Activation of InsP(3) receptors is sufficient for inducing graded intrinsic plasticity in rat hippocampal pyramidal neurons

The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP(3)) receptors (InsP(3)R) in a form of intrinsic plasticity by asking if InsP(3)R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of D-myo-InsP(3) in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP 3 concentration, emphasizing the graded dependence of such plasticity on InsP(3)R activation. Mechanistically, we found that this InsP(3)-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP(3)Rs, the influx of calcium through N-methyl-D-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP(3)Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis.

Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors

This dissertation primarily describes studies of serotonin type 3 (5-HT₃) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT₃A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand’s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.

The third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT₃AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A₃B₂ stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT₃AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT₃AB receptor from these heteromeric sites.

Finally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RARα of the nuclear receptor family of transcription factors.

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT 3 receptors

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors. Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT3A receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. Key Results: 5-HT3 responses were blocked with IC50 values of 13.4 μM, 11.8 μM and 9.36 μM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA2 values of 4.92 (KB=12.0 μM) and 4.97 (KB=16.4 μM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [3H]granisetron with Ki values of 15.0, 24.2 and 35.7 μM. Docking of quinine into a homology model of the 5-HT3 receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT3 receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT3 receptor antagonists.

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.

Upregulation of toll-like receptors in chronic enteropathies in dogs

Background: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. Hypothesis: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. Animals: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. Methods: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. Results: There were significant differences (P small intestine >/= colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. Conclusions and Clinical Importance: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

Functional characterization of two human receptor activity-modifying protein 3 variants

Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM(2) receptor, whereas when paired with the calcitonin receptor AMY(3) receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT((a))). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM(2) and AMY(3(a)) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.

Análise e caracterização da expressão e papel funcional dos potenciais genes da família 2 GPCRs no protostómio Caenorhabditis elegans

Dissertação de Mestrado, Biotecnologia, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

HISTAMINE IN THE POSTERODORSAL MEDIAL AMYGDALA MODULATES CARDIOVASCULAR REFLEX RESPONSES IN AWAKE RATS

Centrally injected histamine (HA) affects heart rate (HR), arterial blood pressure (BP), and sympathetic activity in rats. The posterodorsal medial amygdala (MePD) has high levels of histidine decarboxylase, connections with brain areas involved with the modulation of cardiovascular responses, and is relevant for the pathogenesis of hypertension. However, there is no report demonstrating the role of the MePD histaminergic activity on the cardiovascular function in awake rats. The alms of the present work were: 1) to study the effects of two doses (10-100 nM) of HA microinjected in the MePD on basal cardiovascular recordings and on baroreflex- and chemoreflex-mediated responses; 2) to reveal whether cardiovascular reflex responses could be affected by MePD microinjections of (R)-alpha-methylhistamine (AH(3)), an agonist of the inhibitory autoreceptor H(3); and, 3) to carry out a power spectral analysis to evaluate the contribution of the sympathetic and parasympathetic components in the variability of the HR and BP recordings. When compared with the control group (microinjected with saline, 0.3 mu l), HA (10 nM) promoted an increase in the MAP(50), i.e. the mean value of BP at half of the HR range evoked by the baroreflex response. Histamine (100 nM) did not affect the baroreflex activity, but significantly decreased the parasympathetic component of the HR variability, increased the sympathetic/parasympathetic balance at basal conditions (these two latter evaluated by the power spectral analysis), and promoted an impairment in the chemoreflex bradycardic response. Microinjection of AH(3) (10 mu M) led to mixed results, which resembled the effects of both doses of HA employed here. Present data suggest that cardiovascular changes induced by baroreceptors and chemoreceptors involve the histaminergic activity in the MePD. This neural regulation of reflex cardiovascular responses can have important implications for homeostatic and allostatic conditions and possibly for the behavioral displays modulated by the rat MePD. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Central muscarinic receptor subtypes involved in pilocarpine-induced salivation, hypertension and water intake

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The possible involvement of hyperpolarizing mechanisms in histamine-induced relaxation of the rat portal vein

The present study evaluated the effects of histamine 10 -2 M on longitudinal preparations of rat portal vein. It was observed that histamine 10 -2 M induced relaxation of rat portal vein preparations pre-contracted with phenylephrine 10 -4 M. On the other hand, no pharmacological effects were observed in preparations not pre-contracted. The observed histamine-induced relaxing effect was absent in preparations pre-contracted with KCl (120 mM) or in the presence of depolarizing nutritive solution. However, the histamine-induced relaxation was still present in the endothelium-removed preparations. The histamine-induced relaxation also was not prevented by astemizole (10 -6 M, 10 -5 M and 10 -4 M), cimetidine (10 -5 M, 10 -4 M and 10 -3 M) or thioperamide (10 -6 M, 10 -5 M and 10 -4 M), selective antagonists H 1, H 2 and H 3, respectively. The presence of L-NAME 10 -4 M or L-NAME 10 -4 M plus indomethacin 10 -5 M also did not prevent the histamine-induced relaxation observed in rat portal vein. Thus, the histamine-induced relaxation observed in rat portal vein appears to involve a non-endothelial hyperpolarizing mechanism independent of H 1, H 2 and H 3 receptors.

Effect of intravenous tropisetron on modulation of pain and central hypersensitivity in chronic low back pain patients

The activation of 5-hydroxytryptamine-3 (5-HT-3) receptors in spinal cord can enhance intrinsic spinal mechanisms of central hypersensitivity, possibly leading to exaggerated pain responses. Clinical studies suggest that 5-HT-3 receptor antagonists may have an analgesic effect. This randomized, double-blind, placebo-controlled crossover study tested the hypothesis that the 5-HT-3 receptor antagonist tropisetron attenuates pain and central hypersensitivity in patients with chronic low back pain. Thirty patients with chronic low back pain, 15 of whom were women (aged 53 ± 14 years) and 15 men (aged 48 ± 14 years), were studied. A single intravenous injection of 0.9% saline solution, tropisetron 2mg, and tropisetron 5mg was administrated in 3 different sessions, in a double-blind crossover manner. The main outcome was the visual analogue scale (VAS) score of spontaneous low back pain before, and 15, 30, 60, and 90 minutes after drug administration. Secondary outcomes were nociceptive withdrawal reflexes to single and repeated electrical stimulation, area of reflex receptive fields, pressure pain detection and tolerance thresholds, conditioned pain modulation, and area of clinical pain. The data were analyzed by analysis of variance and panel multiple regressions. All 3 treatments reduced VAS scores. However, there was no statistically significant difference between tropisetron and placebo in VAS scores. Compared to placebo, tropisetron produced a statistically significant increase in pain threshold after single electrical stimulation, but no difference in all other secondary outcomes was found. A single-dose intravenous administration of tropisetron in patients with chronic low back pain had no significant specific effect on intensity of pain and most parameters of central hypersensitivity.

Distribution of muscarinic receptor subtypes and interstitial cells of Cajal in the gastrointestinal tract of healthy dairy cows

OBJECTIVE: To describe the distribution of muscarinic receptor subtypes M(1) to M(5) and interstitial cells of Cajal (ICCs) in the gastrointestinal tract of healthy dairy cows. SAMPLE POPULATION: Full-thickness samples were collected from the fundus, corpus, and pyloric part of the abomasum and from the duodenum, ileum, cecum, proximal loop of the ascending colon, and both external loops of the spiral colon of 5 healthy dairy cows after slaughter. PROCEDURES: Samples were fixed in paraformaldehyde and embedded in paraffin. Muscarinic receptor subtypes and ICCs were identified by immunohistochemical analysis. RESULTS: Staining for M(1) receptors was found in the submucosal plexus and myenteric plexus. Antibodies against M(2) receptors stained nuclei of smooth muscle cells only. Evidence of M(3) receptors was found in the lamina propria, in intramuscular neuronal terminals, on intermuscular nerve fibers, and on myocytes of microvessels. There was no staining for M(4) receptors. Staining for M(5) receptors was evident in the myocytes of microvessels and in smooth muscle cells. The ICCs were detected in the myenteric plexus and within smooth muscle layers. Distribution among locations of the bovine gastrointestinal tract did not differ for muscarinic receptor subtypes or ICCs. CONCLUSIONS AND CLINICAL RELEVANCE: The broad distribution of M(1), M(3), M(5), and ICCs in the bovine gastrointestinal tract indicated that these components are likely to play an important role in the regulation of gastrointestinal tract motility in healthy dairy cows. Muscarinic receptors and ICCs may be implicated in the pathogenesis of motility disorders, such as abomasal displacement and cecal dilatation-dislocation.

Triple-peptide receptor targeting in vitro allows detection of all tested gut and bronchial NETs.

UNLABELLED A high proportion of gut and bronchial neuroendocrine tumors (NETs) overexpresses somatostatin receptors, especially the sst2 subtype. It has also recently been observed that incretin receptors, namely glucagonlike peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) receptors, can be overexpressed in gut and bronchial NETs. However, because not all tumors can express these receptors in sufficient amounts, in vivo imaging with a single radioligand may not always be successful. We therefore evaluated with in vitro methods whether a cocktail of radioligands targeting these 3 receptors would improve tumor labeling. METHODS In vitro receptor autoradiography was performed on 55 NETs, comparing in each successive section of tumor the binding with a single radioligand, either (125)I-Tyr(3)-octreotide, (125)I-GLP-1(7-36)amide, or (125)I-GIP(1-30), with the binding using a cocktail of all 3 radioligands, given concomitantly under identical experimental conditions. RESULTS Using the cocktail of radioligands, all tumors without exception showed moderate to very high binding, with a receptor density corresponding to 1,000-10,000 dpm/mg of tissue; conversely, single-ligand binding, although identifying most tumors as receptor-positive, failed to detect receptors or measured only a low density of receptors below 1,000 dpm/mg in a significant number of tumors. In addition, the cocktail of radioligands always provided a homogeneous labeling of the whole tumor, whereas single radioligands occasionally showed heterogeneous labeling. CONCLUSION The study suggests that the use of a cocktail of 3 radioligands binding to somatostatin receptors, GLP-1 receptors, and GIP receptors would allow detecting virtually all NETs and labeling them homogeneously in vivo, representing a significant improvement for imaging and therapy in NETs.