Biomedical subjects

H Tamir

Publications and source records attributed to H Tamir.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1988

Costorage of serotonin binding protein with serotonin in the rat CNS.

Previous studies have identified two neurectoderm-specific serotonin binding proteins (SBP), one with an apparent Mr of 45 kDa, and one of 56 kDa. The current experiments were undertaken to test the hypothesis that these proteins are specific components of serotonergic neurons. Since actin has been found to bind serotonin, the relationship of the 2 forms of SBP to actin was also investigated. Antisera against purified 45 and 56 kDa SBP were raised in rabbits and shown by analyses of immunoblots and differential absorption to be monospecific and not cross-reactive. Neither antiserum reacted with purified actin and none of 3 different anti-actin sera reacted with purified 45 or 56 kDa SBP. The antisera to 45 and 56 kDa were used for immunocytochemical localization of the proteins, which was compared to that of serotonin. SBP immunoreactivity was found in rat brain and spinal cord; however, no significant differences were observed in the pattern of distribution of 45 and 56 kDa SBP-immunoreactive structures. Immunostaining of neuronal perikarya by either SBP antiserum required pretreatment of animals with colchicine. The distribution of neurons and terminals labeled by each antiserum to SBP was similar to that of neurons and terminals labeled by anti-5-HT sera. SBP-immunoreactive neuronal perikarya were present in the nuclei raphe dorsalis, raphe centralis superior, raphe medianus, raphe magnus, raphe obscurus, raphe pallidus, dorsal to the medial lemniscus in the region of the B9 cell group, near the interpeduncular nucleus, in the area postrema, the pars compacta of the substantia nigra, the dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. SBP-immunoreactive fibers and terminals were present in many additional areas of the brain, as well as the spinal cord, where they paralleled those that were immunostained with antibodies to 5-HT. When double-immunostaining was used, serotonin and 45 and 56 kDa SBP immunoreactivities were found to be colocalized in both the brain and spinal cord. Cells and fibers found to be stained by one immunoreagent were also stained by the others; therefore, serotonergic neurons of the CNS probably contain both 45 and 56 kDa SBP. Moreover, it also seems likely that nonserotonergic neurons contain neither form of SBP. These data strongly suggest that SBP is an intrinsic and specific component of serotonergic neurons that can serve as a serotonergic marker.

Animals↗

PubMed1987

Proteins that bind serotonin in the nervous system of Aplysia californica.

The central nervous system of Aplysia has been found to contain two forms of proteins that bind serotonin with high affinity. These proteins share several similarities with the serotonin binding proteins that have been isolated from the vertebrate nervous system. Their ability to bind serotonin is enhanced by Fe2+ and inhibited by Na+,-SH reagents as well as reserpine and N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophanamide.

Animals↗

PubMed1987

Influence of a serotonin receptor antagonist, 5-HTP-DP-hex, on spinal and thalamic nociceptive neurons in rats.

The antinociceptive properties of a new synthetic dipeptide (N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, or 5-HTP-DP-hex) were studied in rats by an electrophysiological method. After an i.p. injection of alpha-chloralose and urethane, the animals were prepared for stereotaxic approach to the nucleus ventralis posterolateralis of the thalamus. With tungsten microelectrodes, individual nociceptive neurons in the nucleus were identified by the sequence of spikes emitted in response to single-pulse stimulation of the sciatic nerve. In addition to the usual short-latency spikes, a nociceptive neuron fired late spikes at regular intervals within 500 ms following each stimulus. When the spikes were accumulated in poststimulus time histograms, the short-latency spikes compiled an intensity-related (I) peak. The late spikes formed modality-related (M) peaks with spacing characteristic of nociception. Intracarotid infusion of 5-HTP-DP-hex (1 mg/kg) elevated the delayed portion of the I peak and the first M peak. This effect was followed in 25 min by suppression of all M peaks. The control record could be reinstated at any time by 5-hydroxytryptophan (3.5 mg/kg), or by natural recovery in 2.5 h. Responses evoked from a thalamic nociceptive neuron by single-pulse stimulation of the spinothalamic tract were modified by 5-HTP-DP-hex in a similar manner, except that no elevation of the activity peaks was observed. As shown previously, elevation of the delayed I peak and M1 indicated an increased input of A-delta and C fibers, respectively. The increased input lowers the response threshold and may represent hyperalgesia. Suppression of the M peaks may result from altered function of the positive feedback loop in the nociceptive system at the thalamic level, and may represent analgesia. Naloxone, methysergide, as well as ketanserin had no significant effect on the response histograms. These findings suggested that 5-HTP-DP-hex, a known serotonin receptor antagonist, targeted its action on very specific receptors, and thus interfered with particular synaptic activity within the spinal cord and on the thalamic level.

5-Hydroxytryptophan↗

PubMed1987

Prenatal development of serotonin binding protein in relation to other transmitter-related characteristics of central serotonergic neurons.

Serotonin binding protein (SBP) is a neuron-specific protein that binds serotonin (5-HT) with high affinity and is concentrated in synaptic vesicles. 5-HT has been shown to be stored in situ in a macromolecular complex with SBP. We have now investigated the ontogeny of SBP in the rat CNS. The time course of the appearance of SBP was related to the appearance of tryptophan hydroxylase, endogenous 5-HT and monoamine oxidase (MAO; types A and B). Binding of [3H]5-HT by SBP was assayed using molecular sieve chromatography. SBP had appeared by day E16; its activity then rose rapidly and reached adult levels (150 nmol [3H]5-HT/g protein) at days E18-E19. Tryptophan hydroxylase activity was measured by following the accumulation in vivo (30 min) of 5-hydroxytryptophan (5-HTP) in the brains of rat embryos whose mothers were treated with the aromatic L-amino acid decarboxylase inhibitor, NSD-1015, (100 mg/kg; i.p.). Tryptophan hydroxylase activity was first detectable at E15, remained present but at a low level through day E20 and then rapidly increased to reach 75% of the adult level at birth (747 pmol/g brain wet wt.). The development of stores of endogenous 5-HT paralleled the time course of development of tryptophan hydroxylase rather than that of SBP. 5-HT was first detected at E15, remained low until the end of intrauterine life and at birth was 50% of the adult level (2640 pmol/g brain wet wt.). MAO activity was determined in crude mitochondrial fractions by measuring 5-hydroxyindoleacetic acid produced from 5-HT as substrate. This activity was already present prior to day E15 (the activity of type B MAO was higher than that of type A) and reached adult levels at day E20 (55 pmol/mg protein/min; A, B). It is concluded that the potential of neurons to store 5-HT, as measured by the activity of SBP, develops more rapidly than their ability to produce 5-HT. Moreover, although the ratio of its two forms changes, MAO activity appears very early in development.

Animals↗

PubMed1987

A Ca2+-dependent protein kinase activity associated with serotonin binding protein.

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.

Animals↗

PubMed1987

Induction of a neural phenotype in a serotonergic endocrine cell derived from the neural crest.

The thyroid parafollicular cell is an endocrine cell derived from the neural crest that stores 5-hydroxytryptamine (5-HT). In common with serotonergic neurons, but in contrast to 5-HT-storing cells that are not neurectodermal derivatives, parafollicular cells also contain a specific 5-HT binding protein. Despite this similarity to serotonergic neurons, parafollicular cells in situ were found to express an endocrine phenotype with few neural characteristics. Thus, the cells costore 5-HT with calcitonin, not calcitonin gene-related peptide (CGRP), which is the product of the calcitonin gene expressed in neurons, and they do not contain neurofilaments. The ability of adult parafollicular cells to respond to microenvironmental perturbations by expressing neuronal characteristics was examined. Sheep thyroid glands were dissociated, and parafollicular cells were purified by affinity chromatography. The purified parafollicular cells were grown in culture on a variety of substrates in the presence or absence of the beta subunit of nerve growth factor (beta-NGF). Parafollicular cells survived in culture for at least a week but retained a roughly spherical shape. Nevertheless, a subset of the cultured parafollicular cells began to display CGRP immunoreactivity. The addition of beta-NGF to the cultured parafollicular cells induced a number of them to extend neurites and increased the proportion of cells in which CGRP immunoreactivity could be found. Neurite-bearing parafollicular cells appeared not to survive for more than 2 d. While their survival was not enhanced when they were grown on collagen, polylysine, laminin, or reconstituted basal lamina, parafollicular cells that had extended neurites in response to beta-NGF survived for at least a week when cocultured with an explant of aneuronal chick hindgut. The effect of the gut was local and only those neurite-bearing parafollicular cells that were growing in direct contact with the explant survived. The thyroid parafollicular cell therefore resembles another crest-derived endocrine cell, the adrenal chromaffin cell, in being able to manifest neural properties in culture. For the parafollicular cell these neural properties include the processing of RNA encoded by the calcitonin gene to express CGRP and neurite outgrowth in response to beta-NGF.

Animals↗

PubMed1987

Serotonin-storing secretory granules from thyroid parafollicular cells.

The subcellular storage of 5-HT was studied in sheep thyroid parafollicular cells. These cells are neural crest derivatives and were investigated as a serotonergic model system. Light and electron microscopic immunocytochemistry was used to examine the distributions of 5-HT, 45 and 56 kDa forms of 5-HT binding protein (SBP), and calcitonin. A single type of parafollicular cell was found that contained calcitonin, 5-HT, and 45 kDa SBP but not 56 kDa SBP. The secretory granules of parafollicular cells all displayed calcitonin immunoreactivity, and many were also immunoreactive for 5-HT and 45 kDa SBP. Granules were isolated, first by size and then by density, on successive metrizamide gradients. These provided a granular fraction that was enriched in calcitonin, endogenous 5-HT, and 45 kDa SBP. Immunoblots confirmed the presence of 45 kDa SBP in the isolated granules and in suspensions of parafollicular cells that were purified by an affinity chromatographic technique. Parafollicular cell granules did not appear to contain substantial stores of ATP. Granules isolated on Percoll gradients were morphologically homogeneous and took up 3H-5-HT. The specificity of this uptake was confirmed by quantitative electron microscopic radioautography. The granular uptake of 3H-5-HT was inhibited by reserpine (10 microM). It is concluded that parafollicular cell granules are different from other amine-storing vesicles that do contain ATP; nevertheless, since parafollicular cell granules store 5-HT and have the same 45 kDa SBP as is found in serotonergic axon terminals, parafollicular cell granules may be analogous to the synaptic vesicles of serotonergic neurons.

Animals↗

PubMed1985

Serotonin depletion induced by reserpine is attenuated by prophylactic administration of lithium.

Prophylactic administration of lithium significantly attenuated the serotonin depleting effects of reserpine. In rat brain, lithium did not change the capacity of the storage protein, serotonin binding protein to bind the amine either in vivo or in vitro nor did it change its sensitivity to reserpine. However, the chronic administration of lithium significantly decreased reserpine levels in plasma and brain tissue of treated rats compared with rats which were treated only with reserpine. It is concluded that the antagonism between reserpine and lithium does not involve the serotonin binding protein but may be due to the lithium-induced reduction of reserpine levels.

Animals↗

PubMed1985

The enteric neural receptor for 5-hydroxytryptamine.

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.

5-Hydroxytryptophan↗

PubMed1985

Identification, purification, and characterization of two forms of serotonin binding protein from rat brain.

Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5-Hydroxytryptamine (5-HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4-B, Affi-Gel-Blue, hydroxylapatite, and phosphocellulose. The 45K from of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate-gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two-dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5-HT binding capacity; the enhancement of 5-HT binding by Fe2+; and inhibition by--SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross-reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants--56K form showed higher affinity for 5-HT (KD1 = 0.4 nM; KD2 = 32 nM), whereas the 45K form showed lower affinity (KD1 = 9.7 nM; KD2 = 120 nM); (2) ratio of number of 5-HT binding sites with low affinity to those with high affinity--56K (19:1), 45K (10:1); (3) isoelectric point--the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Human serotonectin: a blood glycoprotein that binds serotonin and is associated with platelets and white blood cells.

A glycoprotein that circulates in blood, binds to the surface of platelets, and also binds serotonin with high avidity and specificity, has previously been found in rats. This glycoprotein has been called serotonectin. We now report the purification and characterization of a similar circulating glycoprotein in human blood that we propose be called human serotonectin, to distinguish it from the rat protein. Human serotonectin binds serotonin with high affinity (Kd1 = 36 nM; Kd2 = 1.1 microM). Monospecific antisera were raised in rabbits to purified human serotonectin. These antibodies were used to locate human serotonectin immunocytochemically, for quantitative estimation of the glycoprotein, and for rapid preparation of material purified by affinity chromatography. Evidence was obtained that indicated that human serotonectin circulates in plasma and also binds to the surfaces of white blood cells and platelets but not to red blood cells. In bone marrow it is found on megakaryocytes and on developing white cells of the eosinophil line. The protein can be completely removed by washing with isotonic sucrose or salt solutions from the surfaces of white cells but similar treatment only partially (63% sucrose wash/73% salt wash) removes human serotonectin from platelets. Antibodies to human serotonectin antagonize the uptake of serotonin by platelets but do not inhibit platelet aggregation. These data show that humans, like rats, have a circulating serotonin-binding glycoprotein that is also present as a peripheral membrane protein on platelets. The human also differs from the rat serotonectin in binding to white cells. The material may function in platelet serotonin uptake in both humans and rats; however, its function, if any, with respect to white cells is obscure.

Antibodies↗