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D Scherman

Publications and source records attributed to D Scherman.

At least 127 records · Page 7Linked to original sources
PubMed1986

[Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla].

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Iodobenzylguanidine↗
PubMed1986

The acylphosphate present in chromaffin granule membrane preparations is not associated with the proton-pump.

Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low temperature. A phosphorylated compound was rapidly formed which was stable in 10% trichloroacetic acid at 0 degree C. The lability of this compound in the presence of hydroxylamine or hot trichloroacetic acid indicated an acylphosphate, i.e., an ATPase phosphointermediate. Vanadate but not N-ethylmaleimide inhibited the formation of this derivative. Since the ATP-dependent generation of a transmembrane potential in chromaffin granule vesicles by the H+-pump was inhibited by N-ethylmaleimide but not by vanadate, the acylphosphate should not be associated with the H+-pump, i.e. ATPase I. We suggest that it is associated with ATPase II, an ATPase of unknown function present in chromaffin granule membrane preparations. This hypothesis is supported by the fact that ATPase II is vanadate sensitive and has a molecular mass of 140 kDa, properties similar to those of the phosphorylated intermediate.

Adenosine Triphosphate↗
PubMed1986

Dihydrotetrabenazine binding and monoamine uptake in mouse brain regions.

Binding of [2-3H]dihydrotetrabenazine and uptake of 5-hydroxytryptamine (serotonin) were studied in mouse brain cerebellum, pons-medulla, frontal cortex, hypothalamus, hippocampus, and striatum. Binding of [2-3H]dihydrotetrabenazine to homogenates of these brain areas is stable for several hours and occurs at a homogeneous class of binding sites (KD = 2.4 nM). Subcellular fractionation and regional distribution of [2-3H]dihydrotetrabenazine binding and serotonin uptake showed that the ligand binds to synaptic vesicles. Dihydrotetrabenazine inhibited serotonin uptake with the same inhibitory constant (IC50 = 2.6 nM) for synaptic vesicles from brain regions containing 3,4-dihydroxyphenylethylamine (dopamine) or serotonin and noradrenaline in different proportions. This constant is similar to the KD of [2-3H]dihydrotetrabenazine, which suggests that the latter ligand labels specifically and with the same affinity the monoamine transporter from various monoaminergic synaptic vesicles. Therefore the regional differences in central monoamine depletion induced in vivo by tetrabenazine are not due to regional differences in inhibition of vesicular monoamine uptake. Moreover, vesicular monoamine transporters from the central and peripheral nervous systems of various mammals and from bovine adrenal glands have comparable affinity for substrate and inhibitor (Km values for serotonin and IC50 for dihydrotetrabenazine are about 0.8 microM and 3 nM, respectively) and comparable turnover number (10-35 molecules transported per transporter per minute), which suggests the involvement of a common transporter molecule in the process of monoamine uptake by the various monoaminergic storage vesicles.

Animals↗
PubMed1986

Uptake of meta-iodobenzylguanidine by bovine chromaffin granule membranes.

meta-Iodobenzylguanidine, an adrenal imaging agent used for the scintigraphic detection of human pheochromocytoma, is a substrate for the monoamine uptake system of chromaffin granules. It is accumulated by bovine chromaffin granule membrane vesicles in the presence of ATP, and it can be released by an osmotic shock. The uptake is dependent upon the generation of an H+-electrochemical gradient by an ATP-dependent H+ pump since it is blocked by an H+ ionophore and since meta-iodobenzylguanidine uptake can be driven by imposing an artificial pH gradient (inside acidic) on the membrane vesicles. The transport is saturable and its Km value (2.0 microM at pH 8.0) is similar to that of noradrenaline (5.3 microM). Transport occurs through the monoamine transporter since it is blocked by the same inhibitors, tetrabenazine and reserpine, and also by the transporter substrates noradrenaline and serotonin. Noradrenaline inhibits meta-iodobenzylguanidine uptake competitively (Ki = 13 microM). In addition, meta-iodobenzylguanidine displaces dihydrotetrabenazine and reserpine from their binding sites on chromaffin granule membranes. It is thus likely that, after in vivo administration, [131I] meta-iodobenzylguanidine is ultimately stored in chromaffin granules and that it is translocated by the monoamine transporter.

3-Iodobenzylguanidine↗
PubMed1985

Dicyclohexylcarbodiimide inhibits the monoamine carrier of bovine chromaffin granule membrane.

The monoamine carrier of bovine chromaffin granule membrane catalyzes a H+/neutral amine antiport. Dicyclohexylcarbodiimide (DCCD) inhibits this carrier in a time- and concentration -dependent manner as shown by the following evidence: it inhibits the carrier-mediated pH gradient driven monoamine uptake without collapsing the pH gradient; it affects the binding of the specific inhibitors [2-3H]dihydrotetrabenazine and [3H]reserpine. The DCCD inhibition of the carrier occurs in the same concentration range as that of the ATP-dependent H+ translocase. Saturation isotherms of [2-3H]dihydrotetrabenazine binding indicate that DCCD decreases the number of binding sites without any change of the equilibrium dissociation constant. Kinetic studies of DCCD inactivation indicate that the modification of only one amino acid residue is responsible for the inhibition. Preincubation of the membranes with tetrabenazine protects the carrier against inactivation by DCCD: in this case, [2-3H] dihydrotetrabenazine binding and pH gradient driven monoamine uptake are restored after washing out of DCCD and tetrabenazine. We suggest the existence in the monoamine carrier of a carboxylic acid involved in H+ translocation, similar to those demonstrated not only in F0-F1 ATPases but also in cytochrome c oxidase, mitochondrial cytochrome b-c1 complex, and nucleotide transhydrogenase. Protonation-deprotonation of this group would affect the binding of [2-3H]dihydrotetrabenazine by the carrier.

Adenosine Triphosphatases↗
PubMed1984

Reserpine binding to bovine chromaffin granule membranes. Characterization and comparison with dihydrotetrabenazine binding.

[3H]Reserpine bound reversibly in vitro to chromaffin granule membranes. Binding was temperature-dependent and slow, and had biphasic kinetics. The addition of ATP accelerated the kinetics, which became monophasic and comparable to those of [3H] dihydrotetrabenazine, without affecting the binding equilibrium constants. The ATP effect was related to H+ -electrochemical gradient generation by the granule membrane H+ pump. Binding of reserpine to chromaffin granule membranes occurred on two classes of sites: R1, Bmax = 7 pmoles/mg of protein and KD = 0.7 nM, and R2, Bmax = 60 pmoles/mg of protein and KD = 25 nM. Sites R2 were considered to be equivalent to [3H] dihydrotetrabenazine binding sites, as the densities of the R2 and the [3H] dihydrotetrabenazine binding sites were similar and because tetrabenazine displaced reserpine from R2 sites. Sites R1 were tetrabenazine-resistant; they were involved in monoamine uptake, since their KD values were similar to the KI values of reserpine for noradrenaline uptake. Sites R1 were less abundant than sites R2 on chromaffin granule membranes, but they were present at the same concentration in intact chromaffin granules. We propose that the monoamine carrier exists in two forms: (a) an active form bearing both high- and low-affinity sites for reserpine and (b) an inactive form with only the low-affinity R2 sites.

Adrenal Medulla↗
PubMed1983

Solubilization of the catecholamine carrier of chromaffin granule membranes in a form that binds substrates and inhibitors of uptake.

The component of bovine chromaffin granule membrane responsible for the binding of [2-3H]dihydrotetrabenazine has been solubilized by treatment with detergents. The binding activity of the soluble material was measured either by equilibrium dialysis or by precipitation of the protein by poly(ethylene glycol) and filtration of the precipitate. The best yields and activities were obtained with sodium cholate, which solubilized more than 70% of the binding sites; deoxycholate had deleterious effects on the soluble activity, and among nonionic detergents, octyl beta-glucoside was the most efficient. With cholate, [3H]dihydrotetrabenazine binding occurred on soluble sites as judged by centrifugation of the bound material and by filtration on Sepharose 6B columns. Binding could be measured in the presence of cholate, but high detergent concentrations had a reversible inhibitory effect. The [3H]dihydrotetrabenazine binding characteristics of the soluble material were similar to those of the granule membrane, with one class of binding sites (KD = 23 nM, Bmax = 90 pmol/mg of protein). The association and dissociation rate constants were 0.072 X 10(6) M-1 s-1 and 1.1 X 10(-3) s-1, respectively. The pharmacological properties of the binding sites were also similar to those of the membranes. Our results thus support the view that the catecholamine carrier has been solubilized in an active conformation that binds substrates and inhibitors of uptake.

Adrenal Medulla↗
PubMed1983

Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine.

[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-11b-H-benzo [a]-quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane of purified bovine chromaffin granules. Specific binding was characterized by Kd and Bmax values of 3.1 nM and 62 pmol/mg of membrane protein, respectively. It was reversible, with association and dissociation rate constants of 0.22 x 10(6) M-1 s-1 and 1.8 x 10(-3) s-1, respectively. Binding sites were present in extracts of medulla but not in corticoadrenal extracts; in the medulla they were restricted to chromaffin granule membranes, [2-3H]Dihydrotetrabenazine binding occurred on the catecholamine carrier of the chromaffin granule membrane because it was clearly correlated with inhibition of norepinephrine uptake. In addition, inhibitors and substrates of the uptake reaction displaced [2-3H]dihydrotetrabenazine from its binding sites, and their potency as displacers was qualitatively correlated with their IC50 or Km. These results suggest that use of [2-3H]dihydrotetrabenazine binding might be an interesting technique in the study of the vesicular monoamine carrier.

Adrenal Medulla↗
PubMed1983

The catecholamine carrier of bovine chromaffin granules. Form of the bound amine.

The binding of [2-3H]dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,-4,6,7-hexahydro-11bH-benzo [a]quinolizine), a tetrabenazine derivative which binds to the catecholamine carrier of chromaffin granule membranes, has been studied as a function of the pH. The number of binding sites was constant from pH 6.5 to pH 9.0, whereas the KD decreased to a minimal plateau value, obtained at pH values higher than 7.5, the drug pKa. The pH dependency of the displacement of [3H]dihydrotetrabenazine by noradrenaline was also investigated. Noradrenaline KD values derived from displacement experiments decreased logarithmically when the pH increased from 6.5 to 8.5, i.e., for pH values lower than the pKa of noradrenaline. These pharmacological data support our previous hypothesis based on kinetic data [Scherman and Henry, Eur. J. Biochem. 116:535-539 (1981)] that the monoamine carrier of the chromaffin granule membrane binds and transports neutral amines, a form of low abundancy at physiological pH but for which it has a high affinity.

Animals↗
PubMed1982

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane.

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.

Adenosine Triphosphatases↗
PubMed1982

Acido-basic properties of the catecholamine uptake inhibitors tetrabenazine and dihydrotetrabenazine.

Tetrabenazine (2-oxo-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7 hexahydro-11 bH-benzo (a) quinolizine) and dihydrotetrabenazine (2-hydroxy derivative) are inhibitors of catecholamine uptake by the chromaffin granules of adrenal medulla. In the 6.6 - 8.8 pH range, inhibition by tetrabenazine was pH-independent whereas dihydrotetrabenazine efficiency increased up to pH 8.3. The fluorescence and the buffer-octanol partition coefficient of these drugs was affected by the pH. Analysis of the pH-dependency of these effects indicated the existence of an acido-basic transition characterized by a pKa of 6.0 for tetrabenazine and 7.5 for dihydrotetrabenazine and associated with protonation of the tertiary amine of these molecules. For both compounds, the neutral form was less fluorescent and more soluble in octanol. Comparison of the uptake inhibition constants IC50 and of the neutralization curves showed that this form was the biologically active one. This result implies that the monoamine carrier of chromaffin granule membrane binds either to only deprotonated amines or to the molecules present only in the lipidic phase where the neutral form is largely predominant.

Catecholamines↗
PubMed1982

Internal pH of isolated newly formed and aged neurohypophysial granules.

The pH gradient (delta pH) across the membrane of bovine neurohypophysial granules was estimated by using the [14C]methylamine partition technique. The granule membrane showed high permeability to sugars such as sucrose or sorbitol and to cations. Granules suspended in sucrose medium showed an acidic internal delta pH that decreased with external pH from 1.0 pH unit at pH 7.5 to 0 at pH 4.5. High ionic strength of the external medium destroyed the delta pH, indicating that it originated from a Donnan equilibrium. In a medium constituted to simulate cytoplasm, the delta pH was approximately 0.5 pH unit. Aged neurosecretory granules were more acidic inside than were newly formed granules. The results are discussed in relationship to the nature and the duration of maturation and degradation process of the granule matrix.

Animals↗
PubMed1981

[Binding of a tetrabenazine derivative to the monoamine transporter of the chromaffin granule membrane].

[2-3H] dihydrotetrabenazine (2-hydroxy-3-isobutyl-9-10-dimethoxy-1,2,3,4,6,7 hexahydro-11 bH-benzo [a] quinolizine), a derivative of tetrabenazine obtained by tritiated borohydride reduction, binds to the membrane of purified chromaffin granules. The binding equilibrium has been studied by filtration and by centrifugation of ligand-membrane complexes. Saturable binding sites (190 pmol/mg protein), characterized by a dissociation equilibrium constant KD = 90 nM have been described. The correlation existing between dihydrotetrabenazine site occupancy and noradrenaline uptake inhibition indicates that these sites are located on the monoamine transporter.

Adenosine Triphosphate↗
PubMed1981

pH-dependence of the ATP-driven uptake of noradrenaline by bovine chromaffin-granule ghosts.

The kinetic parameters of noradrenaline uptake by chromaffin granule ghosts have been measured at external pH values between 6.5 and 8.5. The log of the Km for noradrenaline decreased linearly with pH with a slope of -1.0, indicating that the observed affinity increase of originated in deprotonation of a single chemical group. This result is interpreted as showing that the neutral form of monoamines is the true substrate for the amine carrier. The Km of the carrier for the neutral form of noradrenaline was calculated as 0.1 microM. The maximal velocity, V, of the uptake reaction was constant from pH 6.5 to 8.0 and decreased at more alkaline pH values. Since the proton electrochemical gradient delta muH+ generated by the membrane H+-pump was independent of the pH in the range 6.5-9.5, the pH dependence of the maximal velocity of uptake reflects the pH profile of the monoamine transporter.

Adenosine Triphosphatases↗
PubMed1980

Role of the proton electrochemical gradient in monoamine transport by bovine chromaffin granules.

The role of the transmembrane potential (delta phi), the proton concentration gradient (delta pH) and the proton electrochemical gradient (delta gamma H+) in monoamine uptake by bovine chromaffin granules or ghosts was investigated. In presence of ATP the permeant anion SCN- collapsed the delta phi (inside positive) and inhibited monoamine uptake by granules or well buffered ghosts. With lightly buffered ghosts, SCN- induced an acidification which resulted in a low inhibition of uptake. Cation efflux as well as anion influx affected the delta phi, and a transient valinomycin-mediated K+ efflux induced a lag in the uptake. The delta pH-driven noradrenalin uptake was also sensitive to delta phi, since superimposing a positive or a negative delta phi to the delta pH, respectively, increased or decreased the rate of noradrenalin accumulation. A delta pH was required for this increase of uptake rate, adrenalin accumulation. A delta pH was required for this increase of uptake rate, which was proportional to the delta phi. The pH-dependence of the ATP-induced monoamine uptake by granules pointed to the delta gamma H+ as the driving force. In contrast with the rate of uptake, which was not dependent on the anions present, the extent of amine incorporation was decreased when the internal anionic buffer concentration was decreased and, at a low internal buffer concentration, when ATP anion transport was blocked.

Adenosine Triphosphate↗
PubMed1980

Oxonol-V as a probe of chromaffin granule membrane potentials.

The dye, oxonol-V (bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol), can be used to estimate the transmembrane potential of chromaffin granules. The potentials result either from a resting-state Donnan equilibrium (inside negative at pH 6.6) or from an ATP-driven proton pump. The fluorescence and absorption changes generated by ATP addition depended on the pH of the medium and the dye-to-vesicle ratio. Energization resulted in an increase in the number of oxonol-V binding sites, the new binding sites having the same dissociation constant. The rate of dye association was higher with resting than with energized chromaffin granules. The absorption change was associated with a red shift whereas the fluorescence change involved a quenching due to the increase in dye concentration on the membrane. The absorption and fluorescence changes varied linearly with the transmembrane potential difference when the interior potential was positive relative to the medium.

Absorption↗