53 research outputs found
Tamoxifen and its active metabolites inhibit dopamine transporter function independently of the estrogen receptors
As one of the primary mechanisms by which dopamine signaling is regulated, the dopamine transporter (DAT) is an attractive pharmacological target for the treatment of diseases based in dopaminergic dysfunction. In this work we demonstrate for the first time that the commonly prescribed breast cancer therapeutic tamoxifen and its major metabolites, 4âhydroxytamoxifen and endoxifen, inhibit DAT function. Tamoxifen inhibits [3H]dopamine uptake into human DAT (hDAT)âN2A cells via an uncompetitive or mixed mechanism. Endoxifen, an active metabolite of tamoxifen, asymmetrically inhibits DAT function in hDATâN2A cells, showing a preference for the inhibition of amphetamineâstimulated dopamine efflux as compared to dopamine uptake. Importantly, we demonstrate that the effects of tamoxifen and its metabolites on the DAT occur independently of its activity as selective estrogen receptor modulators. This work suggests that tamoxifen is inhibiting DAT function through a previously unidentified mechanism.We demonstrate for the first time that the breast cancer therapeutic tamoxifen and its major metabolites, 4âhydroxytamoxifen and endoxifen, inhibit dopamine transporter function, possibly through an allosteric interaction with the transporter. We demonstrate that these effects occur independently of any activity at the estrogen receptors. This work suggests a previously unidentified mechanism for tamoxifen that may have clinical implications.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136525/1/jnc13955_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136525/2/jnc13955.pd
Regulation of Calmodulin- and Dopamine-Stimulated Adenylate Cyclase Activities by Light in Bovine Retina
Neural retina from most species contains 3,4-dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca 2+ -binding protein, cal-modulin, and that calmodulin can increase dopamine-sen-sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca 2+ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua-nosine-5âČ-(Î,Î-imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn 2+ -, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down-regulation of dopamine receptor activity. The down-regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66410/1/j.1471-4159.1984.tb12753.x.pd
Calmodulin-Sensitive and Calmodulin-Insensitive Components of Adenylate Cyclase Activity in Rat Striatum Have Differential Responsiveness to Guanyl Nucleotides
The interaction between the Ca 2+ -binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal paniculate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine-5âČ-(3,7-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 p.M GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100-fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The cal-modulin-stimulatable component constituted 60% of the total activity. Our two-component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP-binding proteins. The finding that GTP was unable to activate the calmodulin-sensitive component suggests that this component has either a different mode of binding to a GTP-binding protein or inherently higher GTPase activity than has the calmodulin-insensitive component. The results suggest there are two components of adenylate cyclase activity that can be differentiated by their sensitivities to calmodulin and guanyl nucleotides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65160/1/j.1471-4159.1983.tb00838.x.pd
Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum
The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed paniculate fraction from bovine striatum were examined. In the bovine striatal paniculate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC 50 of 110 n M . Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC 50 for GTP from 303 ± 56 n M to 60 ± 10 n M Calmodulin did not alter the maximal response to GTP. The EC 50 for calmodulin in potentiating the GTP response was only 11 n M as compared to 110 n M for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50â60%. The EC 50 for calmodulin in eliciting this response was 35 n M . These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, N s . Mechanisms that involve potentiation of N s -mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 m M MgCl 2 , but not at 1 or 10 m M MgCl 2 . These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65615/1/j.1471-4159.1988.tb01045.x.pd
ROLE OF CALMODULIN IN STATES OF ALTERED CATECHOLAMINE SENSITIVITY
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72490/1/j.1749-6632.1980.tb29620.x.pd
Inhibition of a Low K m GTPase Activity in Rat Striatum by Calmodulin
In rat striatum, the activation of adenylate cyclase by the endogenous Ca 2+ -binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5âČ-(Î, Î-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to âturn-offâ adenylate cyclase activity. We examined the effects of Ca 2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca 2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10 â9 â10 â6 M but had no effect on the hydrolysis of 10 â5 and 10 â6 M GTP, suggesting that calmodulin inhibited a low K m GTPase activity. The inhibition of GTPase activity by calmodulin was Ca 2+ -dependent and was maximal at 0.12 Î M free Ca 2+ . Maximal inhibition by calmodulin was 40% in the presence of 10 â7 M GTP. The IC 50 for calmodulin was 100 n M. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca 2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related. Inhibition of a low K m GTPase activity by calmodulin could represent an action of calmodulin in increasing the association of a GTP-binding protein with the catalytic subunit activity resulting in a reduction of the âturn-offâ GTPase activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66214/1/j.1471-4159.1985.tb05444.x.pd
Desensitization of the dopaminergic system in bovine retina following incubation with high potassium
The effect of potassium depolarization on dopamine D1 receptor activity in bovine retina was investigated. Preincubation of bovine retinas in buffer containing high KC1 (56 mM) as compared to a low KC1 control buffer resulted in a significant decrease in dopamine-stimulated adenylate cyclase with no change in basal or GTP-stimulated adenylate cyclase activity. The apparent Vmax for dopamine was decreased from 102 +/- 15 pmol/min/mg protein in retinas preincubated in high KC1 to 71 +/- 11 pmol/min/mg protein in control retinas (n = 5). The apparent Ka for dopamine stimulation of the enzyme did not change. The potassium-induced desentization could be blocked by preincubation with the dopamine antagonist cis-flupenthixol suggesting that the desentization was caused by the release of dopamine. The rapid desentization was not accompanied by a change in D1 receptor density as assessed by binding of [3H]SCH23390 nor in agonist binding as assessed by competition of the selective D1 agonist, SKF38393, for [3H]SCH23390 binding. The potassium-induced desentization was mimicked by preincubation of retinas in control medium containing isobutylmethylxanthine or dibutyryl cyclic AMP. Incubation of retinas in 56 mM KC1 also fed to a decrease in activation of adenylate cyclase by vasoactive intestinal polypeptide. These results strongly suggest that potassium depolarization leads to a very rapid heterologous desentization of adenylate cyclase in bovine retinas.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28516/1/0000313.pd
Effects of chronic and acute treatment of antipsychotic drugs on calmodulin release from rat striatal membranes
Chronic treatment of rats with the antipsychotic drugs haloperidol and (+)-butaclamol results in supersensitivity of striatal dopamine (DA) receptors. Striatal membranes of these animals have an increased content of an endogenous Ca -binding protein, calmodulin. Both endogenous and protein kinase-induced release of calmodulin from striatal membranes of the antipsychotic drug-treated animals were found substantially lower than that from saline or (-)-butaclamol-treated rats. Acute treatment with the antipsychotic drugs produced no alterations in calmodulin content or calmodulin release from the membranes. The impaired calmodulin release seen in the chronic antipsychotic drug-treated rats could be associated with the supersensitivity of DA receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23306/1/0000244.pd
Increased calmodulin in cultured skin fibroblasts from patients with cystic fibrosis
We have found that the content of an endogenous calcium-binding protein, calmodulin, is greater in cystic fibrosis fibroblasts than in normal cells. Homogenate preparations from CF fibroblasts contained 2.4 +/- 0.2 ng calmodulin/[mu]g protein while normal fibroblasts contained 1.5 +/- 0.1 ng calmodulin/[mu]g protein. Cyclic AMP phosphodiesterase activity, however, was the same in cystic fibrosis and normal fibroblasts. The increased calmodulin in cystic fibrosis cells could be involved in the changes in calcium metabolism and cyclic AMP production found in these cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24178/1/0000437.pd
Quantitative autoradiography of [3H]sulpiride binding sites in rat brain
A technique has been developed to investigate [3H]sulpiride binding in rat brain sections using quantitative autoradiography and tritium-sensitive film. Binding was saturable and reversible with very low nonspecific binding. [3H]Sulpiride bound to an apparent single population of sites in striatum with a Kd of 3.2 nM and Bmax of 447 fmol/mg protein. Binding sites were localized in the lamina glomerulosa of the olfactory bulb, nucleus accumbens, olfactory tubercle, striatum and substantia nigra.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24695/1/0000114.pd
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