Microinjection of thyrotropin-releasing hormone analogue into the central nucleus of the amygdala stimulates gastric contractility in rats

The effect on gastric contractility following bilateral microinjection of thyrotropin-releasing hormone (TRH) analog, RX 77368, into the central nucleus of the amygdala was examined in fasted, urethane-anesthetized rats. Extraluminal force transducers were used to measure gastric corpus contractility. Bilateral microinjection of RX 77368 (0.5 microgram, 1.0 microgram, n = 6 each) stimulated gastric contractility for up to 120 min post-injection, P < 0.05. Gastric contractility was not significantly stimulated by microinjection of 0.1 microgram RX 77368, 0.1% bovine serum albumin (BSA) into the central nucleus or RX 77368 (0.5 microgram, 1.0 microgram) into sites adjacent to the central nucleus. Peak responses (1.0 microgram) occurred 40 min post-injection and represented a 16-26-fold increase over basal values. The frequency of gastric contraction waves was attenuated for 0-90 min in rats receiving central amygdaloid microinjection of RX 77368 (0.1, 0.5 or 1.0 microgram) versus rats microinjected with the vehicle or RX 77368 into sites adjacent to the central nuclei. The stimulatory effect of RX 77368 (1.0 microgram) on gastric contractility was abolished by subdiaphragmatic vagotomy. These results indicate that the TRH analog, RX 77368, acts within the central amygdala to vagally stimulate gastric contractility.     

Cloning and characterization of the chicken thyrotropin-releasing hormone receptor

We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.     

Effects of hyperthyroidism and hypothyroidism on rat growth hormone release induced by thyrotropin-releasing hormone

The official final action method for sodium chloride in cereal foods, 14.129, was found to give erroneously low results because of loss of chloride during ashing. Comparison of the data with values obtained by the official first action potentiometric method, 32.A01-32.A06, which does not require ashing, showed that large and variable losses of chloride occurred. Official ashing methods for other foods specify addition of sodium carbonate to prevent conversion of chloride to volatile forms, but this was not specified in 14.129. In the present study it was found that sodium carbonate did not completely prevent loss of chloride. The official first action potentiometric method, 32.A01-32.A06, has been adopted as official first action for the determination of chloride in cereal foods to replace 14.129, which was repealed, official first action. A cross-reference to 32.A01-32.A06 has been added to 14.096.     

Effects of cimetidine on acute gastric mucosal injury induced by ischemia-reperfusion in rats

We investigated the effects of cimetidine on acute gastric mucosal injury induced by ischemia-reperfusion in rats. Under pentobarbital anesthesia, the celiac artery was clamped for 30 min and reperfused for 60 min. Cimetidine, famotidine and omeprazole caused a dose-dependent suppression in the total area of erosions that were induced by ischemia-reperfusion. Whereas, none of them inhibited the increase in thiobarbituric acid-reactive substances in the stomach, as an index of lipid peroxidation. The inhibitory effect of intraperitoneally administered cimetidine on mucosal damage was abolished by continuous luminal perfusion with HCl solution (pH 1.5, 1 ml/min) during ischemia-reperfusion, while luminal perfusion with the solution containing HCl and cimetidine (3 mmol/l) significantly reduced the total area of erosions compared to luminal perfusion with HCl solution alone. Cimetidine (3 mmol/l) inhibited hydroxyl radical-induced lipid peroxidation of human erythrocyte membranes by 60% in vitro. These results indicate that cimetidine possesses a protective effect against acute gastric mucosal injury induced by ischemia-reperfusion not only due to the suppression in gastric acid secretion, but also due to the antioxidant action when it is present at a high concentration in the intragastric environment.     

Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.     

Pretreatment with antisense oligodeoxynucleotides directed against the NMDA-R1 receptor enhances survival and behavioral recovery following traumatic brain injury in rats

Treatment with N-methyl-D-aspartate (NMDA) receptor antagonists limits tissue damage following CNS ischemia or trauma, supporting the hypothesis that NMDA receptors participate in the pathophysiology of such injuries. An alternative approach for evaluating this hypothesis is to examine the effects of selective inhibition of NMDA receptor synthesis, using antisense oligodeoxynucleotides. In the present studies, the effects of antisense oligodeoxynucleotides directed at NMDA-R1 receptor subunit, administered intracerebroventricularly (i.c.v.) prior to injury, were evaluated in a well-defined traumatic brain injury model in rats. Outcome measures included survival, motor recovery, and histological changes. Administration of antisense oligodeoxynucleotides (15 nmol/ml twice daily x 2 days) did not alter physiological variables or motor function prior to trauma. However, such treatment significantly decreased mortality and improved behavioral recovery at 2 weeks after trauma as compared to animals treated with the corresponding sense oligodeoxynucleotides. Although cell counts in hippocampus did not differ between treatment groups, astrocyte activation as reflected by glial fibrillary astrocytic protein (GFAP) immunocytochemistry was significantly reduced in antisense treated animals. These findings provide additional evidence that NMDA receptors contribute to secondary injury after brain trauma and may suggest an alternative treatment approach.     

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.     

Modulation of arterial baroreflexes by antisense oligodeoxynucleotides to NMDAR1 receptors in the nucleus tractus solitarius

An antisense oligodeoxynucleotide which specifically blocked the production of the glutamate receptor subtype NMDAR1 was administered to the nucleus tractus solitarius (NTS) in order to examine the role of this receptor in baroreflex control of heart rate. Baseline blood pressure and heart rate were unchanged by NTS treatment with the antisense oligodeoxynucleotide to the NMDAR1 receptor subunit. However, the reflex bradycardia evoked in response to the hypertension induced by bolus administration of phenylephrine was significantly attenuated following bilateral NTS antisense oligodeoxynucleotide treatment. Administration of the corresponding mismatched antisense oligodeoxynucleotide did not significantly alter the reflex bradycardia. These data indicate that NMDAR1 receptors are involved in neurotransmission in the baroreflex arc at the level of the NTS. The specificity of antisense oligodeoxynucleotides may prove to be a useful technique to analyze the role of receptor subtypes mediating neurotransmission in central pathways.     

Multisensory deficits in rats produced by acute exposure to cold swim stress.

Cold swim stress and morphine administration produce analgesia. Whether this was accompanied by changes in other senses was investigated. Analgesia was assessed with a tail-flick test; the sensitivity of the other senses was assessed using the ability of mild sensory stimuli (prestimuli) to inhibit the amplitude of a subsequently elicited acoustic startle response. In Experiment 1, auditory prestimuli were used. In Experiment 2, visual prestimuli were used. In each experiment, rats were exposed to cold-water swims followed by behavioral testing, warm swims followed by testing, and testing alone. This was repeated, substituting morphine injections followed by testing, vehicle injections followed by testing, or testing alone. Both cold swim stress and morphine produced reliable analgesia. Only cold swim stress interfered with the ability of the stimuli to inhibit startle. This reflects decreased sensitivity to auditory and visual stimulation caused by cold swim stress, which suggests that the resultant sensory deficits are more global than is currently believed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Telomerase activity is induced in human peripheral B lymphocytes by the stimulation to antigen receptor

To understand the molecular events for the proliferation of B cells, we studied the induction of telomerase activity in vitro after stimulation to B-cell antigen receptor (BCR) on human peripheral B cells. Although unstimulated purified B cells of tonsils and peripheral blood from healthy volunteers do not express detectable telomerase activity, anti-IgM beads induce telomerase activity in these B cells. Soluble anti-IgM antibody (Ab) alone does not induce telomerase activity, but the second signal, given by either one of the cytokines of interleukin-2 (IL-2), IL-4, and IL-13 or by anti-CD40 monoclonal Ab (MoAb), is effective as the costimulation for the induction of the activity. Stimulation with anti-IgM Ab and anti-CD40 MoAb induces telomerase activity in most mature B cells of the tonsils and peripheral blood. The stimuli to both IgM and IgD receptors similarly induce the activity. Induction of telomerase activity is accompanied with the proliferation of B cells, but is not absolutely correlated with the extent of B-cell growth. Phorbol dibutylate (PDB) plus calcium (Ca) ionophore (PDB/Ca), which replace the activation through BCR and the costimulatory molecules, also induce telomerase activity. Moreover, it is suggested that phosphoinositide (PI) 3-kinase plays a role for the induction of telomerase activity in B cells stimulated with anti-IgM Ab and anti-CD40 MoAb. These results suggest that telomerase activity is induced in the B-cell activation of the antigen specific immune response.     

Effect of vagotomy on hyperactivity and increased dopamine turnover induced by intraperitoneal administration of thyrotropin-releasing hormone

A synthetic peptide corresponding to a region encoded by tau gene exon 8 was used to raise an antibody. The antibody, E8, was used to probe normal tau from different species and abnormal tau proteins from subjects with Alzheimer's disease. Immunoblotting and enzyme-linked immunosorbent assays demonstrated that only bovine tau reacted with the E8 antibody. The E8 immunoreactive tau isoform was estimated to represent less than 2% of the bovine tau. Our results indicate that bovine tau is unique in containing isoforms positive with E8, and that PHF formation does not require the presence of PHF-tau with exon 8.     

Use of subunit-specific antisense oligodeoxynucleotides to define developmental changes in the properties of N-methyl-D-aspartate receptors

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.     

PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats

1. Microinjection of peptide YY (PYY, 7-46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY. 2. [Pro34]rat(r)/porcine(p)PYY and [Pro34]human(h)PYY (23-117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68+/-11 and 89+/-31 micromol 90 min(-1) respectively. 3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27+/-8 and 23+/-8 micromol 90 min(-1) respectively) and a higher dose (116 pmol) tended to reduce the response. 4. Pancreatic polypeptide (PP, 4-46 pmol), [Leu31,Pro34]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY3-36, pNPY5-36 and PNPY13-36 (25-168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion. 5. The rank order of potency of PYY > or = [Pro34]r/pPYY = [Pro34]hPYY> r/hNPY = pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu31,Pro34]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.     

Differential changes in induced seizures after hippocampal treatment of rats with an antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit

Binding energy of DNA-Cro protein complexes is analyzed in terms of DNA elasticity, using a sequence-dependent anisotropic bendability (SDAB) model of DNA, developed recently [M.M. Gromiha, M.G. Munteanu, A. Gabrielian and S. Pongor, J. Biol. Phys. 22(1996) 227-243.]. The protein is considered to bind aspecifically to DNA that reduces the freedom of movement in the DNA molecule. In cognate DNA, the Cro protein moves on to form specific interactions and bends DNA. A comparison of the experimental data [Y. Takeda, A. Sarai and V.M. Rivera, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 439-443.] with the calculated DNA stiffness data shows that delta G of the complex formation increases with stiffness of the ligand when the interactions are nonspecific ones, while an opposite trend is observed for specific binding. Both of these trends are in agreement with our approach using the SDAB model. A decomposition of the energy terms suggests that binding energy in the nonspecific case is used maily to compensate the free energy changes due to entropy lost by DNA, while the energy of specific interactions provide enough energy both to bend the DNA molecule and to change the conformation of the Cro protein upon ligand binding.     

Attenuation by genistein of sodium-chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of prolonged administration of genistein, a tyrosine-kinase inhibitor, on sodium-chloride-enhanced induction of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, and the labeling and apoptotic indices and vessel counts in the gastric mucosa and gastric cancers, were investigated in Wistar rats. After 25 weeks of the carcinogen treatment, rats were fed chow pellets containing 10% sodium chloride and were given s.c. injections of genistein at dosages of 15 mg/kg or 30 mg/kg body weight every other day. In week 52, the incidence of gastric cancers was significantly greater in rats fed sodium chloride than in untreated control rats. Prolonged administration of genistein at a dosage of 30 mg/kg, but not 15 mg/kg, body weight significantly reduced the incidence of gastric cancers, which was increased by oral treatment with sodium chloride. Genistein at the higher dose significantly decreased the labeling index and vessel counts of the antral mucosa and the gastric cancers (which were increased by treatment with sodium chloride) and significantly increased the apoptotic index of the antral mucosa and the cancers (which was lowered by the treatment with sodium chloride). These findings suggest that genistein attenuates gastric carcinogenesis promoted by sodium chloride, by inducing increased apoptosis and lower cell proliferation and angiogenesis of antral mucosa and gastric cancers.     

Decrease in gastric sensitivity to distension by 5-HT1A receptor agonists in rats

Using an in vivo model for evaluation of gastric sensitivity in awake rats, we aimed to determine whether 5-hydroxytryptamine 1A (5-HT1A) agonists modify pain threshold and gastric compliance specifically through 5-HT1A receptors. Isobaric gastric distensions were performed with a barostat using steps of 5 mm Hg in male rats equipped with a gastric balloon and electrodes implanted in the neck muscles. Gastric distension at 15 or 20 mm Hg induced a typical posture associated with contractions of the neck muscles. Rats received drugs 30 min before gastric distension. The 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), administered intraperitoneally (0.5 mg/kg) increased gastric pain threshold and gastric tone. These effects were reproduced when administered centrally (0.05 mg/kg) and blocked by intracerebroventricular administration of the 5-HT1A antagonist WAY 100635. Flesinoxan (4 mg/kg, intraperitoneally), another 5-HT1A agonist reproduced the effects of 8-OH-DPAT on pain threshold and gastric tone and the alpha2-receptor antagonist yohimbine did not modify the action of 8-OH-DPAT. Our results indicate that activation of 5-HT1A receptors at the level of the central nervous system increases gastric tone and decreases gastric sensitivity to distension.     

Inhibition of in vitro proliferation of chronic myelogenous leukemia progenitor cells by c-myb antisense oligodeoxynucleotides

Normal hematopoietic progenitors and acute myelogenous leukemia cells show a differential requirement for the encoded product of c-myb proto-oncogene for proliferation. To determine whether c-myb is also differentially required for the proliferation of hematopoietic progenitors of chronic myelogenous leukemia (CML), mononuclear cells derived from both chronic phase and blast crisis were exposed to c-myb antisense oligodeoxynucleotides and assayed for colony-forming ability. Exposure of CML-BC cells from 12 patients to c-myb antisense oligodeoxynucleotides resulted in significant (p<001) inhibition of leukemia colony formation (average inhibition 63%) and was accompanied by down-regulation of c-myb expression. Colonies derived from CML chronic phase progenitors were virtually unaffected in 10 cases, but down-regulation of c-myb expression was not detected. However, in studies conducted with CD34+ leukemia cells, a subset highly enriched for hematopoietic progenitors, colony formation was inhibited at both disease stages, whereas CFU-GM colony formation derived from normal CD34+ cells was not affected by exposure to c-myb antisense oligodeoxynucleotides. These data suggest that CML chronic phase and blast crisis progenitors are both sensitive to the inhibitory effects of c-myb antisense oligomers, and that the lack of inhibition in partially purified CML-chronic phase progenitors is probably due to inefficient penetration of oligodeoxynucleotides into the clonogenic cells. The preferential effect of c-myb antisense oligodeoxynucleotides on colonies arising from the compartment that includes CML-CD34+ progenitors likely reflects the expansion of a cell population with high proliferative potential and elevated c-myb mRNA levels.     

Camostate- and caerulein-induced delay of gastric emptying in the rat: effect of CCK receptor antagonists

The effect of camostate, a potent releaser of endogenous cholecystokinin (CCK), and of caerulein, an amphibian peptide mimicking the biological actions of CCK, as well as of selective CCK receptor antagonists on gastric emptying of liquids was studied in the rat. Oral administration of camostate (200 mg/kg with the liquid test meal preceded by the same dose 10 min before the meal) significantly delayed gastric emptying of saline, an effect which was completely blocked by previous administration of the CCKA receptor antagonist, devazepide, at a dose (1 mg/kg i.v.) unable to modify the emptying rate when administered alone. Caerulein (0.03-30 nmol/kg i.v.) also delayed the emptying rate in a dose-dependent manner, with an ID50 of 3.94 nmol/kg. The effect of the peptide was also inhibited by devazepide. The CCKB receptor antagonist, L365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1, 4-benzodiazepine-3-yl)-N'-(3-methylphenyl)-urea; 3 mg/kg i.v.), was completely unable to modify the CCK (both endogenous and exogenous)-induced delay in gastric emptying. Repeated (7 days) camostate administration did not modify the gastric motor response to endogenous CCK, thus, suggesting that adaptation did not take place. These results demonstrate that endogenous and exogenous CCK delays gastric emptying of liquids through stimulation of CCKA receptors and suggest that adaptation of the gastric motor response to CCK does not occur.