Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain

Characteristics of TRH-receptors were studied in the rat central nervous system (CNS). Ion species, pH and temperature importantly influenced TRH-receptor binding. Subcellular distribution of TRH-receptor binding revealed that synaptic membranes had the greatest percentage of total sites. Scatchard analysis suggested that the rat CNS had two distinct TRH binding sites with apparent dissociation constants (Kd) of 5 X 10(09) M and 13 X 10(-8) M. Receptor activity is sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for the binding of [3H]TRH. Thiol reagents reduced the binding activity of the receptor, suggesting that an intrachain disulfide bond may form an important constituent of the binding site for TRH. The TRH-receptor in the rat brain was successfully solubilized with non-ionic detergent Triton X-100. On gel chromatography with Sepharose 6B column, the solubilized TRH-receptor molecule eluted at the fraction corresponding to an apparent molecular weight of 300,000 daltons, with Stokes' radius of 5.8 nm. The regional distribution of TRH-receptor binding was examined to clarify the site of TRH action. The highest level of binding was in the hypothalamus, cerebral cortex and hippocampus, indicating that TRH affects the CNS function mainly through the limbic system, cerebral cortex and hypothalamus. Moreover, tricyclic anti-depressants and Li+ decreased the binding of [3H]TRH. These findings suggest that endogenous TRH and TRH receptor may play the role of a neurotransmission modulator in the brain to control emotional and mental functions.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Barbiturate competition for TRH receptors in mouse brain: neuromodulation of anesthesia

In vitro thyrotropin releasing hormone (TRH) radioligand binding assays were performed using purified presynaptic and postsynaptic membranes derived from various regions of mouse brain. These studies revealed the pattern of central distribution of specific TRH binding sites. The highest concentrations of both types of membrane receptors were localized in the limbic forebrain. The brain stem contained a high density of only presynaptic receptors, and the cerebral cortex contained a moderate-high level of only postsynaptic receptors. Barbiturate analogues effectively competed for all forebrain and brain stem, but not cortical, TRH receptors, thus implicating these specific receptors in the neuromodulation of barbiturate anesthesia. The results of in vivo radioligand binding assays for [3H] TRH disposition after central infusions concomitant with barbiturate vs. saline challenges further support this viewpoint.     

Metabolites of thyrotropin releasing hormone inhibit angiotensin converting enzyme in vitro

Pyroglutamylhistidylproline and histidylproline, reported metabolites of thyrotropin releasing hormone, were found to competitively inhibit purified rabbit lung angiotensin converting enzyme with K_I values of 0.76 μM and 1.7 mM, respectively. Native thyrotropin releasing hormone and histidylprolinediketopiperazine at concentrations of 10 mM and 5 mM, respectively, had no effect on angiotensin converting enzyme activity. Neither the native hormone nor its deamidated derivative served as substrate for angiotensin converting enzyme.     

Modulation of NRF2 signaling pathway by nuclear receptors: Implications for cancer

Nuclear factor-erythroid 2 p45-related factor 2 (NRF2, also known as Nfe2l2) plays a critical role in regulating cellular defense against electrophilic and oxidative stress by activating the expression of an array of antioxidant response element-dependent genes. On one hand, NRF2 activators have been used in clinical trials for cancer prevention and the treatment of diseases associated with oxidative stress; on the other hand, constitutive activation of NRF2 in many types of tumors contributes to the survival and growth of cancer cells, as well as resistance to anticancer therapy. In this review, we provide an overview of the NRF2 signaling pathway and discuss its role in carcinogenesis. We also introduce the inhibition of NRF2 by nuclear receptors. Further, we address the biological significance of regulation of the NRF2 signaling pathway by nuclear receptors in health and disease. Finally, we discuss the possible impact of NRF2 inhibition by nuclear receptors on cancer therapy.     

FUS contributes to mTOR-dependent inhibition of translation

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.     

Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins

We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat¹-specific transport signal of OEC16 and the Sec passenger protein OEC33, both subunits of the oxygen-evolving system associated with photosystem II. Protein transport experiments performed with isolated pea thylakoids show that the 16/33 chimera is transported in a strictly Tat-dependent manner into the thylakoid vesicles yielding mature OEC33 (mOEC33) in two different topologies. One fraction accumulates in the thylakoid lumen and is thus resistant to externally added protease. A second fraction is arrested during transport in an N-in/C-out topology within the membrane. Chase experiments demonstrate that this membrane-arrested mOEC33 moiety does not represent a translocation intermediate but instead an alternative end product of the transport process. Transport arrest of mOEC33, which is embedded in the membrane with a mildly hydrophobic protein segment, requires more than 26 additional and predominantly hydrophilic residues C-terminal of the membrane-embedded segment. Furthermore, it is stimulated by mutations which potentially affect the conformation of mOEC33 suggesting that at least partial folding of the passenger protein is required for complete membrane translocation.     

Hypo- and hyperthyroidism affect NEI concentration in discrete brain areas of adult male rats

To date, there has been only one in vitro study of the relationship between neuropeptide EI (NEI) and the hypothalamic-pituitary-thyroid (HPT) axis. To investigate the possible relationship between NEI and the HPT axis, we developed a rat model of hypothyroidism and hyperthyroidism that allows us to determine whether NEI content is altered in selected brain areas after treatment, as well as whether such alterations are related to the time of day. Hypothyroidism and hyperthyroidism, induced in male rats, with 6-propyl-1-thiouracil and l-thyroxine, respectively, were confirmed by determination of triiodothyronine, total thyroxine, and thyrotropin levels. All groups were studied at the morning and the afternoon. In rats with hypothyroidism, NEI concentration, evaluated on postinduction days 7 and 24, was unchanged or slightly elevated on day 7 but was decreased on day 24. In rats with hyperthyroidism, NEI content, which was evaluated after 4 days of l-thyroxine administration, was slightly elevated, principally in the preoptic area in the morning and in the median eminence-arcuate nucleus and pineal gland in the afternoon, the morning and afternoon NEI contents being similar in the controls. These results provide the bases to pursue the study of the interaction between NEI and the HPT axis.     

Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta

The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism.     

Biophysics of sphingolipids II. Glycosphingolipids: An assortment of multiple structural information transducers at the membrane surface

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.     

Correlation of the O-intermediate rate with the pKa of Asp-75 in the dark, the counterion of the Schiff base of Pharaonis phoborhodopsin (sensory rhodopsin II)

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppR(M)) and O-intermediates (ppR(O)). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppR(O) of the triple mutant was accelerated approximately 20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppR(O) of mutants (11 species) and those of the wild-type were well-correlated with the pK(a) value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail.     

Endogenous opiates and behavior: 2010

This paper is the thirty-third consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2010 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration (Section 16); and immunological responses (Section 17).