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Biomedical subjects

H Tamir

Publications and source records attributed to H Tamir.

At least 73 records · Page 4Linked to original sources
PubMed1985

Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus.

Research on the role of serotonin (5-hydroxytryptamine, 5-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of 5-HT. Saturable, reversible, high affinity enteric binding sites for 3H-5-HT have recently been characterized and radioautographically located. Affinity for the 3H-5-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-5-HT binding sites have been proposed to be enteric neural 5-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-5-HT with their electrophysiologically determined actions on myenteric neurons. 5-Methoxytryptamine did not inhibit the binding of 3H-5-HT to enteric membranes and neither mimicked nor antagonized the effects of 5-HT on the membrane potential of myenteric neurons. Two dipeptides of 5-hydroxytryptophan, N-acetyl- and N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP and N-hex-5-HTP-DP) inhibited the binding of 3H-5-HT (K1 = 0.25 microM for 5-HTP-DP and 1.19 microM for N-hex-5-HTP-DP). 5-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of 5-HT. 5-HTP-DP also blocked the 5-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, 5-HTP-DP itself did not affect these responses. Moreover, 5-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to acetylcholine, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-5-HT binding sites are enteric neural 5-HT receptors, that dipeptides of 5-hydroxytryptophan are specific antagonists at these receptors, and that 5-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.

5-Hydroxytryptophan↗
PubMed1983

Serotonin-binding glycoprotein of rat platelets.

Platelets, because of their ability to take up serotonin, are often considered as models of serotonergic neurons. We have purified a glycoprotein from rat platelets that specifically binds serotonin (KD = 10 nM) but not [3H]imipramine (up to 0.2 microM). This protein is distinct from fibronectin. Antiserum raised against this protein was shown to be monospecific by double diffusion, rocket immunoelectrophoresis and immunoreactivity of protein transferred from slab gels to nitrocellulose sheets. We have called this protein serotonectin. Serotonectin-like material was localized by light- and electron-microscopic immunocytochemistry in rat platelets, using the unlabelled peroxidase-antiperoxidase complex bridge technique. All serotonectin immunoreactivity was found to be associated with the plasma membrane but no immunoreactivity was found in the surface connecting system. Washing platelets with Krebs solution removed 75% of the extractable serotonectin, indicating that it is a peripheral protein. Serotonectin was found to be circulating in the plasma, as the ease of its removal from platelet membranes suggests it must do; however, none was found in brain synaptosomes. Serotonectin was found to be synthesized in blood-forming organs (spleen, bone marrow) but not in the liver or the mucosa of the gut. Antibodies to serotonectin (4.5 mg/ml) inhibited the uptake of [14H]serotonin (0.2 microM) by rat platelets but not by synaptosomes. Serotonectin did not bind [3H]imipramine and washing the protein off platelet membranes did not impede the binding of [3H]imipramine to the membranes. Thus an action on serotonectin does not seem to be responsible for the antagonism by tricyclic antidepressants of serotonin uptake. These data suggest that serotonectin is present on the external surface of platelet plasma membranes, that it is excluded from the plasma membrane of the surface connecting system, and that it may be in equilibrium with free material in plasma.

Animals↗
PubMed1983

Storage of serotonin in vivo as a complex with serotonin-binding protein in central and peripheral serotonergic neurons.

Serotonin-binding protein (SBP) is a soluble protein found in synaptic vesicles of central and peripheral serotonergic neurons. Experiments were undertaken to determine whether serotonin (5-HT) is physiologically stored as a complex with SBP in vivo. [3H]5-HT was used as a probe. Neurons were allowed to specifically take up the labeled amine and attempts were made to recover the in vivo formed [3H]5-HT X SBP complex. Rats were perfused intraventricularly (3 hr) with [3H]5-HT. Strips of rabbit enteric nervous system (ENS) were incubated with [3H]5-HT in the presence of desipramine. The tissues were then homogenized so as to disrupt synaptic vesicles; protein-bound [3H]5-HT was obtained from the 100,000 X g supernatant by filtration on Sephadex G-50 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Studies with [3H]5-HT added just prior to homogenization indicated that the [3H]5-HT X SBP complex had formed intraneuronally, prior to homogenization. The protein X [3H]5-HT complexes from brain and gut migrated on the gels with apparent molecular weights of 45,000 and 56,000, corresponding to those measured by SDS-PAGE for purified SBP; however, the 45-kilodalton (kd) molecule predominated when the SBP complex was formed in vivo, whereas the 56-kd molecule predominated when the SBP X [3H]5-HT complex was formed with extracted SBP. It is possible that the 56-kd SBP is characteristic of the molecule in perikarya or nonterminal axons, whereas the 45-kd molecule is characteristic of terminal varicosities because radioautographic results show that in both the central nervous system and ENS, [3H]5-HT is mostly concentrated in terminals. In any case, newly taken up [3H]5-HT preferentially labels 45-kd SBP. Depletion of endogenous 5-HT by placing animals on a tryptophan-deficient diet increased the amount of exogenous [3H]5-HT bound to SBP in vivo. This suggests that endogenous 5-HT is normally bound to SBP and competes with the [3H]5-HT probe for available binding sites. The binding of 5-HT to SBP within vesicles may be important to reduce the osmotic pressure that would build up in synaptic vesicles if 5-HT were free in solution.

Animals↗
PubMed1982

Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin.

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.

Animals↗
PubMed1981

Stress and morphine analgesia: alterations following p-chlorophenylalanine.

Recent studies have shown that while the analgesic responses induced by certain stressors appear to be related to morphine analgesia, the analgesic responses to other stressors do not. Para-chlorophenylalanine (PCPA), a potent tryptophan-hydroxylase inhibitor has been shown to decrease both basal pain thresholds and morphine analgesia on the flinch-jump test. To assess further the relationship between morphine and stress-induced analgesia, PCPA's effect upon the analgesic responses to cold-water swims, 2-deoxy-D-glucose, inescapable foot shock and morphine were determined using the flinch-jump and tail-flick tests. PCPA, which produced an 85% depletion of brain serotonin, significantly decreased jump thresholds while significantly increasing tail-flick latencies. Similarly, while morphine analgesia was decreased by PCPA on the flinch-jump test, it was not affected on the tail-flick test. The analgesic jump thresholds induced by cold-water swims and 2-deoxy-D-glucose as well as the increase tail-flick latencies induced by foot shock were unaffected by PCPA. These results are discussed in terms of PCPA's differential effects upon basal nociception and morphine analgesia and in terms of further dissociation between morphine and stress-induced analgesia.

Animals↗
PubMed1981

Separation of dissociated thyroid follicular and parafollicular cells: association of serotonin binding protein with parafollicular cells.

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.

Animals↗
PubMed1981

Intracellular proteins that bind serotonin in neurons, paraneurons and platelets.

1. Properties of serotonin binding proteins (SBP) extracted from neuroectodermal derivatives (brain, myenteric plexus, thyroid parafollicular cells) were found to differ from serotonin binding proteins extracted from cells of mesodermal (platelets) or entodermal (intestinal mucosa) origin. 2. Serotonin binding proteins extracted from brain, myenteric plexus, and thyroid parafollicular cells all had similar properties. 3. Binding of tritiated serotonin to serotonin binding protein is inhibited by a drug that interferes with intracellular storage of serotonin (reserpine) or by exposing the protein to extracellular salt concentrations (Krebs solution). 4. It is proposed that serotonin binding protein is a neuron-specific storage protein for serotonin that serves to decrease intravesicular osmotic pressure, and that serotonin dissociates from a complex with the protein when the lumen of synaptic vesicles is exposed to extracellular fluid at the time of exocytosis.

Animals↗
PubMed1980

Differences between intracellular platelet and brain proteins that bind serotonin.

Two proteins with binding capacity highly specific for serotonin are present in the 100,000 g supernatant obtained from rat platelets: a glycoprotein and albumin. They were purified by means of (NH4)2SO4 fractionation, Sephadex sieve chromatography, and affinity column chromatography. The two proteins differed in most of their physical and chemical properties: (a) migration on 7.5% acrylamide gel of the complex (protein-Fe+2-[3H]serotonin); (b) molecular weight (sodium dodecyl sulfate gels); (c) carbohydrate reaction; (d) binding capacity and binding constants; (e) effect of reserpine; (f) heat stability; and (g) isoelectric point. However, they showed two similar properties: sensitivity to trypsin and dependence on Fe+2 for serotonin binding. The properties of both the glycoprotein and albumin differ considerably from those of serotonin binding protein of brain (SBP), which was not detected in platelets. Since brain serotonin binding protein is thought to be involved in storage of the amine, these results suggest that the storage form of serotonin in rat platelets is different from that in rat brain. These results also imply that the storage of serotonin in platelets may not serve as a model for the storage of serotonin in nerve terminals of the brain.

Albumins↗
PubMed1979

Release of [3H]serotonin and its binding protein from enteric neurons.

The release of [3H]5-HT and its binding protein, SBP, from the guinea pig enteric nervous system was analyzed. Release of both [3H]5-HT and [3H]NE from strips of longitudinal muscle with adherent myenteric plexus preloaded with the respective radioactive amine was evoked by high K+ and the ionophore X537A. However, Ca2+-dependence could not be shown for [3H]5-HT release by either agent or for [3H]NE release by X537A. However, Ca2+-dependence (as well as inhibition of release by high Mg2+ and tetrodotoxin) could be demonstrated for the release of radioactivity evoked by electrical field stimulation of everted segments of ileum preloaded by perfusion through the serosal lumen with [3H]5-HT. Light and electron microscopic radioautography revealed that the sources of released radioactivity were axons, especially axonal varicosities containing a mixture of small clear and large dense-cored vesicles. SPB, but not the cytosol marker protein, lactic dehydrogenase, was spontaneously released from the perfused everted ileum. A marked increase in SBP (but not LDH) release was provoked by electrical field stimulation at 10 Hz, and this increased release (but not the spontaneous release) was Ca2+-dependent. It is concluded that SBP and 5-HT are probably stored together, at least in part in vesicles, and that both can be released by exocytosis from depolarized axon terminals.

Animals↗