Biomedical subjects

R M Lechan

Publications and source records attributed to R M Lechan.

At least 55 records · Page 3Linked to original sources

PubMed1993

Immunocytochemical delineation of thyroid hormone receptor beta 2-like immunoreactivity in the rat central nervous system.

The thyroid hormone receptors (TR) are nuclear proteins that include TR alpha and TR beta subtypes, each encoded by a separate gene. Both TR alpha and TR beta give rise to several isoforms of which three, TR alpha 1, TR beta 1, and TR beta 2 bind T3 and mediate the action of thyroid hormone. Although TR beta 2 was initially thought to be confined to the anterior pituitary, we recently observed small quantities of TR beta 2 messenger RNA (mRNA) by polymerase chain reaction analysis of discrete hypothalamic regions. To further examine the distribution of TR beta 2 in the brain, we performed immunocytochemical studies using a highly specific antiserum to TR beta 2, raised against a unique amino acid sequence (TR beta 2[131-145]) that is not present in the other known TRs. This antiserum immunoprecipitated TR beta 2 but not TR alpha 1 or TR beta 1. Immunoreactive TR beta 2 was widely distributed throughout the brain and primarily localized to the cell nucleus. Particularly intense immunostaining was present in the cerebral cortex, cerebellum, and hypothalamus, including regions where TR beta 2 mRNA had not previously been identified. In addition, immunoprecipitation of nuclear extracts with anti-TR beta 2 reduced total T3 binding capacity by approximately 20%, suggesting that immunoreactive TR beta 2 comprises a substantial portion of the total content of nuclear thyroid hormone binding proteins. These studies demonstrate that immunoreactive TR beta 2 is more widely represented in the central nervous system than previously suspected and may play an important role in mediating the action of T3 in many different regions of the brain. The finding of TR beta 2-like material could be due to a disproportionately high ratio of the TR beta 2 translation product and its mRNA in certain regions of the brain, or could indicate the existence of a novel TR beta 2-related protein that is important for T3 binding.

Amino Acid Sequence↗

PubMed1993

Endotoxin-induced corticotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is mediated centrally by interleukin-1.

In the acute phase of bacterial infection, a variety of cytokines, including interleukin-1 (IL-1), are elicited by bacterial endotoxin in both the periphery and the central nervous system. Bacterial endotoxin has been previously reported to profoundly activate the hypothalamic-pituitary-adrenal axis, resulting in elevated glucocorticoid secretion that may serve an important role as part of the inhibitory feedback mechanisms on the activated immune system. To determine whether IL-1 acts within the brain to mediate endotoxin-induced CRH gene expression in the hypothalamic paraventricular nucleus (PVN), we studied the effect of administering the human IL-1 receptor antagonist (IL-1ra) into the brain, a competitive inhibitor of IL-1, on CRH gene expression in the PVN after systemic lipopolysaccharide (LPS) treatment. Eight hours after the ip administration of LPS, the paraventricular CRH mRNA content was elevated 3-to 4-fold (P < 0.01) compared to the control value, and this elevation could be completely abolished by central IL-1ra pretreatment (P < 0.05 compared to LPS-treated group; P > 0.05 compared to controls). In contrast, systemic IL-1ra administration did not inhibit endotoxin-induced CRH gene expression in the PVN. These studies demonstrate that LPS stimulates hypothalamic CRH by a mechanism that involves the action of IL-1 within the central nervous system and may proceed independently of peripheral actions of IL-1 circulating in the bloodstream.

Animals↗

PubMed1992

Feedback regulation of thyrotropin-releasing hormone gene expression by thyroid hormone in the hypothalamic paraventricular nucleus.

Hypothyroidism caused by chemical or surgical thyroidectomy or hypophysectomy causes a substantial increase in the content of thyrotropin-releasing hormone (TRH) mRNA and proTRH exclusively in cells of the medial and periventricular paravocellular subdivisions of the hypothalamic paraventricular nucleus (PVN). This response may be important to raise the anterior pituitary thyrostat to promote increased secretion of thyroid-stimulating hormone (TSH) and to induce the secretion of a more biologically active TSH. The increase in TRH mRNA can be obliterated by stereotaxic implants of hormonally active L-triiodothyronine (T3) placed into the anterior hypothalamus but not by implants of the hormonally inactive 3,5'-diiodo-L-thyronine (T2); we therefore suggested that T3 has a direct action on TRH-containing cells of the PVN. Ablation of brainstem catecholaminergic projection fields to the PVN (known to stimulate TRH secretion) has no effect on TRH mRNA expression; beta 1 thyroid hormone receptor mRNA is present in extracts of the PVN. Euthyroid levels of serum T3 in hypothyroid animals achieved via intraperitoneally implanted osmotic minipumps are not associated with a return of PVN levels of TRH mRNA to normal unless circulating T3 levels are raised into the hyperthyroid range (1.7 times normal). This requirement is similar to that needed to normalize nuclear thyroid hormone receptor levels in the anterior pituitary of hypothyroid animals, suggesting that in addition to circulating T3 monodeiodination of T4 to T3 within the brain must also contribute to feedback inhibition of TRH mRNA. As Type II deiodinase activity is absent or very low in the PVN and does not rise with hypothyroidism, we propose that an alternative source for T4 monodeiodination exists within the central nervous system.

Animals↗

PubMed1992

Transient thyrotoxicosis and persistent hypothyroidism due to acute autoimmune thyroiditis after interleukin-2 and interferon-alpha therapy for metastatic carcinoma: a case report.

A 59-year-old man with metastatic renal cell carcinoma developed symptomatic thyroid dysfunction following interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) therapy. Thyroid evaluation prior to this therapy revealed evidence of subclinical Hashimoto's thyroiditis. Symptomatic thyrotoxicosis, including atrial fibrillation, developed after the initial two courses of intermittent intravenous bolus therapy with human recombinant IL-2 and IFN-alpha. At 4 weeks after initiation of immunotherapy, the thyroid antimicrosomal antibody (AMA) titer rose from 1:6,400 to 1:25,600; thyroid-stimulating immunoglobulin was negative. A technetium 99m-pertechnetate thyroid scan obtained while the patient was thyrotoxic showed diminished uptake in a symmetrically enlarged gland. The patient was temporarily treated with propranolol, digoxin, and quinidine. The atrial fibrillation quickly resolved, and thyrotoxicosis abated over the following 5 weeks, while the AMA titer rose further to 1:102,400. By 11 weeks after initiation of immunotherapy, hypothyroidism developed and persisted through two subsequent courses of cytokine therapy at Weeks 16 and 18. The tumor metastases partially responded to the immunotherapy. The patient has remained hypothyroid up to 27 weeks of follow-up. This case history suggests that IL-2 and IFN-alpha therapy may precipitate a fulminant autoimmune thyroiditis syndrome in a vulnerable patient with preexisting autoimmune thyroid disease.

Carcinoma, Renal Cell↗

PubMed1992

Expression of thyroid hormone receptor beta 2 in rat hypothalamus.

A polymerase chain reaction based assay was used to evaluate expression of thyroid hormone receptor beta 2 mRNA in rat hypothalamus. Expression was detected in the arcuate, ventromedial and paraventricular nuclei, as well as the median eminence. Trace expression was found in the dorsomedial nucleus, but no expression of thyroid hormone receptor beta 2 was detected in the lateral hypothalamus or the preoptic region. The results indicate that, contrary to previous belief, expression of thyroid hormone receptor beta 2 is not confined to the anterior pituitary.

Animals↗

PubMed1992

Thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is dependent upon feedback regulation by both triiodothyronine and thyroxine.

The biosynthesis of TRH in hypophysiotropic neurons of the paraventricular nucleus (PVN) is inversely regulated by feedback effects of circulating levels of thyroid hormones. As the PVN contains little or no deiodinase activity, the enzyme necessary to convert T4 to biologically active T3, we determined whether feedback inhibition of pro-TRH mRNA in thyroid hormone-sensitive neurons of the PVN is mediated exclusively by circulating levels of T3. The concentration of pro-TRH mRNA in the PVN of hypothyroid male rats receiving constant infusions of T3 over 7 days from ip implanted osmotic minipumps was studied by in situ hybridization histochemistry using computerized image analysis. Pro-TRH mRNA could not be suppressed to euthyroid levels by an infusion of T3 that returned plasma T3 levels to normal and required the infusion of higher concentrations of T3 that elevated plasma T3 into the supranormal range. By regression analysis, the mean concentration of plasma T3 required to suppress pro-TRH mRNA to euthyroid levels was estimated to be 110.3 ng/dl, similar to the amount of T3 estimated to be necessary to suppress TSH secretion from the anterior pituitary (108.7 ng/dl). We conclude that both T3 and T4 contribute to feedback inhibition of TRH biosynthesis in hypophysiotropic neurons of the PVN and propose that the effects of T4 on the PVN could be mediated after its monodeiodination at a different locus within the brain.

Animals↗

PubMed1992

Hypothyroidism increases vasoactive intestinal polypeptide (VIP) immunoreactivity and gene expression in the rat hypothalamic paraventricular nucleus.

Vasoactive intestinal polypeptide (VIP) is produced by neurons in the rat hypothalamic paraventricular nucleus (PVN) and may have an important role as a prolactin-releasing factor. Recent work from our laboratories has shown that thyroid hormone regulates the content of VIP and VIP mRNA in the rat anterior pituitary, but its effect on VIP in the PVN is not known. To determine whether thyroid hormone alters VIP biosynthesis in the PVN, we studied the effect of hypothyroidism on the content of immunoreactive (IR)-VIP and VIP mRNA in PVN neurons using histochemical techniques. By immunocytochemistry, only scattered IR-VIP fibers were present in the PVN of control animals whereas IR-VIP perikarya and fibers were present in hypothyroid rats. By in situ hybridization histochemistry, no labeled neurons were recognized in the PVN in control animals whereas PVN neurons were labeled in hypothyroid rats. These findings raise the possibility that hypothyroidism exerts negative feedback regulation on VIP-producing neurons in the PVN and suggest that this may be important to modulate the stimulatory effects of VIP on anterior and/or posterior pituitary function.

Animals↗

PubMed1991

Muscle denervation increases thyrotropin-releasing hormone (TRH) biosynthesis in the rat medullary raphe.

To determine whether thyrotropin-releasing hormone (TRH) could exert a trophic role in ventral horn motor neurons, we examined the effect of muscle denervation with botulinum toxin A on TRH mRNA in the rat medullary raphe by in situ hybridization histochemistry. Compared to controls, denervated rats showed a significant increase in the number and silver grain density of hybridized medullary raphe neurons. Increased proTRH gene expression in the medullary raphe in response to motor unit perturbation indicates that TRH may be trophic to lower motor neurons.

Action Potentials↗

PubMed1990

Gomori-positive astrocytes in primary culture: effects of in vitro age and cysteamine exposure.

Gomori-positive astrocytes have been identified in the periventricular brain in situ and in diencephalic explants on the basis of their endogenous peroxidase activity, affinity for chrom alum hematoxylin, and orange-red autofluorescence. To facilitate analyses of their functional properties, we sought to identify these cells in dissociated fetal rat brain cultures. Astrocytes containing cytoplasmic inclusions with the above tinctorial and fluorescent properties represented less than 1% of cultured astrocytes at day 10 in vitro (DIV). There was a marked increase in the fraction of Gomori-positive astrocytes and their granule content between 10 and 46 DIV. As in situ, the peroxidase activity appeared to be non-enzyme-mediated insofar as it catalyzed diaminobenzidine oxidation over a wide range of pH (3-11) and could not be inhibited by tissue preheating or the catalase inhibitor, aminotriazole. Metalloporphyrins probably mediate both the pseudoperoxidase activity and autofluorescence in these cells. Cysteamine and cystamine, but not ethanolamine or L-cysteine, induced a massive accumulation of Gomori-positive astrocytes when administered from DIV 6-18. Alterations of the redox microenvironment or induction of porphyrin/heme biosynthetic enzymes may be the mechanisms responsible for this cyst(e)amine effect. Dissociated rat brain culture enriched for Gomori astroglia should provide ample opportunity to investigate the functional properties of these cells.

Animals↗

PubMed1990

proTRH gene expression by fetal pancreatic islets in culture.

The hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), has been detected in neonatal pancreatic tissue and localized by immunocytochemistry in the islets of Langerhans. To determine whether the TRH gene is expressed in islets, we have extracted RNA from cultured rat islets and probed for proTRH mRNA using a [32P]-labeled antisense RNA. Islet proTRH mRNA comigrated with the 1.6 kilobase proTRH mRNA present in the rat hypothalamus. Normalized to total RNA, islets cultured for 7 days contained at least 10 times more proTRH mRNA than day 1 whole pancreas. We conclude that pancreatic TRH is synthesized in situ in the islets of Langerhans. This is the first attempt to characterize and quantify proTRH mRNA using neoformed foetal islets. We propose that quantitative analysis of proTRH mRNA concentrations in this culture system will enable study of the direct regulation of TRH biosynthesis in the pancreas.

Animals↗

PubMed1990

Immunoreactive interleukin-1 beta localization in the rat forebrain.

To determine whether the cytokine, interleukin-1 beta, is present in the rat brain as has been reported in human brain, immunocytochemical studies were performed using an antiserum that recognizes recombinant, rat IL-1 beta. Immunoreaction product was present in the periventricular and medial hypothalamus, mossy fiber distribution in the hippocampus and olfactory tubercle. These studies demonstrate that IL-1 beta is part of a diffuse intrinsic neural system in the rat central nervous system, associated with regions involved with hypophysiotropic, autonomic, limbic and extrapyramidal functions.

Animals↗

PubMed1990

Glial peroxidase activity in the hypothalamic arcuate nucleus: effects of estradiol valerate-induced persistent estrus.

To test the hypothesis that stimulation of glial peroxidase activity by estrogens may play a role in the pathogenesis of the previously reported degenerative changes in the hypothalamic arcuate nucleus that occur in female rats treated with a long-acting estrogen preparation, the cellular localization and number of peroxidase-positive granules in the hypothalamus were determined in adult female rats treated with a single intramuscular injection of 2 mg of estradiol valerate. The persistent estrus state, manifested as persistent vaginal cornification and polycystic ovaries, was induced in 80% of the animals. In comparison with normally cycling controls, the arcuate nuclei of persistent estrus rats exhibited a 3- and 2.8-fold increase in numbers of diaminobenzidine-positive granules and granule clusters, respectively (P less than 0.01 for both comparisons). These peroxidase-positive granules were identified in astrocytes by double-label immunohistochemistry utilizing antiserum to glial fibrillary acidic protein. Diaminobenzidine staining occurred over a pH range of 4-10.5 and was resistant to the catalase inhibitor, aminotriazole, and to tissue pre-heating, indicating that the histochemical reaction was not due to tissue enzyme activity. Rather, these findings indicate that a non-enzymatic, pseudoperoxidation reaction has been induced in these cells by estrogen administration. Possible mediators of this reaction are metallo-porphyrins known to be present in rodent hypothalamus. The mechanisms by which astrocyte peroxidase activity may play a role in estrogen-related neural damage are discussed.

Animals↗

PubMed1990

Estrogen regulates the gene expression of vasoactive intestinal peptide in the anterior pituitary.

Vasoactive intestinal peptide (VIP), a prolactin (PRL)-releasing factor, has been shown to be synthesized within the anterior pituitary. To test the hypothesis that estrogens increase PRL secretion, at least in part, by stimulating VIP secretion, the concentrations of VIP, peptide histidine isoleucine (PHI) and prepro VIP mRNA were measured in the anterior pituitaries of oophorectomized rats treated with 17 beta-estradiol benzoate 25 micrograms/kg/day s.c. for 5 days. For comparison, changes in the hypothalamus were also measured. Estrogen treatment resulted in a marked increase in pituitary VIP content without detectable changes in PHI content, suggesting that estrogen may regulate differentially the enzymes involved in the posttranslational processing of the VIP prohormone. A VIP mRNA-transcript of about 1.7 kilobases was detected in all tissues studied, being most abundant in the cortex, less abundant in the hypothalamus and barely detectable in the untreated pituitary. Estrogen treatment resulted in an increase in VIP gene expression in the pituitary but not in the hypothalamus or cerebral cortex. This marked increase in prepro VIP mRNA rendered possible the demonstration in the estrogen-treated pituitary of a second VIP transcript of about 1.0 kilobase which was present in only very low quantities in the cortex and hypothalamus. We conclude that estrogen regulates the gene expression of VIP in the anterior pituitary. Changes in VIP secretion may contribute to the stimulatory effect of estrogen on PRL secretion.

Animals↗

PubMed1990

Thyrotropin-releasing-hormone-immunoreactive innervation of thyrotropin-releasing-hormone-tuberoinfundibular neurons in rat hypothalamus: anatomical basis to suggest ultrashort feedback regulation.

Thyrotropin-releasing-hormone (TRH)-synthesizing neurons in the medial and periventricular parvocellular subdivisions of the rat hypothalamic paraventricular nucleus (PVN) are involved in regulation of the anterior pituitary. Since ultrashort feedback regulation of TRH in the hypothalamus has been suggested by physiological studies, we sought to identify the presence of TRH synaptic contacts containing TRH on TRH tuberoinfundibular neurons in the PVN. An immunocytochemical study was performed at light- and electron-microscopic levels using antiserum directed to the N-terminal cryptic sequence of the TRH precursor, preproTRH 25-50. At the light-microscopic level, contacts between TRH immunoreactive (IR) fibers and the perikarya and processes of TRH-IR neurons were observed in medial and periventricular subdivisions of the PVN. At the ultrastructural level, TRH-neurons appeared either tightly juxtaposed to TRH-immunopositive perikarya and dendrites or to establish axodendritic and axosomatic contacts suggestive of synaptic associations. These data provide a morphologic basis to support a neuroendocrine role for TRH or processed forms of proTRH in the PVN and in particular suggest their involvement as neuromodulators in an ultrashort feedback regulation of TRH tuberoinfundibular neurons.

Animals↗

PubMed1990

1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems.

We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.

Animals↗

PubMed1990

Neuropeptide-Y-immunoreactive innervation of thyrotropin-releasing hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus.

The association of neuropeptide-Y (NPY)-immunoreactive (IR) axon terminals with TRH-synthesizing neurons in the rat hypothalamic paraventricular nucleus (PVN) has been studied. Immunocytochemical single and double labeling studies were performed at both light and electron microscopic levels using antiserum to NPY and, as a marker of TRH-containing neurons, antisera recognizing the N-terminal flanking peptides of the TRH prohormone, prepro-TRH-(25-50) and prepro-TRH-(53-74). At the light microscopic level, a diffuse group of TRH-IR cell bodies were observed in the anterior parvocellular subdivision of the PVN and became more numerous and densely clustered in the medial and periventricular parvocellular subdivisions. NPY-IR fibers were observed to innervate all subdivisions of the PVN, but were particularly dense in the anterior, medial, and periventricular parvocellular subdivisions of the nucleus, where they appeared to contact TRH-synthesizing perikarya and neuronal processes. At the ultrastructural level, numerous NPY-IR axon terminals containing labeled vesicles were either tightly juxtaposed to TRH-producing neurons or seen to establish both symmetric and asymmetric synaptic contacts with TRH-containing cell bodies and dendrites. Some NPY-IR axon terminals also established synaptic contacts with unlabeled PVN perikarya and processes or were found in close apposition to blood vessels. These data provide a morphological basis to suggest NPY-mediated neuroendocrine regulation over the biosynthesis and/or secretion of TRH in the PVN. Reports of the colocalization of NPY and catecholamines in the same axon terminals raises the possibility of a potential interaction between NPY and catecholamines to influence TRH neurons in the PVN. Morphological evidence for synaptic interactions between NPY-IR axon terminals and non-TRH-containing neurons in the PVN further suggests that this peptide may influence other neuroendocrine systems.

Animals↗

PubMed1990

Intrinsic pituitary interleukin-1 beta is induced by bacterial lipopolysaccharide.

Using a specific antiserum recognizing recombinant rat interleukin-1 beta (IL-1 beta), immunoreactive material was localized to cytoplasmic granules in anterior pituitary endocrine cells and colocalized with TSH in thyrotropes. Authenticity was established by Northern blot hybridization using a specific rat IL-1 beta cRNA probe, revealing a 1.8-kilobase mRNA identical to that in the spleen. The marked increase in anterior pituitary IL-1 beta message after the administration of bacterial lipopolysaccharide, raises the possibility that IL-1 beta may be involved in paracrine or autocrine regulation of pituitary function during infectious challenge.

Animals↗

PubMed1990

Isolated adrenocorticotropin deficiency associated with an autoantibody to a corticotroph antigen that is not adrenocorticotropin or other proopiomelanocortin-derived peptides.

A 44-yr-old man with hypocortisolism was shown to have an undetectable basal plasma ACTH level and absent or subnormal ACTH and beta-lipotropin responses to provocative testing with insulin, vasopressin, and CRH. Endocrine function after glucocorticoid replacement was otherwise normal, thus establishing the diagnosis of isolated ACTH deficiency. This patient's serum was tested immunohistochemically for the presence of an antipituitary antibody by indirect immunofluorescence of rat pituitary tissue. Positive immunostaining was observed in stellate-shaped cells in the anterior and intermediate lobes. Immunopositive cells were shown by immunoelectron microscopy to have ultrastructural characteristics of corticotrophs. Immunoreactivity was concentrated in secretory granules 120-170 nm in diameter. In a double immunolabeling procedure, staining by the patient's serum was shown to colocalize with rabbit antiserum to ACTH, but not with antisera to PRL, GH, beta TSH, or beta LH. Immunoabsorption of the patient's serum with ACTH-(1-24), ACTH-(1-39), gamma MSH, corticotropin-like intermediate lobe peptide, beta-endorphin, or beta-lipotropin failed to diminish immunolabeling in the pituitary. We conclude that the antipituitary antibody in this patient's serum shows immunohistochemical specificity for a rat corticotroph antigen located in secretory granules that is neither ACTH nor any of the proopiomelanocortin (POMC)-derived peptides tested. The autoantigen could be a cell-specific granular factor involved in the posttranslational processing of POMC or secretion of ACTH. We postulate that an autoimmune process may account for this patient's disease, and that his antipituitary antibody could play a pathogenic role by either inhibiting a POMC-processing enzyme or initiating an antibody-dependent cell-mediated cytotoxicity reaction, resulting in the selective destruction of corticotrophs.

Adrenocorticotropic Hormone↗