Biomedical subjects

H Tamir

Publications and source records attributed to H Tamir.

At least 19 recordsLinked to original sources

PubMed1992

Serotonergic signalling between thyroid cells: protein kinase C and 5-HT2 receptors in the secretion and action of serotonin.

Parafollicular (PF) cells of the thyroid gland are neural crest derivatives, which costore the neurotransmitter, 5-hydroxytryptamine (5-HT) with calcitonin. PF cells are located adjacent to follicular (F) cells within the basement membrane of thyroid follicles. It has been proposed that 5-HT serves an intercellular signalling function in the thyroid and that F cells are its target. This proposal was tested by using cell lines derived from PF (medullary thyroid carcinoma [MTC]) and F (FRTL-5) cells to study the mechanisms that mediate the secretion and action of 5-HT. Secretion of 5-HT by MTC cells was evoked by thyroid stimulating hormone, thyrotropin (TSH), elevated extracellular calcium (increases [Ca2+]e), or by agents that increase intracellular cAMP (increases [cAMP]i). When protein kinase C (PKC) was down-regulated by prolonged treatment of MTC cells with phorbol 12-myristate 13-acetate (PMA), or PKC was inhibited by staurosporin, the TSH- or PMA-evoked secretion of 5-HT was blocked; however, interference with PKC function did not affect 5-HT secretion evoked by increases [Ca2+]e or increases [cAMP]i. In the putative targets, FRTL-5 cells, 5-HT increased the turnover of phosphoinositides (PI), cytosolic calcium (increases [Ca2+]i), increases [cAMP]i, and biphasically modified the effect of TSH on cAMP. All of these 5-HT effects were inhibited by 5-HT2 receptor antagonists (spiperone and ketanserin) and by pertussis toxin (PTx), suggesting that the actions of 5-HT are mediated by 5-HT2 receptors, which are coupled to a G protein. This suggestion was supported by the following additional observations: FRTL-5 membranes bound the 5-HT2 agonist, [125I]2,5-dimethoxy-4-iodophenylisopropylamine ([125I]-DOI), and anti-idiotypic antibodies, which recognize 5-HT2 receptors. [125I]-DOI binding was inhibited by guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) and the antibodies were displaced by spiperone. Data are consistent with the hypothesis that 5-HT serves as a PF to F cell messenger.

Alkaloids↗

PubMed1992

Identification and localization of 5-HT1P receptors in the guinea pig pancreas.

Enteropancreatic 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers have recently been described. To test the hypothesis that pancreas contains 5-HT receptors similar to those that characterize the enteric nervous system, binding of 5-[3H]HT to pancreatic membranes was investigated. Membranes were prepared by differential centrifugation from guinea pig pancreas, and binding of 5-[3H]HT was measured by use of a rapid filtration assay. Alternatively, 5-[3H]HT-binding sites were analyzed and located by radioautography on frozen sections of unfixed pancreas. The 5-HT1P receptor-specific antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), was used to define nonspecific binding in all assays. High-affinity reversible binding of 5-[3H]HT to pancreatic membranes was found. Good agreement was observed when Kd was determined from saturation isotherms (Kd = 2.1 +/- 0.4 nM; Bmax = 0.6 +/- 0.07 pmol/mg protein) or from the ratio of association and dissociation constants (2.7 +/- 0.04 nM). As is true of 5-[3H]HT binding to enteric 5-HT1P receptors, binding of 5-[3H]HT to pancreatic membranes was inhibited by 5-HTP-DP, 5- and 6-hydroxyindalpine (agonists at 5-HT1P receptors), and guanine nucleotides. In contrast, binding of 5-[3H]HT to pancreatic membranes was not inhibited by a variety of compounds known to bind to other types of 5-HT receptor, to the neuronal 5-HT transporter, to alpha- or beta-adrenoceptors, or to dopamine receptors. We concluded that pancreas contains 5-HT1P receptors with properties similar to those of bowel. Radioautography and immunocytochemistry with an anti-idiotypic antibody that recognizes 5-HT receptors revealed that pancreatic 5-HT1P receptors (identified with appropriate inhibitors of 5-[3H]HT or antibody binding) were concentrated in ganglia, nerves, and unidentified cells in both exocrine and endocrine pancreas. These observations suggest that future studies of 5-HT's role in pancreatic physiology should concentrate on enteropancreatic innervation and 5-HT1P receptors.

Adenosine Triphosphate↗

PubMed1992

Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity-induced expression of Fos immunoreactivity.

The bowel is the only organ of the body in which neural reflexes can be elicited in the absence of input from the brain or spinal cord. This activity is mediated by the enteric nervous system (ENS), which contains primary afferent neurons. Experiments were carried out to locate the primary afferent neurons of the ENS. Two types of stimulation were used to activate neurons in the wall of the gut in vitro: exposure of the mucosa to cholera toxin or delivery of pressure to the mucosal surface with puffs of N2 from a micropipette. Neurons that became active in response to these stimuli were identified by demonstrating the intranuclear immunoreactivity of Fos, the product of the c-fos protooncogene. No Fos immunoreactivity could be detected in the absence of stimulation; however, application of cholera toxin and puffs of N2 each induced the appearance of Fos immunoreactivity in neurons in both the submucosal and myenteric plexuses. With either stimulus, the induction of Fos immunoreactivity was antagonized by TTX and therefore depended on neuronal activity. The appearance of Fos immunoreactivity could also be prevented by the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide. In contrast, the stimulus-induced expression of Fos immunoreactivity was inhibited, but not abolished, by hexamethonium, which limited the spread of activation within the submucosal plexus and completely prevented expression of Fos immunoreactivity by myenteric neurons in response to mucosal puffs of N2. FluoroGold was injected into single ganglia of the myenteric plexus in order to identify submucosal neurons with myenteric projections. Submucosal neurons in which Fos immunoreactivity was induced by the stimuli were doubly labeled by FluoroGold. A subset of the submucosal, but not myenteric, neurons that expressed Fos immunoreactivity was doubly labeled by antibodies to calbindin. Submucosal calbindin-immunoreactive neurons were found to contain substance P immunoreactivity and could also be immunostained by anti-idiotypic antibodies that react with 5-HT1P receptors. A subset of dynorphin1-8-immunoreactive submucosal neurons (which are known to costore vasoactive intestinal peptide and to be secretomotor in function) expressed nuclear Fos immunoreactivity in response to cholera toxin, but not puffs of N2. These data suggest that intrinsic primary afferent neurons are located in the submucosal plexus, project to the myenteric plexus, and are activated by 5-HT acting on the 5-HT1P receptor subtype. These neurons are probably cholinergic and costore calbindin and substance P.

Animals↗

PubMed1991

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms.

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.

Adenosine Diphosphate Ribose↗

PubMed1991

G-protein beta gamma forms: identity of beta and diversity of gamma subunits.

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.

Adenosine Triphosphate↗

PubMed1991

Identification of serotonin receptors recognized by anti-idiotypic antibodies.

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.

Androstadienes↗

PubMed1990

G protein diversity is increased by associations with a variety of gamma subunits.

Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimeric proteins that transduce extracellular signals into intracellular changes. Functionally different G proteins have been identified by their different alpha subunits. The beta and gamma subunits have been assumed to constitute a common pool shared among various G protein heterotrimers. Two gamma subunits (gamma 3 and gamma 4) have been identified through molecular cloning; these are in addition to two subunits (gamma 1 and gamma 2) that were previously characterized. G proteins purified from a variety of mammalian tissues were examined with antisera specific to three of the gamma subunits. The antisera react with different gamma subunits associated with some of the purified G proteins but not all. This demonstrates that different G protein heterotrimers from different tissues carry structurally distinct members of the gamma-subunit family. Diversity in the structure of the gamma as well as the alpha and beta subunits and preferential associations between members of subunit families increase structural and possibly functional diversity of G proteins.

Amino Acid Sequence↗

PubMed1990

Photoaffinity labeling of the two forms of serotonin binding protein: peptide mapping of the binding sites.

Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm-derived cells that store 5-hydroxytryptamine (5-HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5-HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56-kDa form of SBP may be the precursor of the 45-kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N-(4-azido-2-nitrophenyl)-5-hydroxytryptamine (NAP-5-HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV-visible absorption spectra. A 1 M excess of NAP-5-HT inhibited the binding of [3H]5-HT to SBP by 50%. NAP[3H]5-HT was also synthesized and attached to both high- and low-affinity binding sites on both forms of SBP. The high-affinity constants for 45-kDa and 56-kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low-affinity constants were 0.3 microM and 0.15 microM. When the high-affinity site of partially purified SBP was photoaffinity-labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15-fold molar excess of 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1990

Preparation and characterization of monoclonal antibodies to serotonin binding protein.

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Serotonin-storing secretory vesicles.

Advances have been made in the characterization of 5-HT-storing organelles of neurectodermal cells. The parafollicular cell of the thyroid has been used as a model. This cell stores 5-HT, shares many properties with neurons, and can be induced to change its phenotype from endocrine to neuronal by exposure in vitro to NGF. The membranes of isolated parafollicular 5-HT storage vesicles appear to contain a chloride channel that is gated in response to stimulation of the cells by secretogogues. Opening of this channel permits the interior of the vesicle to acidify in response to the action of a H+ ATPase in the vesicular membrane. Development of a delta psi appears to limit acidification of the vesicular interior when the chloride conductance is low. Transmembrane transport of 3H-5-HT into parafollicular vesicle is inhibited by dissipating the delta pH across the granular membranes. The physiological significance of the ability of parafollicular vesicles to modify the internal pH of their 5-HT-storing organelles remains to be determined. Like the synaptic vesicles of central and peripheral serotonergic neurons parafollicular vesicles contain a specific 5-HT binding protein, SBP. 5-HT storage organelles and SBP have been found in medullary thyroid carcinoma (MTC) cells, a tumor line derived from parafollicular cells. The cell biology of SBP is now under study utilizing the MTC cells.

Animals↗

PubMed1990

Multiple signal transduction mechanisms leading to the secretion of 5-hydroxytryptamine by MTC cells, a neurectodermally derived cell line.

Parafollicular (PF) cells of the thyroid gland are neural crest derivatives. These cells remain plastic even in adult animals and can be induced to exhibit neural properties when exposed to NGF in vitro. A human cell line derived from PF cells, medullary thyroid carcinoma (MTC), has previously been shown to synthesize and store 5-HT, a serotonin-binding protein (SBP), and several neuropeptides; moreover, when grown in impoverished media, MTC cells display neural properties. The purpose of the current study was to utilize MTC cells as a neurally relevant model system to investigate factors involved in mediating 5-HT secretion. Electron microscopic immunocytochemistry revealed that secretory vesicles of MTC cells costore immunoreactive 5-HT with SBP and calcitonin. The cAMP derivative, N6-2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP; 1.0 mM) increased the concentration of 5-HT in MTC cells and almost doubled the rate of synthesis of 5-HT from L-tryptophan. Dibutyryl-cAMP also significantly increased the secretion of 5-HT. Cycloheximide (20 micrograms/ml) and anisomycin (20 microM) inhibited the dibutyryl-cAMP-induced increase of 5-HT release, suggesting that this action of dibutyryl-cAMP requires protein synthesis. Cholera toxin (1.0 microgram/ml) and forskolin (0.05 mM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1.0 mM) both increased 5-HT biosynthesis and secretion. Attempts were made to identify a ligand that stimulates cAMP-mediated secretion of 5-HT. Both thyroid-stimulating hormone (TSH: 50 mU/ml) and elevated [Ca2+]e (7.0 mM), each of which acts as a secretogogue for PF cells, stimulated the secretion of 5-HT. The effect of TSH was Ca2(+)-dependent. Immunocytochemistry with monoclonal antibodies to the TSH receptor confirmed that these receptors are present on MTC cells. Neither TSH nor elevated [Ca2+]e elevated cAMP levels. Measurements of Fura-2 fluorescence, however, indicated that both TSH and elevated [Ca2+]e increased cytosolic calcium ([Ca2+]i), as did elevation of [K+]e. It is concluded that exocytosis can be triggered in MTC cells by multiple signal transduction mechanisms. Either cAMP or elevated [Ca2+]i can stimulate secretion; however, a secretogogue that increases cAMP has yet to be identified.

Animals↗

PubMed1989

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens.

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.

Adrenal Medulla↗

PubMed1989

Localization of serotonin receptors in the rat thalamus by electrophysiology and the action of 5-HTP-DP-hex.

This study was designed to pinpoint the site at which N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP-hex) exerts its previously reported effect on thalamic neurons in rats. The animals were prepared under chloralose-urethane anesthesia for a stereotaxic approach to either the nucleus ventralis posterolateralis (nVPL) or the centrum medianum-parafascicularis complex (CM-Pf) of the thalamus. Individual neurons in these nuclei were separately activated by single-pulse stimulation of the sciatic nerve or the thalamic fibers that form reciprocal connections between the CM-Pf and the second somatosensory (SII) region of the nVPL. Poststimulus time histograms were constructed from computer readouts of the stimulus-evoked responses of a neuron during a 500-ms period accumulated in a digital computer 100X. In addition, the number of spikes accumulated in each histogram was compared to the number of spikes accumulated under identical conditions on the same neuron after intracarotid infusion of 5-HTP-DP-hex. The effect of the drug was reversed by the infusion of 5-HTP. Statistical evaluation of the accumulated spike counts indicated that 5-HTP-DP-hex suppressed only the excitation of CM-Pf neurons from the SII of the nVPL; the input of the sciatic nerve into the CM-Pf remained unaltered. Furthermore, no effect was exerted by this dipeptide on the afferent excitation of neurons in the SII of the nVPL.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptophan↗

PubMed1989

Human medullary thyroid carcinoma: characterization of the serotonergic and neuronal properties of a neurectodermally derived cell line.

Parafollicular (PF) cells have been found to be a good model system for the study of serotonergic cellular mechanisms relevant to neurons. PF cells are derived from the same region of the neural crest that gives rise to the neurons of the gut and are capable of extending neurofilament-bearing neuritic processes. PF cells also synthesize 5-hydroxytryptamine (5-HT) and costore 5-HT in the same vesicles as the specific 5-HT-binding protein, 45 kDa SBP. A hypothesis has been advanced that PF cells and enteric neurons share a common developmental precursor. The present investigation was undertaken in order to determine whether a human medullary thyroid carcinoma (MTC) cell line, which is derived from PF cells, sufficiently mimics PF cells that it can be substituted for them in investigations of serotonergic cellular biology. In contrast to PF cells, MTC cells can be propagated in vitro to provide adequate amounts of material for biochemical studies. MTC cells were found to contain neuropeptides, including calcitonin, calcitonin gene-related peptide, and somatostatin, which have also been reported to be present in PF cells and enteric neurons. MTC cells also were observed to store endogenous 5-HT, to be able to synthesize 3H-5-HT from 3H-L-tryptophan, and to take up 3H-5-HT from the ambient medium by a carrier-mediated mechanism very similar to that of serotonergic neurons. In addition, the longterm accumulation of 3H-5-HT in MTC cells was antagonized by reserpine, suggesting that the cells contain 5-HT storage vesicles that, like the synaptic vesicles of serotonergic neurons, are characterized by a reserpine-sensitive transporter of biogenic amines. MTC cells also contain type A, but not type B, monoamine oxidase. Finally, MTC cells were found to contain both 45 and 56 kDa SBP. MTC cells thus retain a great many of the properties of PF cells, and, like PF cells, they are serotonergic cells with characteristics similar to serotonergic neurons. Substantial differences were found in the content of immunoreactive 5-HT and neuropeptides in individual MTC cells. Moreover, the release of newly synthesized 5-HT to the medium exceeded the ability of the cells to store the amine. Studies of the ultrastructure of the MTC cells revealed a limited and highly variable number of secretory granules, probably accounting for their limited 5-HT storage capacity and for the heterogeneity of immunostaining with antisera to 5-HT or neuropeptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Ultrastructural immunocytochemical studies of the localization and distribution of somatostatin, calcitonin, calcitonin gene-related peptide, and substance P in the bat thyroid follicle.

Electron microscope immunocytochemistry was used to determine the intracellular localization and distribution among follicular elements of four peptides: calcitonin, somatostatin, calcitonin gene-related peptide (CGRP), and substance P in the thyroid glands of bats captured in the prehibernation phase of their annual life cycle. Previous studies have shown that this period of the hibernation-activity cycle is characterized by the accumulation and storage of secretory granules in parafollicular cells. Sites of binding of primary antisera to each of the four peptides were identified by means of affinity-purified secondary antisera directly coupled to colloidal gold particles. Calcitonin and somatostatin immunoreactivities were found in all parafollicular cells examined and in every secretory granule within these cells. CGRP was also found in all parafollicular cells examined (n = 75) but only in about half of their secretory granules. In contrast to these peptides, substance P immunoreactivity was not found in any parafollicular cells, but was localized exclusively in nerve endings within the basement membrane of the follicle.

Animals↗

PubMed1988

Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane.

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.

Adenosine Triphosphate↗

PubMed1988

Serotonin and morphogenesis. I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo.

The possible involvement of the neurotransmitter serotonin (5-HT) in morphogenesis of the craniofacial region in the mouse embryo has been investigated using the method of whole-embryo culture. Day-12 embryos were incubated for 3-4 h in the presence of 5-HT or its precursors L-tryptophan (L-TRP) or 5-hydroxytryptophan (5-HTP), followed by fixation, sectioning and staining with a specific antiserum to 5-HT. Sites of 5-HT immunoreactivity were found in a variety of locations in tissues of the head and neck, which are either epithelia derived from the non-neural ectoderm or are non-neuronal midline brain structures. These sites include the surface epithelia of the head, face, nasal prominences, branchial arches, oral cavity and associated parts of the nasal epithelium, the epithelium covering the eye, parts of the otic vesicle, the epiphysis and roof of the diencephalon. With the exception of the oral cavity, sites of immunoreactivity for serotonin-binding protein were identified in the mesenchyme adjacent to these sites. This mesenchyme consists of ectodermally derived neural crest cells, which are known to receive inductive influences from the epithelia with which they interact during their migration through the craniofacial region. The presence of 5-HT uptake sites in epithelia and adjacent sites of SBP in the underlying mesenchyme raises the possibility that 5-HT might be involved in those epithelial-mesenchymal interactions known to be important for the development of structures in the craniofacial region.

5-Hydroxytryptophan↗