Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Falcarinol is a covalent cannabinoid CB1 receptor antagonist and induces pro-allergic effects in skin

The skin irritant polyyne falcarinol (panaxynol, carotatoxin) is found in carrots, parsley, celery, and in the medicinal plant Panax ginseng. In our ongoing search for new cannabinoid (CB) receptor ligands we have isolated falcarinol from the endemic Sardinian plant Seseli praecox. We show that falcarinol exhibits binding affinity to both human CB receptors but selectively alkylates the anandamide binding site in the CB(1) receptor (K(i)=594nM), acting as covalent inverse agonist in CB(1) receptor-transfected CHO cells. Given the inherent instability of purified falcarinol we repeatedly isolated this compound for biological characterization and one new polyyne was characterized. In human HaCaT keratinocytes falcarinol increased the expression of the pro-allergic chemokines IL-8 and CCL2/MCP-1 in a CB(1) receptor-dependent manner. Moreover, falcarinol inhibited the effects of anandamide on TNF-alpha stimulated keratinocytes. In vivo, falcarinol strongly aggravated histamine-induced oedema reactions in skin prick tests. Both effects were also obtained with the CB(1) receptor inverse agonist rimonabant, thus indicating the potential role of the CB(1) receptor in skin immunopharmacology. Our data suggest anti-allergic effects of anandamide and that falcarinol-associated dermatitis is due to antagonism of the CB(1) receptor in keratinocytes, leading to increased chemokine expression and aggravation of histamine action.

Concurrent activation of dopamine D1 and D2 receptors is required to evoke neural and behavioral phenotypes of cocaine sensitization

Repeated exposure to psychomotor stimulants produces a striking behavioral syndrome involving repetitive, stereotypic behaviors that occur if an additional exposure to the stimulant is experienced. The same stimulant exposure produces specific alterations in gene expression patterns in the striatum. To identify the dopamine receptor subtypes required for the parallel expression of these acquired neural and behavioral responses, we treated rats with different D1-class and D2-class dopamine receptor agonists and compared the responses of drug-naive rats with those of rats given previous intermittent treatment with cocaine. In rats exposed to repeated cocaine treatment, the effects of a subsequent challenge treatment with either a D1-class agonist (SKF 81297) or a D2-class agonist (quinpirole) were not significantly different from those observed in drug-naive animals: the drugs administered singly did not induce robust stereotyped motor behaviors nor produce significantly striosome-predominant expression of early genes in the striatum. In contrast, challenge treatment with the D1-class and D2-class agonists in combination led to marked and correlated increases in stereotypy and striosome-predominant gene expression in the striatum. Thus, immediately after repeated psychomotor stimulant exposure, only the concurrent activation of D1 and D2 receptor subclasses evoked expression of the neural and behavioral phenotypes acquired through repeated cocaine exposure. These findings suggest that D1-D2 dopamine receptor synergisms underlie the coordinate expression of both network-level changes in basal ganglia activation patterns and the repetitive and stereotypic motor response patterns characteristic of psychomotor stimulant sensitization.

Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor activities

Vitamin A is a nutrient with remarkable effects on adipose tissue and skeletal muscles, and plays a role in controlling energy balance. Retinoic acid (RA), the carboxylic form of vitamin A, has been associated with improved glucose tolerance and insulin sensitivity. In contrast, elevated glucocorticoids have been implicated in the development of insulin resistance and impaired glucose tolerance. Here, we investigated whether RA might counteract glucocorticoid effects in skeletal muscle cells by lowering 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local glucocorticoid activation and/or activation of glucocorticoid receptor (GR). We found a dose-dependent down-regulation of 11beta-HSD1 mRNA expression and activity upon incubation of fully differentiated mouse C2C12 myotubes with RA. In addition, RA inhibited GR transactivation by an 11beta-HSD1-independent mechanism. The presence of RA during myogenesis did not prevent myotube formation but resulted in relatively glucocorticoid-resistant myotubes, exhibiting very low 11beta-HSD1 expression and GR activity. The use of selective retinoic acid receptor (RAR) and retinoid X receptor ligands provided evidence that these effects were mediated through RARgamma. Importantly, short hairpin RNA against RARgamma abolished the effect of RA on 11beta-HSD1 and GR. In conclusion, we provide evidence for an important role of RA in the control of glucocorticoid activity during myogenesis and in myotubes. Disturbances of the nutrient and hormonal regulation of glucocorticoid action in skeletal muscles might be relevant for metabolic diseases.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

TIME DEPENDENT ALTERATIONS IN CENTRAL NERVOUS SYSTEM DOPAMINE RECEPTORS INDUCED BY DOPAMINE AGONIST TREATMENT

Levodopa, the precursor of dopamine, is currently the drug of choice in the treatment of Parkinson's disease. Recently, two direct dopamine agonists, bromocriptine and pergolide, have been tested for the treatment of Parkinson's disease because of reduced side effects compared to levodopa. Few studies have evaluated the effects of long-term treatment of dopamine agonists on dopamine receptor regulation in the central nervous system. Thus, the purpose of this study was to determine whether chronic dopamine agonist treatment produces a down-regulation of striatal dopamine receptor function and to compare the results of the two classes of dopaminergic drugs.^ Levodopa with carbidopa, a peripheral decarboxylase inhibitor, was administered orally to rats whereas bromocriptine and pergolide were injected intraperitoneally once daily. Several neurochemical parameters were examined from 1 to 28 days.^ Levodopa minimally decreased striatal D-1 receptor activity but increased the number of striatal D-2 binding sites. Levodopa increased the V(,max) of tyrosine hydroxylase (TH) in all brain regions tested. Protein blot analysis of striatal TH indicated a significant increase in the amount of TH present. Dopamine-beta-hydroxylase (DBH) activity was markedly decreased in all brain regions studied and mixing experiments of control and drug-treated cortices did not show the presence of an increased level of endogenous inhibitors.^ Bromocriptine treatment decreased the number of D-2 binding sites. Striatal TH activity was decreased and protein blot analysis indicated no change in TH quantity. The specificity of bromocriptine for striatal TH suggested that bromocriptine preferentially interacts with dopamine autoreceptors.^ Combination levodopa-bromocriptine was administered for 12 days. There was a decrease in both D-1 receptor activity and D-2 binding sites, and a decrease in brain HVA levels suggesting a postsynaptic receptor action. Pergolide produced identical results to the combination levodopa-bromocriptine studies.^ In conclusion, combination levodopa-bromocriptine and pergolide treatments exhibited the expected down-regulation of dopamine receptor activity. In contrast, levodopa appeared to up-regulate dopamine receptor activity. Thus, these data may help to explain, on a biochemical basis, the decrease in the levodopa-induced side effects noted with combination levodopa-bromocriptine or pergolide therapies in the treatment of Parkinson's disease. ^

Prominence of the dopamine D2 short isoform in dopaminergic pathways

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.

Receptors induce chemotaxis by releasing the βγ subunit of Gi, not by activating Gq or Gs

Many chemoattractants cause chemotaxis of leukocytes by stimulating a structurally distinct class of G protein-coupled receptors. To identify receptor functions required for chemotaxis, we studied chemotaxis in HEK293 cells transfected with receptors for nonchemokine ligands or for interleukin 8 (IL-8), a classical chemokine. In gradients of the appropriate agonist, three nonchemokine Gi-coupled receptors (the D2 dopamine receptor and opioid μ and δ receptors) mediated chemotaxis; the β2-adrenoreceptor and the M3-muscarinic receptor, which couple respectively to Gs and Gq, did not mediate chemotaxis. A mutation deleting 31 C-terminal amino acids from the IL-8 receptor type B quantitatively impaired chemotaxis and agonist-induced receptor internalization, but not inhibition of adenylyl cyclase or stimulation of mitogen-activated protein kinase. To probe the possible relation between receptor internalization and chemotaxis, we used two agonists of the μ-opioid receptor. Morphine and etorphine elicited quantitatively similar chemotaxis, but only etorphine induced receptor internalization. Overexpression of two βγ sequestering proteins (βARK-ct and αt) prevented IL-8 receptor type B-mediated chemotaxis but did not affect inhibition of adenylyl cyclase by IL-8. We conclude that: (i) Nonchemokine Gi-coupled receptors can mediate chemotaxis. (ii) Gi activation is necessary but probably not sufficient for chemotaxis. (iii) Chemotaxis does not require receptor internalization. (iv) Chemotaxis requires the release of free βγ subunits.

Molecular determinants of tissue selectivity in estrogen receptor modulators

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (A) the presence of phenolic hydroxyls, (B) different substituents on the basic amine, (C) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (D) the imposition of a carbonyl “hinge” between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.

Most central nervous system D2 dopamine receptors are coupled to their effectors by Go

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPγS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPγS to frozen brain sections, with no significant left–right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [35S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.

Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor–FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.

Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.

Numerous immature thymocytes undergo apoptosis and are rapidly engulfed by phagocytic thymic macrophages. The macrophage surface receptors involved in apoptotic thymocyte recognition are unknown. We have examined the role of the class A macrophage scavenger receptor (SR-A) in the engulfment of apoptotic thymocytes. Uptake of steroid-treated apoptotic thymocytes by thymic and inflammatory-elicited SR-A positive macrophages is partially inhibited by an anti-SR-A mAb and more completely by a range of scavenger receptor ligands. Thymic macrophages from mice with targeted disruption of the SR-A gene show a 50% reduction in phagocytosis of apoptotic thymocytes in vitro. These data suggest that SR-A may play a role in the clearance of dying cells in the thymus.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.