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1.
J Biol Chem ; 275(13): 9136-42, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10734047

ABSTRACT

Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracellular Ca(2+) evoked by depolarizing agents. Subcellular studies revealed that reserpine and tetrabenazine at concentrations near their K(i) values not only could increase cytoplasmic catecholamines but also could displace Ca(2+) from vesicles. Furthermore, transient exposure to tetrabenazine and reserpine, but not methyl reserpate and reserpic acid, induced exocytotic release of catecholamines. Reserpine induced a rise in intracellular Ca(2+), as detected by whole-cell measurements with Fura-2. It could induce exocytosis, albeit at a lower frequency, in Ca(2+)-free solutions, supporting an internal Ca(2+) source. Depletion of endoplasmic reticulum and mitochondrial Ca(2+) pools did not eliminate the reserpine-activated release. These results indicate that vesicular Ca(2+) can play an important role in exocytosis and under some conditions may be involved in initiating this process.


Subject(s)
Calcium/metabolism , Exocytosis , Membrane Transport Proteins , Neuropeptides , Animals , Biological Transport , Catalysis , Catecholamines/metabolism , Cattle , Cells, Cultured , Cytoplasm/metabolism , Exocytosis/drug effects , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , Potassium/metabolism , Reserpine/pharmacology , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport Proteins
2.
Neurochem Int ; 30(4-5): 411-5, 1997.
Article in English | MEDLINE | ID: mdl-9106255

ABSTRACT

Tetrabenazine and dihydrotetrabenzaine (TBZOH) are potent inhibitors of substrate transport by the predominant forms of the vesicular monoamine transporter (VMAT) present in bovine brain synaptic vesicles and bovine adrenal medullary chromaffin vesicles. Radiolabeled TBZOH binds to these preparations with apparent dissociation constants in the low nanomolar range. However, tetrabenazine is a much less potent inhibitor of transport by rVMAT1, a form of the transporter cloned from a rat pheochromocytoma (PC12) cDNA library and expressed in CHO cells. Reported attempts to observe binding of [3H]TBZOH to rVMAT1 have not been successful. We examined binding of [3H]TBZOH to a crude membrane fraction from PC12 cells. Computerized nonlinear least squares curve fitting revealed two classes of binding sites (Kd1 = 1.5 nM, R1 = 0.2 pmol/mg protein, Kd2 = 340 nM, R2 = 15.2 pmol/mg protein). While the identity of the higher affinity sites is not certain, their high affinity for TBZOH suggests that they may be associated with rVMAT2. The lower affinity sites are likely to be associated with rVMAT1 on the basis of their affinity for TBZOH and sensitivity to inhibition of TBZOH binding by transporter substrates and inhibitors. NGF-treated PC12 cells also exhibited two classes of sites (Kd1 = 1.9 nM, R1 = 0.18 pmol/mg protein; Kd2 = 370 nM, R2 = 23.7 pmol/mg protein). While there were no significant differences between control and NGF-treated cells in binding capacity of the higher affinity sites, nor in apparent dissociation constants for either class of sites, there was a highly significant increase in number of lower affinity binding sites in the NGF-treated cells (p = 0.001). These results provide direct evidence that the differential sensitivity of rat brain and adrenal catecholamine stores to depletion by tetrabenazine and its derivatives is due to a much lower affinity of rVMAT1 for these compounds, and that NGF treatment may increase levels of rVMAT1 expression in PC12 cells.


Subject(s)
Membrane Transport Proteins , Nerve Growth Factors/pharmacology , Neuropeptides , Tetrabenazine/analogs & derivatives , Animals , Binding Sites , Biological Transport/drug effects , Cattle , Kinetics , Membrane Glycoproteins/metabolism , Neurotransmitter Agents/metabolism , PC12 Cells , Rats , Tetrabenazine/metabolism , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport Proteins
3.
Biochem Pharmacol ; 48(1): 87-91, 1994 Jul 05.
Article in English | MEDLINE | ID: mdl-8043034

ABSTRACT

There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgG1 kappa, 2 were IgG2a kappa, and 3 were IgG2b kappa isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2-3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously.


Subject(s)
Adrenal Glands/immunology , Antibodies, Monoclonal/immunology , Biogenic Monoamines/immunology , Carrier Proteins/immunology , Chromaffin Granules/immunology , Adrenal Glands/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Binding Sites, Antibody , Carrier Proteins/isolation & purification , Cattle , Chromaffin Granules/metabolism , Chromatography, Affinity/methods , Enzyme-Linked Immunosorbent Assay , Female , Mice , Mice, Inbred BALB C
4.
J Pharmacol Exp Ther ; 261(2): 574-9, 1992 May.
Article in English | MEDLINE | ID: mdl-1533666

ABSTRACT

In vivo voltammetry was used to assess the change in stimulated striatal dopamine overflow in response to various treatments with the dopamine receptor antagonist haloperidol. Dopamine overflow was induced with stimulating electrodes implanted in the medial forebrain bundle of anesthetized rats while dopamine concentrations were monitored with Nafion-coated, carbon-fiber microelectrodes implanted in the striatum. An acute challenge of haloperidol (0.5 mg kg-1, i.p.) given to naive animals caused stimulated overflow to increase at all stimulation frequencies (10-60 Hz), with the greatest change, 5-fold, occurring at 30 Hz. These results have been compared to those obtained in a different group of rats given daily injections of haloperidol (0.5 mg kg-1, s.c.) for 30 consecutive days. On the 30th day, dopamine striatal tissue levels and uptake kinetics were not altered by this treatment, but 3,4-dihydroxyphenylacetic acid tissue levels were elevated almost 2-fold. A challenge dose of haloperidol (0.5 mg kg-1, i.p.) administered to the animals treated with chronic haloperidol did not elicit a change in stimulated dopamine overflow. In two other groups, rats were withdrawn from 30-day haloperidol treatment for 3 days or 14 days before experimentation. Stimulated dopamine overflow concentrations in both groups were not significantly different from naive animals. When the withdrawn animals were given a haloperidol challenge (0.5 mg kg-1, i.p.), 15- and 12-fold increases in overflow for 3-day and 14-day withdrawal groups, respectively, were observed at a stimulation frequency of 30 Hz. Thus, chronic treatment with haloperidol induces long-lasting effects on the capacity of dopamine receptors to modulate dopamine release.


Subject(s)
Corpus Striatum/drug effects , Dopamine/metabolism , Haloperidol/pharmacology , Animals , Corpus Striatum/metabolism , Dose-Response Relationship, Drug , Electric Stimulation , Haloperidol/administration & dosage , Injections, Subcutaneous , Kinetics , Male , Rats , Rats, Inbred Strains , Receptors, Dopamine/drug effects , Receptors, Dopamine D2 , Synapses/drug effects , Synapses/metabolism
5.
Proc Natl Acad Sci U S A ; 88(23): 10754-8, 1991 Dec 01.
Article in English | MEDLINE | ID: mdl-1961743

ABSTRACT

Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.


Subject(s)
Adrenal Medulla/physiology , Catecholamines/metabolism , Chromaffin Granules/physiology , Adrenal Medulla/drug effects , Adrenal Medulla/metabolism , Animals , Carbachol/pharmacology , Cattle , Cells, Cultured , Chromaffin Granules/drug effects , Electric Stimulation , Kinetics , Mathematics , Models, Biological , Nicotine/pharmacology , Potassium/pharmacology , Time Factors
6.
Mol Pharmacol ; 40(6): 889-94, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1661839

ABSTRACT

A high affinity binding site for [3H]dihydrotetrabenazine is thought to be present on the monoamine transport protein from chromaffin granules. We describe a procedure for purification of this binding activity from frozen bovine adrenal tissue, and we partially characterize the purified preparation. Binding activity solubilized with sodium cholate and soybean lecithin was fractionated on wheat germ lectin-Sepharose, phenyl-Sepharose, Mono Q, and hydroxylapatite. Denaturing electrophoresis of the purified binding activity, followed by silver staining, revealed a single broad band centered at an apparent molecular weight of 85,000. This preparation bound [3H]dihydrotetrabenazine with an apparent dissociation constant of 2.7 nM and had a site density of 10 nmol/mg. Treatment of the purified protein with neuraminidase reduced the apparent molecular weight by 9000, indicating the presence of terminal sialic acids on the oligosaccharide portion of this molecule.


Subject(s)
Adrenal Medulla/chemistry , Carrier Proteins/isolation & purification , Tetrabenazine/analogs & derivatives , Animals , Anion Exchange Resins , Binding Sites , Carrier Proteins/metabolism , Cattle , Chromatography, Liquid , Durapatite , Electrophoresis, Polyacrylamide Gel , Hydroxyapatites/metabolism , Kinetics , Protein Binding , Resins, Synthetic , Sepharose , Sodium Dodecyl Sulfate , Tetrabenazine/isolation & purification , Tetrabenazine/metabolism , Tritium , Wheat Germ Agglutinins
7.
J Neurochem ; 56(6): 1855-63, 1991 Jun.
Article in English | MEDLINE | ID: mdl-2027003

ABSTRACT

Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cation-exchange polymer. Catecholamine secretion is induced by nicotine (10-500 microM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotine-induced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.


Subject(s)
Adrenal Medulla/metabolism , Catecholamines/metabolism , Chromaffin System/metabolism , Acetylcholine/physiology , Adrenal Medulla/cytology , Adrenal Medulla/drug effects , Animals , Catecholamines/antagonists & inhibitors , Cells, Cultured , Chromaffin System/cytology , Chromaffin System/drug effects , Nicotine/pharmacology , Osmolar Concentration , Parasympathomimetics/pharmacology , Potassium/pharmacology
8.
J Biol Chem ; 265(25): 14736-7, 1990 Sep 05.
Article in English | MEDLINE | ID: mdl-2394692

ABSTRACT

Nicotinic receptor-mediated secretion of catecholamines from individual cultured bovine adrenal medullary chromaffin cells was measured and characterized with a voltametric microelectrode placed adjacent to the cells. Nicotine-induced secretion is associated with a large increase in chemical spikes that is temporally resolved into the apparent secretion of discrete packets of attomole quantities of easily oxidized molecules. These data are consistent with direct chemical measurement of single exocytotic events.


Subject(s)
Adrenal Medulla/physiology , Catecholamines/metabolism , Nicotine/pharmacology , Receptors, Nicotinic/physiology , Adrenal Medulla/drug effects , Animals , Cattle , Cells, Cultured , Electric Conductivity , Receptors, Nicotinic/drug effects
10.
Eur J Pharmacol ; 172(3): 317-9, 1989 Aug 15.
Article in English | MEDLINE | ID: mdl-2776847

ABSTRACT

High-affinity dihydrotetrabenazine binding activity was solubilized by treatment of crude bovine striatal synaptosomes with cholate and incubated with lectins immobilized on sepharose. Both concanavalin A and wheat germ lectin sepharose removed dihydrotetrabenazine binding activity from the cholate extract, and this removal was prevented by inclusion of the appropriate hapten sugars. These results imply that the dihydrotetrabenazine binding subunit of the synaptic vesicle catecholamine/serotonin (5-HT) transporter, or a vesicle component tightly associated with it, is glycosylated.


Subject(s)
Lectins/metabolism , Synaptosomes/metabolism , Tetrabenazine/analogs & derivatives , Adsorption , Animals , Cattle , Concanavalin A/metabolism , Corpus Striatum/metabolism , In Vitro Techniques , Tetrabenazine/metabolism , Wheat Germ Agglutinins/metabolism
11.
J Pharmacol Exp Ther ; 245(3): 921-7, 1988 Jun.
Article in English | MEDLINE | ID: mdl-3385647

ABSTRACT

Uptake of dopamine (DA) by chopped tissue prepared from rat corpus striatum has been examined to determine whether one or two kinetically distinct uptake sites exist. Two methods were used: direct measurement of accumulated [3H]DA and determination of the rate of formation of 3,4-dihydroxyphenylacetic acid (DOPAC) after exposure to DA. The rate of formation of DOPAC in the latter experiments is a direct function of the rate of DA uptake. The rates of [3H]DA uptake and DOPAC formation are both linear with time in the presence of 10 microM substrate. Studies of [3H]DA accumulation into chopped tissue reveal two apparent components with Km values of 160 nM and 3.8 microM, whereas similar experiments with striatal homogenate or synaptosomes yield a single uptake component with a Km equivalent to the lower value found in chopped tissue. Evaluation of DA uptake via the rate of DOPAC formation gave a Km value of 2.3 microM. (High substrate values were used, so a lower value for Km is not apparent in the data.) The high Km-value component was absent in animals with a lesioned striatum induced by prior nigral injections of 6-hydroxydopamine. Several pharmacologic agents (benztropine, amfonelic acid, bupropion, nomifensine and ouabain) were evaluated. All reduced the uptake of DA in chopped tissue, but with reduced potencies compared with the effect of these agents in synaptosomes. The high Km activity in chopped tissue, as well as the apparent reduced potency of uptake inhibitor, appears to arise from the diffusional barrier present inside more intact tissue. This barrier is not present in homogenates or synaptosomes, and, thus, a single uptake process is seen.


Subject(s)
Corpus Striatum/metabolism , Dopamine/pharmacokinetics , Synaptosomes/metabolism , 3,4-Dihydroxyphenylacetic Acid/biosynthesis , Animals , In Vitro Techniques , Male , Nomifensine/pharmacology , Ouabain/pharmacology , Rats
12.
J Neurochem ; 50(3): 824-30, 1988 Mar.
Article in English | MEDLINE | ID: mdl-3123608

ABSTRACT

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.


Subject(s)
Corpus Striatum/metabolism , Synapses/metabolism , Synaptic Vesicles/metabolism , Tetrabenazine/analogs & derivatives , Animals , Binding Sites , Cattle , Cell Membrane/analysis , Cell Membrane/metabolism , Corpus Striatum/ultrastructure , Ligands/analysis , Membrane Proteins/metabolism , Subcellular Fractions/metabolism , Synapses/ultrastructure , Synaptophysin , Tetrabenazine/antagonists & inhibitors , Tetrabenazine/metabolism , Tissue Distribution , Tritium
13.
Mol Pharmacol ; 30(3): 252-7, 1986 Sep.
Article in English | MEDLINE | ID: mdl-3748008

ABSTRACT

[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo[a]quinolizine) bound to a single class of binding sites in synaptic vesicles isolated from bovine corpus striatum, with an apparent dissociation constant (Kd) of 2.3 nM and a Bmax of 15.1 pmol/mg of protein determined at equilibrium. Kinetic determination of the equilibrium dissociation constant yielded a value of 5.4 nM. ATP had no effect on the apparent Kd or Bmax, nor did it alter the kinetics of association or dissociation. Dopamine, serotonin, and other substrates for transport into synaptic vesicles inhibited binding at concentrations that were several orders of magnitude higher than their Km values for transport in the presence of ATP. The potent uptake blocker reserpine inhibited with a Ki of 340 nM in the absence of ATP, but biphasic inhibition, with Ki values of 3.2 and 345 nM, was observed in the presence of ATP. With incubation times of 24 hr, the potency of reserpine as an inhibitor of binding in the absence of ATP is increased by 1 to 2 orders of magnitude, implying a slow association rate for reserpine in the absence of nucleotide. These results suggest that dihydrotetrabenazine interacts with the catecholamine/serotonin porter in synaptic vesicles, although the binding site is probably not identical to that involved in active transport of substrate.


Subject(s)
Corpus Striatum/metabolism , Synaptic Vesicles/metabolism , Tetrabenazine/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Binding Sites , Biological Transport , Cattle , Dose-Response Relationship, Drug , In Vitro Techniques , Kinetics , Reserpine/pharmacology , Tetrabenazine/metabolism , Tritium
14.
J Pharmacol Exp Ther ; 232(2): 388-94, 1985 Feb.
Article in English | MEDLINE | ID: mdl-3968641

ABSTRACT

Carbon-fiber voltammetric electrodes have been used to measure the release of dopamine in the caudate nucleus of an anesthetized rat. Release is induced by electrical stimulation of the medial forebrain bundle. The amplitude of the observed release is attenuated by i.p. injection of amphetamine. A similar attenuation is induced by reserpine; however, at a slower rate. The combined regimen of amphetamine (1 or 10 mg/kg) and electrical stimulation does not deplete striatal dopamine levels and thus the decreased release of dopamine is not a result of depleted dopamine stores. Benztropine (25 mg kg-1) is able to cause a short term inhibition of the action of amphetamine (1 mg kg-1). The dopamine agonist pergolide (0.5 mg kg-1) does not affect the stimulated release. Haloperidol (1.0 mg kg-1) increases the amount of DA release, but is unable to attenuate the inhibition caused by amphetamine. Thus, it appears that the actions induced by amphetamine are a result of interaction with the neuronal uptake carrier and subsequent transport of dopamine from a functional to nonfunctional pool. In isolated striatal synaptic vesicles, amphetamine is found to block dopamine uptake and induce its release. This in vitro evidence provides a possible mechanism for the observed in vivo actions of amphetamine.


Subject(s)
Caudate Nucleus/metabolism , Dextroamphetamine/pharmacology , Dopamine/metabolism , Animals , Benztropine/pharmacology , Biological Transport/drug effects , Cattle , Caudate Nucleus/drug effects , Electric Stimulation , Electrochemistry , Ergolines/pharmacology , Haloperidol/pharmacology , In Vitro Techniques , Male , Pergolide , Rats , Reserpine/pharmacology , Synaptic Vesicles/metabolism
15.
Biochem Int ; 8(2): 265-70, 1984 Feb.
Article in English | MEDLINE | ID: mdl-6236819

ABSTRACT

Synaptic vesicles prepared from bovine corpus striatum exhibit an ATPase activity that is insensitive to ouabain and specific inhibitors of mitochondrial ATPase, but that is stimulated by proton ionophores and inhibited by sulfhydryl reagents. Low concentrations of orthovanadate, DCCD and tributyl tin are also ineffective as inhibitors of the vesicle-associated activity. The properties of the synaptic vesicle enzyme suggest that this ATPase may be similar to that of clathrin-coated vesicles, and to one of the activities described in preparations of adrenal chromaffin granule membranes.


Subject(s)
Adenosine Triphosphatases/metabolism , Corpus Striatum/enzymology , Synaptic Vesicles/enzymology , Adenosine Triphosphatases/antagonists & inhibitors , Animals , Cattle , Kinetics
16.
FEBS Lett ; 158(1): 31-5, 1983 Jul 11.
Article in English | MEDLINE | ID: mdl-6862032

ABSTRACT

Tritiated reserpine binds to synaptic vesicles from bovine caudate with high affinity (Kappd = 1.25 nM, Bmax = 3.3 pmol/mg protein). This interaction is both ATP-dependent and sensitive to the protonophores CCCP and nigericin, suggesting that a proton electrochemical gradient is required for binding. Dopamine, epinephrine, norepinephrine and serotonin all inhibit reserpine binding at concentrations similar to those required for inhibition of dopamine uptake. Treatment with saponin to release vesicle contents results in complete loss of accumulated dopamine but retention of bound reserpine. These results indicate that reserpine binds to the catecholamine transport system of synaptic vesicles with high affinity and specificity.


Subject(s)
Carrier Proteins/metabolism , Caudate Nucleus/metabolism , Membrane Transport Proteins , Reserpine/metabolism , Synaptic Vesicles/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport, Active , Catecholamine Plasma Membrane Transport Proteins , Cattle , Electrochemistry , In Vitro Techniques , Protein Binding
17.
Neurochem Res ; 7(7): 831-7, 1982 Jul.
Article in English | MEDLINE | ID: mdl-7121724

ABSTRACT

The addition of GTP (50 muM), MnCl2 (1 mM) or EDTA (2 mM) had no effect on the affinity or capacity of bovine striatal plasma membranes for [3H]spiperone. However, GTP caused a decrease in the potency of dopamine as an inhibitor of [3H]spiperone binding under all conditions tested. Manganese enhanced the potency of dopamine both in the presence and absence of GTP, but NaCl (100 mM) had no effect. Neither manganese nor GTP caused any change in the affinity or capacity of bovine striatal membranes for the tritiated agonists dopamine, apomorphine or ADTN. GPPNHP, a nonhydrolyzable analog of GTP, was also ineffective. However, in identical experiments using rat striatal membranes, 50 muM GTP caused a decrease in affinity for all three tritiated agonists and this effect was observed both in the presence and absence of manganese (1 mM). In addition, binding capacities for [3H]dopamine and [3H]ADTN were doubled when manganese was present. In light of this and other reports that GTP inhibits tritiated agonist binding in rat striatum, it is suggested that the absence of such inhibition in bovine striatal membranes may reflect a fundamental difference between the two species with regard to their receptors for dopamine agonists.


Subject(s)
Butyrophenones/metabolism , Corpus Striatum/drug effects , Guanosine Triphosphate/pharmacology , Receptors, Dopamine/drug effects , Spiperone/metabolism , Animals , Apomorphine/metabolism , Binding, Competitive/drug effects , Cattle , Caudate Nucleus/drug effects , Caudate Nucleus/metabolism , Dopamine/metabolism , Dopamine/pharmacology , Receptors, Dopamine/metabolism , Tetrahydronaphthalenes/metabolism
20.
Biochim Biophys Acta ; 587(4): 522-39, 1979 Nov 01.
Article in English | MEDLINE | ID: mdl-228750

ABSTRACT

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.


Subject(s)
Cyclic AMP/analogs & derivatives , Kidney/metabolism , Animals , Binding Sites , Cell Membrane/metabolism , Chromatography, DEAE-Cellulose , Chromatography, Gel , Cyclic AMP/metabolism , Cytosol/metabolism , Diazonium Compounds/metabolism , Light , Molecular Weight , Protein Kinases/metabolism , Swine
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