Pituitary and peripheral thyroid hormone responses to thyrotropin-releasing hormone during sustained sleep deprivation in freely moving rats

Sleep deprivation is associated with poor cognitive ability and impaired physical health, but the ways in which the brain and body become compromised are not understood. In sleep-deprived rats, plasma total T4 and T3 concentrations decline progressively to 78% and 47% below baseline values, respectively, brown adipose tissue 5'-deiodinase type II activity increases 100-fold, and serum TSH values are unknown. The progressive decline in plasma thyroid hormones is associated with a deep negative energy balance despite normal or increased food intake and malnutrition-like symptoms that eventuate in hypothermia and lethal systemic infections. The purpose of the present experiment was to evaluate the probable causes of the low plasma total T4 during sleep deprivation by measuring the free hormone concentration to minimize binding irregularities and by challenging the pituitary-thyroid axis with iv TRH to determine both 1) the pituitary release of TSH and 2) the thyroidal response of free T4 (FT4) and free T3 (FT3) release to the TSH increment. Sleep-deprived rats were awake 91% of the total time compared with 63% of the total time in yoked control rats and 50% of the total time during the baseline period. Cage control comparison rats were permitted to sleep normally. Sustained sleep deprivation resulted in a decline from baseline in plasma FT4 of 73 +/- 6% and FT3 of 45 +/- 12%, which were similar to the declines in total hormone concentrations observed previously; nonstimulated TSH was unchanged. In the yoked and cage control groups, FT4 also declined, but much less than that of the sleep-deprived group. The relative changes in free compared with total hormone concentrations over the study were also less parallel than those in the sleep-deprived group. The plasma TSH response to TRH was similar in all groups across experimental days. The plasma FT4 and FT3 concentrations in sleep-deprived rats increased after TRH-stimulated TSH release to an extent comparable to control values. Taken together, low basal FT4 and FT3 hormone concentrations and unchanged TSH and thyroidal responses to TRH suggest a pituitary or hypothalamic contribution to the hypothyroxinemia during sleep deprivation.     

Differential regulation of discrete apoptotic pathways by Ras

The products of the ras genes are known to regulate cell proliferation and differentiation; recently, they have been found to play a role in apoptosis. The expression of oncogenic p21(ras) in a number of cell types, including Jurkat (a human T lymphoblastoid cell line) and murine fibroblasts, makes the cells susceptible to apoptosis following suppression of protein kinase C (PKC) activity (PKC/Ras-mediated apoptosis). Engagement of Fas antigen, a potent effector of apoptosis, activates cellular p21(ras), which may be required for completion of the cell death program. To further investigate the role of p21(ras) in the regulation of apoptosis, the cellular mechanisms employed in these two apoptotic processes in which Ras activity is involved (PKC/Ras-related and Fas-triggered apoptosis), was explored. Increasing p21(ras) activity by expressing v-ras or by treatment with an antisense oligonucleotide to the GTPase-activating protein was found to accelerate the Fas-mediated apoptotic process in Jurkat and mouse LF cells. PKC/Ras-related apoptosis was associated with, and required, cell cycle progression, accompanied by the expression of the G1/S cyclins. In contrast, Fas engagement, although inducing a vigorous and PKC-independent activation of endogenous p21(ras), did not alter cell cycle progression, nor did it require such progression for apoptosis. Both the protein synthesis inhibitor cycloheximide and cyclin E antisense oligonucleotides partially abolished PKC/Ras-mediated apoptosis but had only a moderate effect on Fas-induced apoptosis. In contrast, the CED-3/interleukin-1beta-converting enzyme (ICE) protease inhibitor Z-VADfmk efficiently suppressed Fas-induced apoptosis and only marginally inhibited PKC/Ras-mediated apoptosis. Induction of both pathways resulted in activation of the Jun NH2-terminal kinase/JUN signaling system. These results suggest that different cell death programs, such as PKC/Ras-mediated and Fas-mediated apoptosis, may be interconnected via p21(ras) and perhaps Jun NH2-terminal kinase/JUN. In response to various death stimuli, p21(ras) may act as a common intermediate regulator in the transduction of apoptotic signals.     

Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor

The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.     

Pituitary adenylate cyclase activating polypeptide induces multiple signaling pathways in rat peritoneal mast cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is a high-affinity ligand for at least two types of G-protein coupled receptors, the PACAP type 1 and type 2 receptor. In this study it is demonstrated that the C-terminal PACAP-fragment PACAP(6-27) stimulates serotonin release from rat peritoneal mast cells with higher potency (EC50: 0.2 vs. 2.0 microM) than the PACAP receptor ligand PACAP(1-27). PACAP-induced degranulation of rat peritoneal mast cells was abolished by pertussis toxin and by benzalkonium chloride (IC50: 9.1 microg/ml) indicating the involvement of heterotrimeric G-proteins of the Gi-type. The PACAP effect was also reduced by inhibitors of the phosphatidylinositol specific phospholipase C ((U73122), IC50: 4 microM; (ET-18-O-CH3), IC50: 18 microM), by D609, a specific inhibitor of the phosphatidylcholine specific phospholipase C (IC50: 41 microM), by the protein kinase C-inhibitor staurosporine (IC50: 0.6 microM) and by the lipoxygenase inhibitor nordihydroguaiaretic acid (NGDA) but not by indomethacin. It is concluded that PACAP peptides stimulate secretion in rat peritoneal mast cells in a PACAP receptor-independent manner, probably via direct activation of heterotrimeric G-proteins of the Gi-type; these G-proteins may lead to a sequential activation of different signaling cascades (see above), which may converge at the level of one or more staurosporine-sensitive protein kinase.     

Is it appropriate to treat thyroid nodules with thyroid hormone?

Local transendoscopic endobronchial antigen challenge, which has proved to be a valuable clinical and research technique in the study of human pulmonary hypersensitivity, was evaluated in control and asymptomatic COPD--affected horses. Transendoscopic endobronchial challenges with phosphate-buffered saline (PBS), Micropolyspora faeni extract at 60 and 600 micrograms/ml and mouldy hay extract elicited neutrophilic airway inflammatory responses in control (N = 5-7) and asymptomatic COPD-affected (N = 5-7) horses, as determined by cytological examinations of bronchoalveolar lavage fluid (BALF) harvested from the challenged lung segments. Endobronchial challenges with 600 micrograms M. faeni extract/ml induced a significant BALF neutrophilia only in horses with asymptomatic COPD, when compared with PBS challenges. However, as the BALF neutrophil ratios of COPD-affected horses after this M. faeni challenge did not differ significantly from those of control horses, this finding has little clinical diagnostic value. The BALF neutrophilia induced in control and asymptomatic COPD-affected horses by 60 micrograms M. faeni extract/ml and mouldy hay extract challenges was not significantly different from that induced by PBS challenge. Endoscopically visible bronchial changes were observed in some of the control and COPD-affected horses within 5 mins and at 5 h after PBS, M. faeni and mouldy hay extract challenges. We conclude that this technique is of no value in the investigation of equine COPD.     

Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.     

Retinoic acid and thyroid hormone may function through similar and competitive pathways in regenerating axolotls

The objective of this study was to determine whether thyroid hormone (TH) would interfere with retinoic acid (RA), which proximalizes axolotl larvae regenerate limb pattern. RA and TH are ligands for members of the steroid hormone thyroid hormone nuclear binding protein superfamily which form functional homodimers, but may also form stable heterodimers with the RXR protein and may recognize identical DNA sequences. TH alone does not affect limb pattern but induces metamorphosis in regenerating animals. Coinjected animals do not metamorphose, and when compared to RA controls regenerate more proximal and in some cases anteroposterior (AP) and dorsoventral (DV) duplicate limb structures. In addition, the tissues that are normally lost or changed during metamorphosis appear to be sensitized resulting in the formation of (1) new dorsal gill lamellae accompanied by bifurcation and broadening of the original gill lamellae, (2) partial resorption of the tail fin, and (3) changes in eye position and snout morphology. Bifurcation of gill lamellae tips, but not the formation of supernumerary gills, is also observed in animals treated with RA alone. These results indicate that the molecular mechanism of RA and TH function through similar and perhaps competitive pathways.     

Selective pituitary resistance to thyroid hormone produced by expression of a mutant thyroid hormone receptor beta gene in the pituitary gland of transgenic mice

Phosphine (PH3), from hydrolysis of metal phosphides, is an important insecticide (aluminum phosphide) and rodenticide (zinc phosphide) and is considered genotoxic and cytotoxic in mammals. This study tests the hypothesis that PH3-induced genotoxicity and cytotoxicity are associated with oxidative stress by examining liver (Hepa 1c1c7) cells for possible relationships among cell death, increases in reactive oxygen species (ROS) and lipid peroxidation, and elevated 8-hydroxyguanine (8-OH-Gua) in DNA. PH3 was generated from 0.5 mM magnesium phosphide (Mg3P2) to give 1 mM PH3 as the nominal and maximal concentration. This level causes 31% cell death at 6 h, measured by lactate dehydrogenase leakage, with appropriate dependence on concentration and time. The intracellular ROS level is elevated within 0.5 h following exposure to PH3, peaking at 235% of the control by about 1 h. Lipid peroxidation (measured as malondialdehyde plus 4-hydroxyalkenals) is increased up to 504% by PH3 at 6 h in a time-dependent manner. The level of 8-OH-Gua in DNA, a biomarker of mutagenic oxidative DNA damage analyzed by GC/MS, increases to 259% at 6 h after PH3 treatment. Antioxidants significantly attenuate the PH3-induced ROS formation, lipid peroxidation, 8-OH-Gua formation in DNA, and cell death, with the general order for effectiveness of GSH (5 mM) and D-mannitol (10 mM) (hydroxyl radical scavengers), then Tempol (2.5 mM) and sodium azide (3 mM) (superoxide anion and singlet oxygen scavengers, respectively). These studies support the hypothesis that PH3-induced mutagenic and cytotoxic effects are due to increased ROS levels, probably hydroxyl radicals, initiating oxidative damage.     

Chronically denervated rat Schwann cells respond to GGF in vitro

C-erbB receptor/neuregulin signalling plays a significant role in Schwann cell function. In vivo, Schwann cells up-regulate expression of c-erbB receptors in the first month after injury, but receptor expression is down-regulated with time to levels that are not detectable immunohistochemically. The inability of chronically denervated Schwann cells to respond adequately to signals derived from regenerating axons may be one reason why delayed repair of an injured peripheral nerve frequently fails. We have examined the effects of GGF on denervated Schwann cells in vitro. A modified delayed dissociation technique was used to obtain adult rat Schwann cells from the distal stumps of transected sciatic nerves which had been acutely (7 days) or chronically (2-6 month) denervated. We found that in vitro denervated Schwann cells invariably expressed p75NTR and c-erbB receptors. There was a progressive decrease in total cell yield and the percentage of cells with Schwann cell phenotype (p75NTR and/S-100 or/laminin or /GFAP or/c-erbB positive); proliferation rate; migratory potential; and expression of the cell adhesion molecules N-CAM and N-cadherin, with increasing time of denervation. Addition of GGF2 had a significant stimulatory effect upon Schwann cell proliferation and migration, and an increased proportion of Schwann cells expressed N-CAM and N-cadherin, suggesting that these responses were mediated via GGF/c-erbB signalling. Our results support the view that it may be possible to manipulate chronically denervated Schwann cells so that they become more responsive to signals derived from regrowing axons.     

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.     

Pituitary enlargement due to primary hypothyroidism: growth hormone response to GHRH, GHRP-6 and GHRH plus GHRP-6

While intake of clear fluids 2-3 h before surgery is considered safe as it does not influence gastric content, it is not known if the same applies to a light breakfast meal. We therefore studied gastric emptying of a light breakfast in healthy, female volunteers without evidence of gastrointestinal motility disorders. The test meal consisted of one slice of buttered toast with jam, one cup of coffee without milk or sugar and one glass of pulp-free orange juice taken together with a paracetamol mixture. Using gastric ultrasonography, the stomach was identified without problems in all subjects, and gastric emptying curves using changes in gastric antral area and serum-paracetamol were obtained. Emptying of the fluid phase started immediately after intake of the meal. All subjects had solid particles in the stomach 120 min after the meal, 3 patients were considered empty after 180 min, 6 after 210 min and all after 240 min. The gastric antral area returned to fasting value significantly faster than the disappearance of solid particles; median 150 min versus 210 min; P = 0.01. Our results show that in healthy subjects the stomach cannot be considered empty for solid particles the first 4 h after a light breakfast meal. To secure some safety limits, we suggest a 6-h mandatory preoperative fast after a light breakfast.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.