D5 Dopamine Receptor Knockout Mice and Hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D₁, D₂, D₃, D₄, and D₅) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D₁-like receptors (D₁ and D₅) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D₁-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D₁-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D₁ receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D₅ receptor is also important in blood pressure regulation. Disruption of the D₅ or the D₁ receptor gene in mice increases blood pressure. However, unlike the D₁ receptor, the hypertension in D₅ receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V₁ vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.     

D1-Like Dopamine Receptor-Mediated Function in Congenic Mutants with D1 vs. D5 Receptor “Knockout”

Current understanding of the functional roles of individual dopamine D₁-like [D₁, D₅] and D₂-like [D_2L/S, D₃, D₄] receptor subtypes remains incomplete. In particular, the lack of pharmacological agonists and antagonists able to distinguish between D₁ and D₅ receptors means that any differential roles in the regulation of behavior are poorly understood. Mutant mice with targeted gene deletion (“knockout”) of individual dopamine receptor subtypes offer an important alternative approach to resolving these functional roles. In congenic D₁ mutants examined ethologically, progressive increases in specific topographies of behavior over wildtypes were considerably greater than those in D₁ mutants on a mixed genetic background; D₁ knockout appears to influence the neuronal substrate(s) of habituation to disrupt sculpture of the changing topography of behavior from initial exploration through to quiescence. Similarly, the D₁ receptor appears to regulate specific topographies of orofacial movement in the mouse as these are “sculpted” in a time-dependent manner. Although the well-recognized role of the D₁-like family in regulating several aspects of behavioral topography has been assumed to involve primarily D₁ receptors, this presumption may require modification to accommodate a subtle but not negligible role for their D₅ counterparts as evidenced in the phenotype of congenic D₅ mutants.     

Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn²⁺ into the prefrontal cortex indicated that DAT KO mice have a truncated Mn²⁺ distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn²⁺ transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here.     

Serotonin/Dopamine Interactions in a Hyperactive Mouse: Reduced Serotonin Receptor 1B Activity Reverses Effects of Dopamine Transporter Knockout

Knockout (KO) mice that lack the dopamine transporter (SL6A3; DAT) display increased locomotion that can be attenuated, under some circumstances, by administration of drugs that normally produce psychostimulant-like effects, such as amphetamine and methylphenidate. These results have led to suggestions that DAT KO mice may model features of attention deficit hyperactivity disorder (ADHD) and that these drugs may act upon serotonin (5-HT) systems to produce these unusual locomotor decreasing effects. Evidence from patterns of brain expression and initial pharmacologic studies led us to use genetic and pharmacologic approaches to examine the influence of altered 5-HT_1B receptor activity on hyperactivity in DAT KO mice. Heterozygous 5-HT_1B KO and pharmacologic 5-HT_1B antagonism both attenuated locomotor hyperactivity in DAT KO mice. Furthermore, DAT KO mice with reduced, but not eliminated, 5-HT_1B receptor expression regained cocaine-stimulated locomotion, which was absent in DAT KO mice with normal levels of 5-HT_1B receptor expression. Further experiments demonstrated that the degree of habituation to the testing apparatus determined whether cocaine had no effect on locomotion in DAT KO or reduced locomotion, helping to resolve differences among prior reports. These findings of complementation of the locomotor effects of DAT KO by reducing 5-HT_1B receptor activity underscore roles for interactions between specific 5-HT receptors and dopamine (DA) systems in basal and cocaine-stimulated locomotion and support evaluation of 5-HT_1B antagonists as potential, non-stimulant ADHD therapeutics.     

Signaling Mechanisms of the D3 Dopamine Receptor

A substantial body of evidence shows the capacity of the dopamine D₃ receptor to couple functionally to G proteins when expressed in an appropriate milieu in heterologous expression systems. In these systems, activation of D₃ receptors inhibits adenylate cyclase, modulates ion flow through potassium and calcium channels, and activates kinases, most notably mitogen-activated protein kinase. Coupling to G_i/G_o is implicated in many of these effects, but other G proteins may contribute. Studies with chimeric receptors implicate the third intracellular loop in the mediation of agonist-induced signal transduction. Finally, D₃-preferring drugs modulate expression of c-fos in neuronal cultures and brain. Signaling mechanisms of the D₃ receptor in brain, however, remain to be definitively determined.     

Adolescent Maturation of Dopamine D1 and D2 Receptor Function and Interactions in Rodents

Adolescence is a developmental period characterized by heightened vulnerability to illicit drug use and the onset of neuropsychiatric disorders. These clinical phenomena likely share common neurobiological substrates, as mesocorticolimbic dopamine systems actively mature during this period. Whereas prior studies have examined age-dependent changes in dopamine receptor binding, there have been fewer functional analyses. The aim of the present study was therefore to determine whether the functional consequences of D1 and D2-like activation are age-dependent. Adolescent and adult rats were given direct D1 and D2 agonists, alone and in combination. Locomotor and stereotypic behaviors were measured, and brains were collected for analysis of mRNA expression for the immediate early genes (IEGs), cfos and arc. Adolescents showed enhanced D2-like receptor control of locomotor and repetitive behaviors, which transitioned to dominant D1-like mechanisms in adulthood. When low doses of agonists were co-administered, adults showed supra-additive behavioral responses to D1/D2 combinations, whereas adolescents did not, which may suggest age differences in D1/D2 synergy. D1/D2-stimulated IEG expression was particularly prominent in the bed nucleus of the stria terminalis (BNST). Given the BNST’s function as an integrator of corticostriatal, hippocampal, and stress-related circuitry, and the importance of neural network dynamics in producing behavior, an exploratory functional network analysis of regional IEG expression was performed. This data-driven analysis demonstrated similar developmental trajectories as those described in humans and suggested that dopaminergic drugs alter forebrain coordinated gene expression age dependently. D1/D2 recruitment of stress nuclei into functional networks was associated with low behavioral output in adolescents. Network analysis presents a novel tool to assess pharmacological action, and highlights critical developmental changes in functional neural circuitry. Immature D1/D2 interactions in adolescents may underlie their unique responses to drugs of abuse and vulnerability to psychopathology. These data highlight the need for age-specific pharmacotherapy design and clinical application in adolescence.     

G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

During conditions of moderate sodium excess, the dopaminergic system regulates blood pressure and water and electrolyte balance by engendering natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D₃ receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D₁ receptor, another dopamine receptor. This study evaluated the role of GRK4 on D₃ receptor function in human proximal tubule cells. D₃ receptor co-segregated in lipid rafts and co-immunoprecipitated and co-localized in human proximal tubule cells and in proximal and distal tubules and glomeruli of kidneys of Wistar Kyoto rats. Bimolecular fluorescence complementation and confocal microscopy revealed that agonist activation of the receptor initiated the interaction between D₃ receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-γ and GRK4-α mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D₃ receptor, respectively. Inhibition of GRK activity with heparin or knockdown of GRK4 expression via RNA interference completely abolished p44/42 phosphorylation and mitogenesis induced by D₃ receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-γ and GRK4-α isoforms, phosphorylates the D₃ receptor and is crucial for its signaling in human proximal tubule cells.During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs).² The dopamine receptors are classified into two subtypes based on their ability to increase cAMP levels, sequence similarity, G protein coupling, and pharmacological profiles (3, 4). The D₁-like dopamine receptors activate adenylyl cyclase by coupling to stimulatory Gα_s/Gα_olf and include the D₁ (D₁R) and D₅ receptors (D₅R). The D₂-like dopamine receptors inhibit adenylyl cyclase by coupling to Gα_i/Gα_o and consist of the D₂ (D₂R), D₃ (D₃R), and D₄ (D₄R) receptors. The D₃R has also been shown to couple to Gα_o, Gβγ, and to the stimulatory Gα_s (5, 6).The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8–10). GRK2 and GRK3 phosphorylate the β-adrenergic receptor and belong to the β-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-α is considered the full-length version, whereas GRK4-β, -γ, and -δ are shortened versions of GRK4-α (14). The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to essential hypertension and/or salt sensitivity in various ethnic groups (17).The D₃R gene is found at 3q13.3 (18), a locus that is also linked to essential hypertension (19, 20). Sequence analysis of the D₃R gene shows the presence of several single nucleotide polymorphisms, which do not correlate with either essential hypertension among Japanese (21) or with blood pressure levels and diabetic nephropathy among Finns (22). However, D₃R knock-out mice develop a renin-dependent form of hypertension and fail to excrete a sodium load (23).The D₃R has a long third intracellular loop that contains several putative GRK phosphorylation sites (24). A previous study evaluated the ability of GRK2 and GRK3 to phosphorylate D₃R and showed that co-transfection of GRK3, but not GRK2, resulted in a weak phosphorylation of the heterologously expressed, dopamine-stimulated D₃R in HEK-293 (25), a human embryonic kidney cell line. We tested the hypothesis that GRK4 is required in D₃R signaling in terminally differentiated human renal proximal tubule cells (hPTCs) by determining the spatiotemporal dynamics of the interaction of D₃R and GRK4 through their subfractionation in membrane microdomains and subcellular co-localization via confocal microscopy and bimolecular fluorescence complementation assay (BiFC). We also identified which of the GRK4 splice variants are involved in D₃R phosphorylation and evaluated the physiological roles of GRK4 in D₃R signaling in the hPTCs. We now report that D₃R and GRK4 co-fractionate in lipid rafts and co-localize in both hPTCs and WYK kidneys. Moreover, D₃R is phosphorylated by GRK4-γ and GRK4-α isoforms, and the absence of GRK4 impairs D₃R-mediated mitogenesis and activation of p44/42 in hPTCs.     

Analysis of Human Dopamine D3 Receptor Quaternary Structure

The dopamine D₃ receptor is a class A, rhodopsin-like G protein-coupled receptor that can form dimers and/or higher order oligomers. However, the molecular basis for production of these complexes is not well defined. Using combinations of molecular modeling, site-directed mutagenesis, and homogenous time-resolved FRET, the interfaces that allow dopamine D₃ receptor monomers to interact were defined and used to describe likely quaternary arrangements of the receptor. These were then compared with published crystal structures of dimeric β₁-adrenoreceptor, μ-opioid, and CXCR4 receptors. The data indicate important contributions of residues from within each of transmembrane domains I, II, IV, V, VI, and VII as well as the intracellular helix VIII in the formation of D₃-D₃ receptor interfaces within homo-oligomers and are consistent with the D₃ receptor adopting a β₁-adrenoreceptor-like quaternary arrangement. Specifically, results suggest that D₃ protomers can interact with each other via at least two distinct interfaces: the first one comprising residues from transmembrane domains I and II along with those from helix VIII and a second one involving transmembrane domains IV and V. Moreover, rather than existing only as distinct dimeric species, the results are consistent with the D₃ receptor also assuming a quaternary structure in which two transmembrane domain I-II-helix VIII dimers interact to form a ”rhombic” tetramer via an interface involving residues from transmembrane domains VI and VII. In addition, the results also provide insights into the potential contribution of molecules of cholesterol to the overall organization and potential stability of the D₃ receptor and possibly other GPCR quaternary structures.     

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.     

Quantification of Beta Adrenergic Receptor Subtypes in Beta-Arrestin Knockout Mouse Airways

In allergic asthma Beta 2 adrenergic receptors (β₂ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β₂AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β₂AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β₂AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β₂ARs predominate over β₁ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β₂AR expression. These data provide further evidence that β₂ARs are expressed in greater abundance than β₁ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β₂AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.     

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.     

Striatal Dopamine D2/3 Receptor Availability in Treatment Resistant Depression

Several studies demonstrated improvement of depressive symptoms in treatment resistant depression (TRD) after administering dopamine agonists which suggest abnormal dopaminergic neurotransmission in TRD. However, the role of dopaminergic signaling through measurement of striatal dopamine D_2/3 receptor (D2/3R) binding has not been investigated in TRD subjects. We used [¹²³I]IBZM single photon emission computed tomography (SPECT) to investigate striatal D2/3R binding in TRD. We included 6 severe TRD patients, 11 severe TRD patients on antipsychotics (TRD AP group) and 15 matched healthy controls. Results showed no significant difference (p = 0.75) in striatal D2/3R availability was found between TRD patients and healthy controls. In the TRD AP group D2/3R availability was significantly decreased (reflecting occupancy of D2/3Rs by antipsychotics) relative to TRD patients and healthy controls (p<0.001) but there were no differences in clinical symptoms between TRD AP and TRD patients. This preliminary study therefore does not provide evidence for large differences in D2/3 availability in severe TRD patients and suggests this TRD subgroup is not characterized by altered dopaminergic transmission. Atypical antipsychotics appear to have no clinical benefit in severe TRD patients who remain depressed, despite their strong occupancy of D2/3Rs.     

Beta-Adrenergic Stimulation Maintains Cardiac Function in Serca2 Knockout Mice

Previous studies on Serca2 knockout (KO) mice showed that cardiac function is sustained in vivo for several weeks after knockout, whereas SERCA protein levels decrease and calcium dynamics are significantly impaired. In this study, we reconcile observed cellular and organ level contractile function using a cardiac multiscale model. We identified and quantified the changes in cellular function that are both consistent with observations and able to compensate for the decrease in SERCA. Calcium transients were used as input for multiscale computational simulations to predict whole-organ response. Although this response matched experimental pressure-volume (PV) measurements in healthy mice, the reduced magnitude calcium transients observed in KO cells were insufficient to trigger ventricular ejection. To replicate the effects of elevated catecholamine levels observed in vivo, cells were treated with isoproterenol. Incorporation of the resulting measured β-adrenergically stimulated calcium transients into the model resulted in a close match with experimental PV loops. Changes in myofilament properties, when considered in isolation, were not able to increase tension development to levels consistent with measurements, further confirming the necessity of a high β-adrenergic state. Modeling additionally indicated that increased venous return observed in the KO mice helps maintain a high ejection fraction via the Frank-Starling effect. Our study shows that increased β-adrenergic stimulation is a potentially highly significant compensatory mechanism by which cardiac function is maintained in Serca2 KO mice, producing the increases in both systolic and diastolic calcium, consistent with the observed contractile function observed in experimental PV measurements.     

Dopamine D2 Receptor Stimulation Potentiates PolyQ-Huntingtin-Induced Mouse Striatal Neuron Dysfunctions via Rho/ROCK-II Activation

Background

Huntington''s disease (HD) is a polyglutamine-expanded related neurodegenerative disease. Despite the ubiquitous expression of expanded, polyQ-Huntingtin (ExpHtt) in the brain, striatal neurons present a higher susceptibility to the mutation. A commonly admitted hypothesis is that Dopaminergic inputs participate to this vulnerability. We previously showed that D2 receptor stimulation increased aggregate formation and neuronal death induced by ExpHtt in primary striatal neurons in culture, and chronic D2 antagonist treatment protects striatal dysfunctions induced by ExpHtt in a lentiviral-induced model system in vivo. The present work was designed to elucidate the signalling pathways involved, downstream D2 receptor (D2R) stimulation, in striatal vulnerability to ExpHtt.

Methodology/Principal Findings

Using primary striatal neurons in culture, transfected with a tagged-GFP version of human exon 1 ExpHtt, and siRNAs against D2R or D1R, we confirm that DA potentiates neuronal dysfunctions via D2R but not D1R stimulation. We demonstrate that D2 agonist treatment induces neuritic retraction and growth cone collapse in Htt- and ExpHtt expressing neurons. We then tested a possible involvement of the Rho/ROCK signalling pathway, which plays a key role in the dynamic of the cytoskeleton, in these processes. The pharmacological inhibitors of ROCK (Y27632 and Hydroxyfasudil), as well as siRNAs against ROCK-II, reversed D2-related effects on neuritic retraction and growth cone collapse. We show a coupling between D2 receptor stimulation and Rho activation, as well as hyperphosphorylation of Cofilin, a downstream effector of ROCK-II pathway. Importantly, D2 agonist-mediated potentiation of aggregate formation and neuronal death induced by ExpHtt, was totally reversed by Y27632 and Hydroxyfasudil and ROCK-II siRNAs.

Conclusions/Significance

Our data provide the first demonstration that D2R-induced vulnerability in HD is critically linked to the activation of the Rho/ROCK signalling pathway. The inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option aimed at forestalling the onset of the disease.     

多巴胺D5受体转基因小鼠的建立

目的建立人多巴胺D5受体突变基因F173 L（D5F173L）,S390G（D5S390G）及正常人D5基因（hD5 WT）的转基因小鼠,利用该转基因动物模型来研究D5受体在原发性高血压中的发病机制。方法利用显微注射的技术将插入CMV启动子下游的D5F173L,D5S390G及hD5 WT基因的转基因载体注射入C57BL/6小鼠体内,建立多巴胺D5F173L,D5S390G及hD5 WT的转基因小鼠。通过PCR鉴定转基因小鼠的基因型。利用Western Blotting方法鉴定该受体蛋白在肾脏的表达情况。使用智能无创血压测量仪测量转基因小鼠的血压值。结果分别建立了多巴胺D5F173L,D5S390G及hD5 WT转基因C57BL/6小鼠,Western Blotting方法鉴定结果显示,与非转基因C57BL/6小鼠比较,D5转基因小鼠D5受体在肾脏有较高的表达。3-6月龄D5 F173L转基因小鼠的收缩压、舒张压和平均动脉血压均明显高于多巴胺D5S390G及hD5 WT转基因小鼠（n=6-8,P〈0.05）。结论多巴胺D5受体在原发性高血压发病中具有重要作用,但作用机理还有待于进一步研究。     

Dopamine Metabolism and Receptor Function After Acute and Chronic Ethanol

Abstract: Acute ethanol treatment in rats elicits a selective increase in dihydroxyphenylacetic acid (DOPAC) content in striatum. In contrast, striatal DOPAC concentration does not differ from normal values after chronic ethanol treatment. Chronic administration of ethanol however causes a selective increase of specific [3H]spiroperidol binding and met-enkephalin content in the striatum. Kinetic analysis of [3H]spiroperidol binding data shows that after chronic ethanol treatment there is a significant increase in the affinity constant rather than in the number of binding sites for the ligand. Our results support the hypothesis that dopaminergic mechanisms at both pre- and postsynaptic level may be involved in the mediation of some of the central effects observed after ethanol consumption.     

Modulation of Bleomycin-Induced Lung Fibrosis by Pegylated Hyaluronidase and Dopamine Receptor Antagonist in Mice

Hyaluronidases are groups of enzymes that degrade hyaluronic acid (HA). To stop enzymatic hydrolysis we modified testicular hyaluronidase (HYAL) by activated polyethylene oxide with the help of electron-beam synthesis. As a result we received pegylated hyaluronidase (pegHYAL). Spiperone is a selective D₂ dopamine receptor antagonist. It was demonstrated on the model of a single bleomycin damage of alveolar epithelium that during the inflammatory phase monotherapy by pegHYAL or spiperone reduced the populations of hematopoietic stem /progenitor cells in the lung parenchyma. PegHYAL also reduced the levels of transforming growth factor (TGF)-β, interleukin (IL)-1β, tumor necrosis factor (TNF)-α in the serum and lungs, while spiperone reduced the level of the serum IL-1β. Polytherapy by spiperone and pegHYAL caused the increase of the quantity of hematopoietic stem/ progenitor cells in the lungs. Such an influx of blood cell precursors was observed on the background of considerable fall level of TGF-β and the increase level of TNF-α in the serum and lungs. These results show pegHYAL reduced the bleomycin-induced fibrosis reaction (production and accumulation of collagen) in the lung parenchyma. This effect was observed at a single and repetitive bleomycin damage of alveolar epithelium, the antifibrotic activity of pegHYAL surpassing the activity of testicular HYAL. The antifibrotic effect of pegHYAL is enhanced by an additional instillation of spiperone. Therapy by pegHYAL causes the flow of CD31^‒CD34^‒CD45^‒CD44⁺CD73⁺CD90⁺CD106⁺-cells into the fibrous lungs. These cells are incapable of differentiating into fibroblast cells. Spiperone instillation separately or together with pegHYAL reduced the MSC-like cells considerably. These data enable us to assume, that pegHYAL is a new and promising instrument both for preventive and therapy of toxic pneumofibrosis. The blockage of D₂ dopamine receptors with the following change of hyaluronan matrix can be considered as a new strategy in treatment of pneumofibrosis.