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D Scherman

Publications and source records attributed to D Scherman.

At least 91 records · Page 5Linked to original sources
PubMed1994

Autoradiographic distribution of the eye monoaminergic synaptic vesicles.

The ligand [3H]dihydrotetrabenazine ([3H]TBZOH), a tritiated derivative of the monoamine depleting agent tetrabenazine, has been reported to bind specifically to the transporter present on the membrane of monoaminergic synaptic vesicles. We have studied the pharmacological characteristics of [3H]TBZOH binding to rabbit eye sections and the anatomical distribution of [3H]TBZOH binding sites in rabbit, rat and human eyes by in vitro autoradiography. In rabbit eye sections, [3H]TBZOH binding bound to a single class of binding sites (Kd = 4.1 nM, Bmax = 30.6 fmol/mg protein). When animals were kept for various times after death before removal of the eyes, the mean density of [3H]TBZOH binding sites decreased to 94 and 93% of control values after 24 and 48 h, respectively. In both species, the topographical distribution of [3H]TBZOH sites shows a pattern of distribution similar to that of monoaminergic innervation of the eye. The present data demonstrate that [3H]TBZOH can be used for the identification and the quantitation of monoamine-containing neurons in the mammalian eye.

Aged↗
PubMed1994

High lipophilicity decreases drug transport across intestinal epithelial cells.

It is generally admitted in drug research that the passage of molecules across cellular barriers increases with lipophilicity and that the most lipophilic compounds will have the highest intestinal absorption. Using two in vitro models of intestinal epithelium, we presently demonstrate that this concept is not always valid and that highly lipophilic compounds display a low transepithelial permeability. We used epithelial cell lines grown on permeable filters to measure in vitro the transepithelial permeability of various molecules. The octanol/buffer distribution coefficient of the drugs (Do/b) was taken as an index of lipophilicity. When log Do/b values were lower than 3.5, the transepithelial permeability coefficient increased with the lipophilicity. But for log Do/b values ranging from 3.5 to 5.2, the transepithelial permeability coefficient decreased with increasing lipophilicity. Identical results were observed with two differentiated intestinal epithelial cell lines (HT29-18-C1 and Caco-2) and in unstirred or agitated conditions. The results show that an octanol/buffer distribution coefficient value around 3000 corresponds to an optimal transepithelial passage of drugs and that too high a lipophilicity can result in low intestinal epithelial permeability and in low oral absorption.

Biological Transport↗
PubMed1993

HT29-18-C1 intestinal cells: a new model for studying the epithelial transport of drugs.

A polarized differentiated subclone of HT29, a human colon carcinoma cell line, was used to measure the passage of drugs across the intestinal epithelium. These cells, HT29-18-C1, when grown on permeable filters, formed tight monolayers of high electrical resistance (> 400 omega cm2). Electron micrographs revealed the presence of numerous apical microvilli and well developed junctional complexes. The transepithelial passage of various drugs was studied: the transepithelial permeability coefficients ranged from 0.8 x 10(-6) cm/s for fluorescein, to 4.8 x 10(-5) cm/s for the most lipophilic molecule, testosterone. For the five compounds tested, there was a coarse correlation between the apparent transepithelial permeability coefficient and the octanol/buffer distribution coefficient. The HT29-18-C1 subclone thus represents a new in-vitro model for studying the intestinal absorption of drugs.

1-Octanol↗
PubMed1993

Axonal transport of dopamine-containing vesicles labelled in vivo with [3H]reserpine.

Axonal transport of the vesicular monoamine transporter was assayed in the rat brain by in vivo binding of the specific ligand [3H]reserpine. Because of the marked localization of reserpine binding sites in dopaminergic cell bodies and nerve terminals, the dopaminergic nigrostriatal pathway was chosen for the study of the axonal transport of the monoamine carrier present in the membrane of synaptic vesicles. When labelled reserpine was injected into the substantia nigra, a delayed accumulation of radioactivity in the ipsilateral striatum was observed approximately 4 h after the injection. Similarly, injection into the right striatum was followed by a substantial accumulation of radioactivity in the ipsilateral substantia nigra, which was prevented by peripheral injection of unlabelled reserpine or tetrabenazine. This process was rapid and dependent on microtubules. In senescent rats, the amount of retrogradely transported [3H]reserpine was significantly reduced. These results demonstrate that labelled reserpine may be used to monitor in vivo fast axonal transport in central neurons.

Aging↗
PubMed1992

Dopamine transport: pharmacological distinction between the synaptic membrane and the vesicular transporter in rat striatum.

The pharmacological properties of the monoamine transporters in the synaptic vesicles and of the dopamine transporters in the synaptic plasma membrane were compared. Tetrabenazine, an inhibitor of the vesicular transporter did not block ligand binding to the plasma membrane transporter. Various potent cocaine analogues and other compounds active at the plasma membrane transporter did not block ligand binding to the vesicular transporter. These data indicate pharmacological differences between the vesicular and synaptic membrane transporters.

Animals↗
PubMed1992

Regulation of the chromaffin granule catecholamine transporter in cultured bovine adrenal medullary cells: stimulus-biosynthesis coupling.

The transsynaptic induction of the monoamine transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Synaptin/synaptophysin, p65 and SV2: their presence in adrenal chromaffin granules and sympathetic large dense core vesicles.

The subcellular distribution of three proteins of synaptic vesicles (synaptin/synaptophysin, p65 and SV2) was determined in bovine adrenal medulla and sympathetic nerve axons. In adrenals most p65 and SV2 is confined to chromaffin granules. Part of synaptin/synaptophysin is apparently also present in these organelles, but a considerable portion is found in a light vesicle which does not contain significant concentrations of typical markers of chromaffin granules (cytochrome b-561, dopamine beta-hydroxylase or the amine carrier). An analogous finding was obtained for sympathetic axons. The large dense core vesicles contain most p65 and also SV2 but only a smaller portion of synaptin/synaptophysin. A lighter vesicle containing this latter antigen and some SV2 has also been found. These results establish that in adrenal medulla and sympathetic axons three typical antigens of synaptic vesicles are not restricted to light vesicles. Apparently, a varying part of these antigens is found in chromaffin granules and large dense core vesicles. On the other hand, the light vesicles do not contain significant concentrations of functional antigens of chromaffin granules. Thus, the biogenesis of small presynaptic vesicles which contain all three antigens as well as functional components like the amine carrier is likely to involve considerable membrane sorting.

Adrenal Medulla↗
PubMed1990

Postnatal development of the monoamine vesicular transporter in mesencephalic and telencephalic regions of the rat brain: a quantitative autoradiographic study with [3H]dihydrotetrabenazine.

[3H]Dihydrotetrabenazine [3H]TBZOH, a high affinity ligand of the monoamine vesicular transporter, has been used to study by autoradiography the development of monoaminergic synaptic vesicles from birth to adulthood, in the rat brain. The study was focused on dopaminergic areas. Binding sites for [3H]TBZOH were already detectable in 1-day-old rats, and the affinity for the ligand was identical in the striatum of 8-day-old and adult rats. The density of binding sites almost attained the adult level at day 20 postnatal in regions rich in dopaminergic cell bodies (substantia nigra pars compacta and ventral tegmental area), as well as in the lateral septum. In the striatum, nucleus accumbens, and olfactory tubercle, the increase in binding sites density was progressive from birth to adulthood. This increase was more pronounced in the olfactory tubercle.

Aging↗
PubMed1990

[3H]Dihydrotetrabenazine, a new marker for the visualization of dopaminergic denervation in the rat striatum.

[3H]Dihydrotetrabenazine ([3H]TBZOH), a derivative of the monoamine depleting agent, tetrabenazine, has been shown to bind to the vesicular monoamine transporter. We studied the effect of unilateral nigral lesion with 6-hydroxydopamine on [3H]TBZOH binding in the striatum by means of quantitative autoradiography. Topographical analysis of striatal [3H]TBZOH binding showed in control rats a pattern similar to the known distribution of dopaminergic innervation, and allowed to visualize dopaminergic denervation in lesioned rats. A good correlation was found between striatal [3H]TBZOH binding levels and tyrosine hydroxylase activities in similar areas on adjacent slices following 6-OHDA lesions with varying severity. These data reveal that autoradiography of [3H]TBZOH binding is a good index for the visualization and quantitation of dopaminergic nerve terminals integrity in the striatum.

Animals↗
PubMed1990

Regulation of neurotensin-containing neurons in the rat striatum. Effects of unilateral striatal lesions with quinolinic acid and ibotenic acid on neurotensin content and its binding site density.

Recently, we reported bilateral increases in striatal neurotensin (NT) levels following unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic pathway. In the present study, the effect of unilateral striatal lesions with quinolinic acid (QA, 300 nmol) or ibotenic acid (IBO, 130 nmol) on striatal NT levels and binding site densities were analyzed in order to investigate other possible regulations of NT systems. QA and IBO injection decreased gamma-aminobutyric acid (GABA) levels and [125I]iodosulpride (a specific D2 receptor antagonist) binding site densities in the lesioned striatum, indicating degeneration of striatal intrinsic neurons. Striatal dopaminergic terminals were not altered by QA as shown by the lack of changes in [3H]dihydrotetrabenazine [( 3H]TBZOH, a specific ligand of the vesicular monoamine transporter) binding site densities. Moreover, QA lesion induced an increase in NT levels and a decrease in NT binding sites in the lesioned striatum without any change in the contralateral structure. In contrast to QA, IBO might destroy a certain proportion of dopaminergic terminals in the lesioned striatum, as shown by a 54% decrease in [3H]TBZOH binding. Furthermore, IBO lesion enhanced striatal NT levels bilaterally, while NT binding sites decreased in the lesioned striatum and increased in the contralateral side. The present results suggest that not only dopaminergic neurons but also striatal intrinsic neurons may control NT systems in the striatum.

Animals↗
PubMed1990

Regulation of neurotensin-containing neurons in the rat striatum and substantia nigra. Effects of unilateral nigral lesion with 6-hydroxydopamine on neurotensin content and its binding site density.

The effect of unilateral lesion of the rat substantia nigra with 6-hydroxydopamine (6-OHDA) was investigated on the endogenous contents of neurotensin (NT) and its binding site densities in the striatum and substantia nigra. Tyrosine hydroxylase (T-OH) activity, gamma-aminobutyric acid (GABA) content, binding site densities of dihydrotetrabenazine (TBZOH), a marker of dopaminergic synaptic vesicles, and of iodosulpiride, a ligand for dopamine D2 receptors, were also determined. Fourteen days following nigral lesions, these markers were analyzed quantitative autoradiography for NT, TBZOH and iodosulpiride binding site densities. Unilateral nigral lesion with 6-OHDA provoked only ipsilateral modifications in dopamine markers. T-OH activity and TBZOH binding site densities significantly decreased in both the ipsilateral striatum and substantia nigra. Iodosulpiride binding sites decreased in the substantia nigra and increased in the striatum on the ipsilateral side. In contrast to these unilateral changes observed for dopamine markers, dramatic increased in NT contents were found in both the ipsi- and contralateral striata. No change was found in nigral NT levels on either side. On the other hand, NT binding sites decreased in the ipsilateral striatum and substantia nigra, which reflected the destruction of dopaminergic elements in these regions. The present results strongly suggest a dopaminergic control of striatal NT systems and demonstrate that a unilateral loss of this control may lead to strong bilateral alterations in NT levels.

Animals↗
PubMed1990

Induction of the vesicular monoamine transporter by elevated potassium concentration in cultures of rat sympathetic neurons.

The expression of the vesicular monoamine transporter was studied in newborn rat sympathetic neurons and compared to that of the catecholamine biosynthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase. The vesicular monoamine transporter was assayed using the specific ligand [3H]dihydrotetrabenazine. In cultures grown for 10 days in the presence of 35 mM K+, tyrosine hydroxylase activity and the density of [3H]dihydrotetrabenazine binding sites were increased by a similar 2-3-fold factor, while dopamine-beta-hydroxylase activity and protein level were unchanged. Under these conditions, choline acetyltransferase activity was depressed by 90%. The induction of the vesicular monoamine transporter by high K+ was dependent upon Ca2+ entry through slow calcium channels since it was inhibited by the diphenylbutylpiperidine antagonist fluspirilene and by 20 mM Mg2+, and was enhanced by the dihydropyridine agonist, Bay K8644. The induction of the vesicular monoamine transporter by neuronal depolarization indicates the existence of a Ca2(+)-dependent mechanism of coregulation for this intrinsic component of monoaminergic synaptic vesicles and tyrosine hydroxylase. On the other hand, the apparent absence of dopamine-beta-hydroxylase induction is probably due to the continuous secretion of this intravesicular enzyme by the depolarized sympathetic neurons, an effect already observed in trans-synaptically stimulated adult sympathetic ganglion and adrenal medulla.

Animals↗
PubMed1990

Sympathetic axons and nerve terminals: the protein composition of small and large dense-core and of a third type of vesicles.

Homogenates of bovine splenic nerve and of vas deferens were subjected to differential and density gradient centrifugation to investigate their noradrenaline-storing organelles. The subcellular fractions obtained were analysed by immunoblotting in order to define the presence of various antigens in small dense-core and large dense-core vesicles. In both large granule and microsomal fractions from splenic nerve only one type of noradrenaline-storing vesicle was found, which represents the large dense-core vesicles. These organelles contained chromogranin A, chromogranin B, cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, dopamine beta-hydroxylase and the monoamine carrier which are also present in adrenal chromaffin granules. The subcellular distribution of synaptin/synatophysin was more complex since this protein was apparently present in two organelles: in a light vesicle which did not contain significant amounts of antigens found in large dense-core vesicles (dopamine beta-hydroxylase, cytochrome b-561 and the monoamine carrier) and in the dense fractions of the gradient, possibly within large dense-core vesicles. In the microsomal gradient from vas deferens several markers (catecholamines, synaptin/synaptophysin and dopamine beta-hydroxylase) were found in a bimodal distribution, which is consistent with their presence in small and large dense-core vesicles. When the larger granules were removed with higher centrifugation speed a microsomal fraction containing only light vesicles was obtained. After gradient centrifugation of this fraction several components (catecholamines, dopamine beta-hydroxylase, cytochrome b-561, the monoamine carrier and synaptin/synaptophysin) were concentrated in a peak at low density; apparently only small dense-core vesicles were now present.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules.

The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells. Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine beta-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

Adrenal Cortex↗
PubMed1990

Axonal transport of synaptic vesicles and muscarinic receptors: effect of protein synthesis inhibitors.

The effect of cycloheximide, a protein synthesis inhibitor, was studied on the axonal transport of noradrenergic synaptic vesicles and presynaptic muscarinic receptors, identified by in vitro binding of [3H]dihydrotetrabenazine and [3H]quinuclidinylbenzilate, respectively, in rat sciatic nerve. Cycloheximide (1.5 mg/kg) administered subcutaneously 2 h before ligation decreased by approximately 50% the accumulation of vesicles and receptors in the proximal segment above the ligature placed on the nerve; its action was detectable after a lag period of 10 h and disappeared 96 h after administration. Double ligatures were placed on the nerve at various time intervals between the first (distal) and the second (proximal) ligature, and the accumulation of vesicles and receptors proximal to the second ligature was measured; the first ligature diminished the accumulation above the second ligature. At an interval of 96 h between the first and the second ligature, cycloheximide completely prevented the accumulation of vesicles and receptors proximal to the second ligature. The effects of double ligatures and the response to cycloheximide treatment can best be explained on the assumption that an important proportion of synaptic vesicles and presynaptic receptors is being recycled in the nerve cell bodies after retrograde transport.

Animals↗
PubMed1989

Reserpine binding to chromaffin granules suggests the existence of two conformations of the monoamine transporter.

The binding of [3H]reserpine ([3H]RES) to purified bovine chromaffin granule membranes has been studied at low membrane concentration. Saturation isotherms indicated a dissociation equilibrium constant KD of 30 pM and a density of binding sites of 8 pmol/mg of protein at 30 degrees C. The association rate constant was 4.0 X 10(5) M-1 s-1, and the calculated dissociation rate constant was 1.2 X 10(-5) s-1, corresponding to a half-lifetime of about 16 h. Although this dissociation was too low to be measured directly, [3H]RES binding was indeed reversible since it was lost after addition of the detergent Triton X-100. Dihydrotetrabenazine (TBZOH) inhibited [3H]RES binding in a time-dependent manner, EC50 varying from 37 nM after a 1-h incubation to 600 nM after 16 h. On the contrary, [3H]RES binding inhibition by the substrate noradrenaline was time independent. It is proposed that the transporter exists in two different conformations which bind exclusively either tetrabenazine (TBZ) or RES and which are in equilibrium. The effects of detergents were consistent with this two-conformation model. The transporter solubilized by cholate bound [3H]TBZOH, but not [3H]RES. On the other hand, addition of cholate to membrane-bound [3H]RES solubilized the membrane without releasing the ligand from its binding site. It is proposed that the TBZ-binding conformation is obtained by solubilization with cholate and that RES stabilizes the RES-binding conformation, allowing its solubilization by this detergent.

Amines↗