Activation of Human D3 Dopamine Receptor Inhibits P/Q-Type Calcium Channels and Secretory Activity in AtT-20 Cells

The D3 dopamine receptor is postulated to play an important role in the regulation of neurotransmitter secretion at both pre- and postsynaptic terminals. However, this hypothesis and the underlying mechanisms remain untested because of the lack of D3-selective ligands, paucity of appropriate model secretory systems, and the weak and inconsistent coupling of D3 receptors to classical signal transduction pathways. The absence of ligands that selectively discriminate between D3 and D2 receptor

Sucrase-isomaltase is an adenosine 3',5'-cyclic monophosphate–dependent epithelial chloride channel

BACKGROUND & AIMS: We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals. METHODS: Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody. RESULTS: Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody. CONCLUSIONS: Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time

Functional Analysis of Capsaicin Receptor (Vanilloid Receptor Subtype 1) Multimerization and Agonist Responsiveness Using a Dominant Negative Mutation

The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for

Micro RNA expression profile and functional analysis reveal that mi R ‐382 is a critical novel gene of alcohol addiction

Alcohol addiction is a major social and health concern. Here, we determined the expression profile of microRNAs (miRNAs) in the nucleus accumbens (NAc) of rats treated with alcohol. The results suggest that multiple miRNAs were aberrantly expressed in rat NAc after alcohol injection. Among them, miR‐382 was down‐regulated in alcohol‐treated rats. In both cultured neuronal cells in vitro and in the NAc in vivo , we identified that the dopamine receptor D1 ( Drd1 ) is a direct target gene of miR‐382. Via this target gene, miR‐382 strongly modulated the expression of DeltaFosB. Moreover, overexpression of miR‐382 significantly attenuated alcohol‐induced up‐regulation of DRD1 and DeltaFosB, decreased voluntary intake of and preference for alcohol and inhibited the DRD1‐induced action potential responses. The results indicated that miRNAs are involved in and may represent novel therapeutic targets for alcoholism. The underlying molecular causes of alcohol addiction remain unclear. Many miRNAs are found modulated in the nucleus accumbens of rats chronically treated with alcohol. Specifically, miR‐382 is shown to regulate alcohol intake via DRD1 and DeltaFosB.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/1/emmm201201900-sm-0001-Review_Process_File.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/2/emmm201201900.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/3/emmm201202100-sm-0006-SourceData-S5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/4/emmm201201900-sm-0002-SuppData-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/5/emmm201202100-sm-0005-SourceData-S4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/6/emmm201202100-sm-0004-SourceData-S3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/7/emmm201202100-sm-0003-SourceData-S2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99635/8/emmm201202100-sm-0007-SourceData-S6.pd

Dopamine D3 Receptor Mediates Preadolescent Stress-Induced Adult Psychiatric Disorders.

Several studies have shown that repeated stressful experiences during childhood increases the likelihood of developing depression- and anxiety-related disorders in adulthood; however, the underlying mechanisms are not well understood. We subjected drd3-EGFP and drd3-null mice to daily, two hour restraint stress episodes over a five day period during preadolescence (postnatal day 35 to 39), followed by social isolation. When these mice reached adulthood (post-natal day > 90), we assessed locomotor behavior in a novel environment, and assessed depression-related behavior in the Porsolt Forced Swim test. We also measured the expression and function of dopamine D3 receptor in limbic brain areas such as hippocampus, nucleus accumbens and amygdala in control and stressed drd3-EGFP mice in adulthood. Adult male mice subjected to restraint stress during preadolescence exhibited both anxiety- and depression-related behaviors; however, adult female mice subjected to preadolescent restraint stress exhibited only depression-related behaviors. The development of preadolescent stress-derived psychiatric disorders was blocked by D3 receptor selective antagonist, SB 277011-A, and absent in D3 receptor null mice. Adult male mice that experienced stress during preadolescence exhibited a loss of D3 receptor expression and function in the amygdala but not in hippocampus or nucleus accumbens. In contrast, adult female mice that experienced preadolescent stress exhibited increased D3 receptor expression in the nucleus accumbens but not in amygdala or hippocampus. Our results suggest that the dopamine D3 receptor is centrally involved in the etiology of adult anxiety- and depression-related behaviors that arise from repeated stressful experiences during childhood

Effect of repeated restraint stress and social isolation during preadolescence on function and expression of dopamine D3 receptors in three limbic brain regions of adult male <i>drd3</i>-EGFP mice.

<p>Male <i>drd3</i>-EGFP mice (<b>A-D,</b> n = 5 per treatment group) were either not stressed (blue) or subjected to preadolescent restraint stress and social isolation (red). In adulthood, the two groups of mice were administered saline or 0.05 mg/kg PD128907, s.c., a D3 receptor agonist. The three brain regions nucleus accumbens (<b>A</b>), hippocampus (<b>B</b>) and amygdala (<b>C</b> and <b>D</b>) were harvested ten minutes after the injection. The levels of phosphorylated ERK and total ERK were measured using western blot and expressed as a ratio (<b>A-C</b>). A significant increase in PD128907-induced ERK phosphorylation was observed in the nucleus accumbens and hippocampus of non-stressed and stressed mice but only in the amygdala of non-stressed mice (*, p<0.05). There was also a significant reduction of D3 receptor protein levels in the amygdala of stressed mice (<b>D</b>, # p<0.05). Error bars represents ± SEM.</p

Effect of D3 receptor antagonist on preadolescent stress-induced anxiety-related behaviors in adult <i>drd3</i>-EGFP mice.

<p>Adult male <i>drd3</i>-EGFP mice were either not stressed (blue, n = 11) or subjected to repeated restraint stress and social isolation during preadolescence (red, n = 11). Horizontal locomotor activity (<b>A</b>) and number of center zone entries (<b>B</b>) were measured in an Open Field test in adulthood. Significant reduction in locomotor activity and center zone entries were observed in stressed <i>drd3</i>-EGFP mice (*, p<0.05). Administration of 10 mg/kg SB277011-A (green cross hatch; n = 6) but not saline (pink cross hatch, n = 6) to <i>drd3</i>-EGFP mice ten minutes before each restraint stress episode during preadolescence prevented the reduction of locomotor activity (<b>A</b>) and center zone entries observed in adulthood (<b>B</b>) (**, p<0.001 compared to saline-injected stressed mice and +, p<0.05 compared to non-injected stressed mice). Error bars represents ± SEM.</p

Effect of repeated restraint stress and social isolation during preadolescence on locomotor activity in an open field arena test by adult <i>drd3</i>-null mice.

<p>Adult male (<b>A</b> and <b>B</b>) and female (<b>C</b> and <b>D</b>) <i>drd3</i>-null mice were either not stressed (blue, n = 5) or subjected to repeated restraint stress and social isolation during preadolescence (red, n = 5). Horizontal locomotor activity were measured in an Open Field test in adulthood. Compared to non-stressed <i>drd3</i>-null male mice, preadolescent stressed male <i>drd3</i>-null mice exhibited significant increase in locomotor activity (<b>A</b>) only during the initial 15 minute period following the placement of the mice in the arena (*, p<0.05, two-way repeated measure ANOVA, post-hoc SNK test). The total locomotor activity over the entire 60 minute observation period was not significantly altered in stressed male <i>drd3</i>-null mice (p>0.05, Student’s <i>t</i>-test). The stress-induced changes in locomotor activity was absent in adult female <i>drd3</i>-null mice subjected to preadolescent stress and social isolation (<b>C</b> and <b>D</b>). Error bars represents ± SEM.</p

Effect of repeated restraint stress and social isolation during preadolescence on number of center zone entries in an open field arena by adult <i>drd3</i>-EGFP mice.

<p>Adult male (<b>A</b> and <b>B</b>) and female (<b>C</b> and <b>D</b>) <i>drd3</i>-EGFP mice were either not stressed (blue, n = 11) or subjected to repeated restraint stress and social isolation during preadolescence (red, n = 11). Number of center zone entries were measured in an Open Field test in adulthood. Significant reduction in number of entries was observed in stressed male <i>drd3</i>-EGFP mice during the initial 20 minute period following the placement of the mice in the arena (*, p<0.05, two-way repeated measure ANOVA, post-hoc SNK test; <b>A</b>). The total number of center zone entries over the entire 60 minute observation period was also significantly reduced in stressed male <i>drd3</i>-EGFP mice (*, p<0.05, Student’s <i>t</i>-test). The stress-induced changes in locomotor activity was absent in adult female <i>drd3</i>-EGFP mice subjected to preadolescent stress and social isolation (<b>C</b> and <b>D</b>). Error bars represents ± SEM.</p