for these products is suggested.

Pyroglutamyl-phenylalanyl-proline amide attenuates thyrotropin-releasing hormone-stimulated insulin secretion in perifused rat islets and insulin-secreting clonal beta-cell lines.

TRH immunoreactivity has been detected in the pancreas of man and rat and localized to the islets of Langerhans. We studied the effect of synthetic TRH and the related tripeptide pyroglutamyl-phenylalanyl-proline amide (EFP) on isolated perifused rat islets and the glucose-responsive clonal cell lines HIT-T15 and RIN5AH. TRH at 10 nM potentiated [0.5 +/- 0.1 (control) vs. 0.8 +/- 0.1 (TRH) pmol/10(6) cells per 120 min; mean +/- SEM; n = 6; P < 0.001; n = 15], whereas EFP from 1 nM upwards suppressed glucose-stimulated insulin secretion [0.8 +/- 0.1 (control) vs. 0.5 +/- 0.1 (EFP) pmol/10(6) cells per 120 min; P < 0.001; n = 12) in the cell lines. Further, EFP reversed TRH-stimulated insulin release. Similar responses were observed in perifused isolated rat islets at the tested dose of 1 microM. Gel permeation chromatography of rat adult and neonatal whole pancreas, isolated islets, and HIT cell extracts demonstrated the elution of total TRH-like immunoreactivity (t-TRH-LI) in the same position as synthetic TRH. Cation exchange analysis of the t-TRH-LI from rat adult pancreas and HIT cell extracts showed that neutral TRH-like peptides corresponding to synthetic EFP were also present. Reverse-phase fast protein liquid chromatographic analysis of t-TRH-LI in the unbound fraction of these extracts subjected to anion exchange columns, also demonstrated peaks corresponding to synthetic EFP. We conclude that TRH potentiates, whereas EFP inhibits, glucose-stimulated insulin release in isolated perifused islets and the cell lines. In addition, EFP reversed the stimulatory effect of TRH. The presence of EFP-LI in rat adult and neonatal pancreas and HIT cell extracts suggests it may contribute in the modulation of pancreatic endocrine function.

Thyrotropin-releasing hormone-immunoreactive varicosities synapse on rat phrenic motoneurons.

The relationship between retrogradely labelled or intracellularly filled phrenic motoneurons and varicosities containing thyrotropin-releasing hormone immunoreactivity was investigated in rats by light and electron microscopy. Phrenic motoneurons were identified via retrograde tracing from the diaphragm with cholera toxin B subunit, which was followed by immunocytochemistry to visualise retrogradely labelled motoneurons and thyrotropin-releasing hormone-immunoreactive nerve fibres in their vicinity. At the light microscopic level, varicose thyrotropin-releasing hormone-immunoreactive nerve fibres were distributed sparsely in the phrenic motor nucleus, with some axons surrounding retrogradely labelled motoneurons. In separate intracellular experiments, four phrenic motoneurons identified by antidromic activation from the C5 phrenic nerve root were subsequently filled with Neurobiotin, and nerve fibres that contained thyrotropin-releasing hormone immunoreactivity were identified by immunocytochemistry. The numbers and locations of thyrotropin-releasing hormone-immunoreactive varicosities that were closely appeared to the intracellularly labelled motoneurons were mapped using a camera lucida technique. Close appositions by thyrotropin-releasing hormone-immunoreactive varicosities were seen on somata as well as on proximal and distal dendrites. The closely apposed varicosities were usually present in tight clusters, which were formed by single varicose axons. However, the distribution was nonuniform, in that some dendrites did not receive any close appositions. Ultrastructural analysis of random ultrathin sections through retrogradely labelled neurons showed that varicosities with thyrotropin-releasing hormone immunoreactivity made 1.8% of all synapses and direct contacts on somata and 2.3% of synapses and contacts with dendrites of the retrogradely labelled phrenic motoneurons. The results of these experiments suggest that thyrotropin-releasing hormone-immunoreactive varicosities provide similar numbers of inputs to both the somata and dendrites of phrenic motoneurons. These thyrotropin-releasing hormone-containing inputs seen via light and electron microscopy could modulate the excitability of phrenic motoneurons.