RESUMO
New classes of chemistries are needed to control insecticide resistant populations of mosquitoes and prevent transmission of vector-borne diseases (VBDs). Organismal screens of chemical collections have played an important role in the search for new vector insecticides and the identification of active ingredients (AIs) that cause rapid mortality of mosquitoes. Advances in image-based screening offer an opportunity to identify chemistries that operate via novel biochemical modes and investigate the range of phenotypes exhibited by mosquitoes following exposure to lethal and sub-lethal chemical dose. An automated, high throughput phenotypic screen (HTS) employing high-content imaging of first instar (L1) Aedes aegypti larvae was developed to identify chemistries associated with mortality and atypical morphological phenotypes. A pilot screen of the Library of Pharmacologically Active Compounds (LOPAC1280) identified 92 chemistries that disrupted larval activity and development, including conventional insecticides and chemistries known to modulate G protein-coupled receptors (GPCRs) and other molecular targets in mammalian systems. Secondary assay series were used to evaluate a selection of chemistries for impacts on mosquito activity, survival and development. Ritodrine hydrochloride reduced mobility of larvae but had no observable effect on survival and development of mosquitoes. High doses of metergoline suppressed larval activity and sub-lethal dose resulted in pupal mortality. Assay data support the utility of phenotypic screening and diverse entomological end-points for discovery of novel insecticidal chemical scaffolds. The insecticide discovery process must consider how multi-modal efficacy spectra contribute to vector and VBD control.
Assuntos
Aedes , Inseticidas , Animais , Inseticidas/química , Inseticidas/toxicidade , Larva , Controle de Mosquitos/métodos , Mosquitos Vetores , FenótipoRESUMO
Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D1 expressing MSNs of the direct pathway, as well as the D2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gαs-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.
Assuntos
Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Discinesias/genética , Discinesias/metabolismo , Mutação com Ganho de Função/fisiologia , Variação Genética/fisiologia , Inibidores de Adenilil Ciclases/farmacologia , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismoRESUMO
CB(1) cannabinoid (CB(1)) and D(2) dopamine (D(2)) receptors are known to couple to the G protein Galpha(i/o). It has been reported that concurrent activation of D(2) receptors and CB(1) receptors, in primary striatal neuronal culture, promotes functional CB(1) receptor coupling to Galpha(s) resulting in elevations in intracellular cyclic AMP levels. We now report that in the absence of D(2) receptors, acute activation of CB(1) receptors inhibits cyclic AMP accumulation, whereas the presence of D(2) receptors promotes CB(1)-stimulated cAMP accumulation, presumably through Galpha(s). This Galpha(s) subunit switching was not prevented by pertussis toxin treatment and occurred in the presence and absence of D(2) receptor activation. Thus, coexpression of the D(2) receptor with the CB(1) receptor was sufficient to switch the coupling of the CB(1) receptors from Galpha(i/o) to Galpha(s). Persistent activation of D(2) receptors resulted in heterologous sensitization of adenylate cyclase to subsequent stimulation by forskolin, whereas the persistent activation of CB(1) receptors did not. Additional studies in human embryonic kidney cells cotransfected with D(2) and CB(1) receptors revealed that persistent activation (18 h) of D(2) receptors induced a switch of CB(1) receptor coupling from Galpha(s) to Galpha(i/o). This D(2) receptor-induced effect allowed for CB(1) receptor-mediated inhibition of cyclic AMP accumulation. The present studies suggest D(2) receptors may have a significant modulatory role in determining the G protein coupling specificity of CB(1) receptors.
Assuntos
AMP Cíclico/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas , HumanosRESUMO
Whereas acute stimulation of Galphai/o-coupled receptors inhibits the activity of adenylyl cyclase, a delayed consequence of persistent activation of the receptors is heterologous sensitization, an enhanced responsiveness of adenylyl cyclase to activators such as forskolin or agonists of Galphas-coupled receptors. Galphas-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization requires Galphas-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D2L dopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimulated cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D2 receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three Galphas-insensitive mutants (F379L, R1021Q, and F1093S) in HEK293 cells stably expressing the D2L receptor demonstrated that all three mutants had little or no responsiveness to beta-adrenergic receptor-mediated activation of Galphas but that the mutants retained sensitivity to forskolin and to D2L receptor-mediated inhibition. Transiently expressed adenylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpirole. In contrast, the Galphas-insensitive mutants displayed no sensitization of forskolin-stimulated cyclic AMP accumulation, indicating that responsiveness to Galphas is required for the expression of heterologous sensitization.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Receptores de Dopamina D2/metabolismo , Adenilil Ciclases/genética , Agonistas Adrenérgicos beta/farmacologia , Células Cultivadas , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Humanos , Isoenzimas/genética , Isoproterenol/farmacologia , MutaçãoRESUMO
A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.
Assuntos
Receptores de Dopamina D2/química , Sódio/metabolismo , Sequência de Aminoácidos , Asparagina/genética , Sítios de Ligação , Células Cultivadas , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Serina/genéticaRESUMO
Adenylate cyclase activity in NS20Y cells expressing D2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D2 receptors. Furthermore, chronic 18 h activation of D2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Galpha(s) or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.
Assuntos
Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos/farmacologia , Neuroblastoma/metabolismo , Receptores de Dopamina D2/metabolismo , Toxina Adenilato Ciclase , Antagonistas Adrenérgicos beta/farmacologia , Animais , Colforsina/farmacologia , AMP Cíclico/metabolismo , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Ativação Enzimática/efeitos dos fármacos , Epinefrina/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Camundongos , Norepinefrina/farmacologia , Toxina Pertussis , Receptores de Dopamina D2/genética , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Persistent activation of Galpha(i/o)-coupled receptors results in an enhanced responsiveness of drug-stimulated adenylate cyclase activity through an unknown mechanism. This agonist-induced heterologous sensitization of drug-stimulated cyclic AMP accumulation has been proposed to be a mechanism by which cells adapt to prolonged Galpha(i/o) activation. Heterologous sensitization was examined in human embryonic kidney 293 cells stably expressing D(2L) dopamine receptors in combination with recombinant isoforms of adenylate cyclase. The ability of each isoform to be differentially regulated by G protein subunits and other signaling intermediates allowed us to identify potential mechanisms that are involved in heterologous sensitization of adenylate cyclase. We now report that both short- and long-term activation of D(2L) dopamine receptors resulted in a marked degree of sensitization of ACI, ACII, ACV, and ACIX, but not ACVIII. The effects of agonist treatment on ACI, ACII, and ACVIII appeared to be dependent upon the ability of these adenylate cyclase isoforms to synergistically respond to selective activators in the presence of activated Galpha(s). Sensitization of ACV was characterized by enhanced cyclic AMP accumulation following Galpha(s) or forskolin stimulation. Furthermore, agonist pretreatment enhanced the basal levels of cyclic AMP accumulation in ACV/D(2L) cells, an effect that was not observed with the other adenylate cyclase isoforms. ACIX, which has no known activators other than Galpha(s), showed robust agonist-induced sensitization of isoproterenol-stimulated cyclic AMP accumulation. In summary, heterologous sensitization appeared to be related to the ability of each adenylate cyclase isoform to be modulated by Galpha(s).
Assuntos
Adenilil Ciclases/metabolismo , Rim/metabolismo , Receptores de Dopamina D2/metabolismo , Proteínas Recombinantes/metabolismo , Adenilil Ciclases/genética , Agonistas Adrenérgicos beta/farmacologia , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/análise , AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ionóforos/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Isoproterenol/farmacologia , Rim/citologia , Quimpirol/farmacologia , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/genética , Proteínas Recombinantes/genética , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A series of fluorinated analogues of the hallucinogenic tryptamines N,N-diethyltryptamine (DET), 4-hydroxy-N,N-dimethyltryptamine (4-OH-DMT, psilocin), and 5-methoxy-DMT was synthesized to investigate possible explanations for the inactivity of 6-fluoro-DET as a hallucinogen and to determine the effects of fluorination on the molecular recognition and activation of these compounds at serotonin receptor subtypes. The target compounds were evaluated using in vivo behavioral assays for hallucinogen-like and 5-HT(1A) agonist activity and in vitro radioligand competition assays for their affinity at 5-HT(2A), 5-HT(2C), and 5-HT(1A) receptor sites. Functional activity at the 5-HT(2A) receptor was determined for all compounds. In addition, for some compounds functional activity was determined at the 5-HT(1A) receptor. Hallucinogen-like activity, evaluated in the two-lever drug discrimination paradigm using LSD-trained rats, was attenuated or abolished for all of the fluorinated analogues. One of the tryptamines, 4-fluoro-5-methoxy-DMT (6), displayed high 5-HT(1A) agonist activity, with potency greater than that of the 5-HT(1A) agonist 8-OH-DPAT. The ED(50) of 6 in the two-lever drug discrimination paradigm using rats trained to discriminate the 5-HT(1A) agonist LY293284 was 0.17 micromol/kg, and the K(i) at [(3)H]8-OH-DPAT-labeled 5-HT(1A) receptors was 0.23 nM. The results indicate that fluorination of hallucinogenic tryptamines generally has little effect on 5-HT(2A/2C) receptor affinity or intrinsic activity. Affinity at the 5-HT(1A) receptor was reduced, however, in all but one example, and all of the compounds tested were full agonists but with reduced functional potency at this serotonin receptor subtype. The one notable exception was 4-fluoro-5-methoxy-DMT (6), which had markedly enhanced 5-HT(1A) receptor affinity and functional potency. Although it is generally considered that hallucinogenic activity results from 5-HT(2A) receptor activation, the present results suggest a possible role for involvement of the 5-HT(1A) receptor with tryptamines.
Assuntos
Flúor/química , Alucinógenos/síntese química , Agonistas do Receptor de Serotonina/síntese química , Triptaminas/síntese química , Células 3T3 , Animais , Ligação Competitiva , Células CHO , Colforsina/farmacologia , Cricetinae , AMP Cíclico/biossíntese , Aprendizagem por Discriminação/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Alucinógenos/química , Alucinógenos/farmacologia , Humanos , Hidrólise , Camundongos , Fosfatidilinositóis/metabolismo , Ensaio Radioligante , Ratos , Receptor 5-HT2A de Serotonina , Receptor 5-HT2C de Serotonina , Receptores de Serotonina/metabolismo , Receptores de Serotonina/fisiologia , Receptores 5-HT1 de Serotonina , Agonistas do Receptor de Serotonina/química , Agonistas do Receptor de Serotonina/farmacologia , Relação Estrutura-Atividade , Triptaminas/química , Triptaminas/farmacologiaRESUMO
1. Decline in beta-adrenoceptor (beta-AR)-mediated function occurs with increasing age, as well as in multiple disease conditions. The mechanisms responsible for this decline include alterations in beta-AR itself, beta-AR coupling proteins, such as G-proteins, or other beta-AR-linked proteins, such as G-protein receptor kinases and/or phosphatases. 2. The present study examines the physiological effects of in vitro transfer of constitutively activated G alpha s (G alpha s-Q227L) to both cultured vascular smooth muscle cells (VSMC) and whole aortic tissue of 6-month-old (adult) animals via a replication-deficient Herpes simplex virus (HSV) vector. These studies were conducted to provide a model for future examination of the role of G alpha s in the age-related decline in beta-AR-mediated vasorelaxation. 3. Gene transfer was confirmed by western blotting for specific proteins. Aortic tissue infected with HSV-G alpha s-Q227L had reduced phenylephrine-induced contraction and enhanced isoproterenol-stimulated vasorelaxation. Infection of cultured VSMC with HSV-G alpha s-Q227L increased both basal- and isoproterenol-stimulated cAMP accumulation, whereas forskolin-stimulated cAMP production was unchanged. 4. These results implicate G alpha s as a target for further investigation in age-related changes in vascular reactivity and support the use of viral-mediated gene transfer as an effective tool to study adrenergic signal transduction and physiology in vascular tissue.
Assuntos
AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Transferência de Genes , Músculo Liso Vascular/metabolismo , Vasodilatação/fisiologia , Animais , Aorta/metabolismo , Células Cultivadas , AMP Cíclico/genética , Proteínas de Ligação ao GTP/genética , Vetores Genéticos/genética , Masculino , Ratos , Ratos Endogâmicos F344 , Simplexvirus/genética , Vasodilatação/genéticaRESUMO
Beta-adrenergic-mediated vasorelaxation declines with maturation and aging. Available data suggest that impaired stimulatory G-protein function could explain this deficit. We have previously found a loss of cholera toxin (CT)-stimulated adenosine diphosphate (ADP) ribosylation with age in rat aortic membrane preparations, without evidence for loss of the stimulatory alpha subunit of G protein (Gsalpha) by immunoblotting. The purpose of this investigation was to determine if cholera toxin-mediated vasorelaxation was also impaired with age. Aortic ring segments from 6 weeks, 6 months, 12 months, and 24 months old male F-344 rats were used. Contraction to KCl and phenylephrine was assessed along with relaxation to cholera toxin (azide-free), isoproterenol and forskolin. There were no age-related changes to KCl or phenylephrine contraction. There was a significant decrease with age in relaxation to isoproterenol. This loss with age was significantly greater with KCl-preconstricted vessels than phenylephrine-preconstricted vessels. There were no age-related changes in the relaxation to forskolin. There was a significant decrease with age in the maximal relaxation to cholera toxin as well as a rightward shift in the dose-response curve. Cholera toxin-stimulated adenosine 3', 5'-cyclic phosphate (cAMP) levels were measured and there was no increase in cAMP levels surrounding the time period associated with relaxation induced by cholera toxin. These data suggest that different preconstricting agents markedly affect the age-related changes in beta-adrenergic-mediated vasorelaxation. Furthermore, they suggest that the mechanism of cholera toxin-mediated vasorelaxation may not be mediated through increases in cAMP concentration.
Assuntos
Envelhecimento/fisiologia , Aorta Torácica/efeitos dos fármacos , Toxina da Cólera/farmacologia , Vasodilatadores/farmacologia , Difosfato de Adenosina/metabolismo , Agonistas Adrenérgicos beta/administração & dosagem , Agonistas Adrenérgicos beta/farmacologia , Animais , Toxina da Cólera/administração & dosagem , Colforsina/administração & dosagem , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/fisiologia , Isoproterenol/administração & dosagem , Isoproterenol/farmacologia , Masculino , Fenilefrina/administração & dosagem , Fenilefrina/farmacologia , Cloreto de Potássio/administração & dosagem , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos F344 , Vasoconstritores/administração & dosagem , Vasoconstritores/farmacologia , Vasodilatadores/administração & dosagemRESUMO
The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.
Assuntos
Adenilil Ciclases/metabolismo , Receptores de Dopamina D2/metabolismo , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Humanos , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D4RESUMO
D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Neurônios/fisiologia , Receptores de Dopamina D2/fisiologia , Toxina Adenilato Ciclase , Animais , Colforsina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Vetores Genéticos , Camundongos , Neuroblastoma , Toxina Pertussis , Quimpirol/farmacologia , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Simplexvirus/genética , Transfecção , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The role of drug efficacy in agonist-induced desensitization was studied in C-6 glioma cells transfected with the monkey dopamine D1A (mD1A) receptor. Dopamine pretreatment for 2 hr produced greater than 80% loss of responsiveness in the stimulation of cAMP accumulation that was blocked by the D1 antagonist SCH23390. A series of full and partial D1 agonists from structurally dissimilar classes were then examined. Three full agonists (dihydrexidine, SKF82958, A77636) desensitized the receptor to the same extent as dopamine, whereas two other full agonists (dinapsoline and A68930) and all the partial agonists tested (SKF38393, pergolide and d-lysergic acid diethylamide tartrate) produced only partial desensitization (i.e., 50% that of dopamine). Whereas partial agonists (i.e., SKF38393, pergolide and d-lysergic acid diethylamide tartrate) caused no alteration in ligand-accessible mD1A receptors, four of the full agonists (dopamine, dihydrexidine, dinapsoline, A68930) caused a 30 to 40% reduction in receptor number. One full agonist, A77636, caused nearly an 80% decrease in receptor number, despite the fact that the degree of functional desensitization was similar to the other full agonists. The desensitization of the D1 receptor was homologous, not affecting beta-2 adrenergic receptors endogenous to C-6 cells. Neither incubation with cAMP analogs, nor inhibition of protein kinase A, affected dopamine-induced desensitization, suggesting a cAMP-independent mechanism in this cell line. Together, these data suggest that functional desensitization of the mD1A receptor expressed in C-6 glioma cells is a cAMP-independent mechanism, cannot be predicted reliably from agonist efficacy for stimulating adenylate cyclase and can occur in the absence of changes in receptor number.
Assuntos
Receptores de Dopamina D1/fisiologia , Animais , Benzazepinas/metabolismo , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Macaca mulatta , Transtornos Relacionados ao Uso de Substâncias/etiologiaRESUMO
The D2-like dopamine receptors couple to a variety of signal transduction pathways, including inhibition of adenylate cyclase, mitogenesis, and activation of potassium channels. Although these effects are mediated via pertussis toxin-sensitive G proteins, G(i/o), it is likely that some of these effects are influenced by the release of G protein betagamma subunits. Type II adenylate cyclase (ACII) is highly regulated by multiple biochemical stimuli, including protein kinase C, forskolin, G protein alpha subunits, and G protein betagamma subunits. The ability of betagamma subunits to activate this enzyme in the presence of activated alpha(s) has been particularly well characterized. Although stimulation by betagamma subunits has been described as conditional on the presence of activated alpha(s), betagamma subunits also potentiate ACII activity after activation of protein kinase C. We created stable cell lines expressing ACII and the D2L receptor, the D3 receptor, or the D4.4 receptor. Activation of D2L or D4.4 receptors, but not D3 receptors, potentiated beta-adrenergic receptor/Gs-stimulated activity of ACII, as measured by the intracellular accumulation of cAMP. Similarly, stimulation of D2L or D4.4 receptors potentiated phorbol ester-stimulated ACII activity in the absence of activated alpha(s), whereas stimulation of D3 receptors did not. The effect of D2-like receptor stimulation was blocked by pretreatment with pertussis toxin and by inhibition of protein kinase C. We propose that activation of both D2L and D4.4 dopamine receptors potentiated phorbol-12-myristate-13-acetate-stimulated ACII activity through the release of betagamma subunits from pertussis toxin-sensitive G proteins. In contrast, the lack of D3 receptor-mediated effects suggests that stimulation of D3 receptors does not result in an appreciable release of betagamma subunits.
Assuntos
Adenilil Ciclases/metabolismo , Receptores de Dopamina D2/fisiologia , Linhagem Celular , AMP Cíclico/metabolismo , Dopamina/farmacologia , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Isoproterenol/farmacologia , Proteína Quinase C/fisiologia , Quimpirol/farmacologia , Receptores de Dopamina D3 , Receptores de Dopamina D4 , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
In an effort to define further the active geometry of the beta-phenyldopamine pharmacophore of certain dopamine D1 agonists, the title compounds have been synthesized as conformationally restricted homologues of the potent benzophenanthridine dopamine D1 agonist dihydrexidine 4a. The dihydroxy secondary amine 5b was evaluated as a potential agonist, whereas the N-methyl compounds 5a and 5c were hypothesized to be antagonists. Surprisingly, none of the three compounds had high affinity for dopamine D1 or D2 receptors. A comparison of the low-energy conformations of these molecules shows that the pendant phenyl ring of 5b is twisted about 28 degrees relative to that of the corresponding ring of 4a. Further, the additional methylene used to expand the C ring of 5b projects toward the alpha face of the molecule, perhaps suggesting that steric protrusion in this region of the molecule is not tolerated. Finally, the phenethylamine fragment incorporated into these molecules deviates about 30 degrees from the antiperiplanar conformation postulated to be necessary for agonist activity. On the other hand, the potential antagonist molecules 5a and 5c were compared with the dopamine D1 antagonist SCH 39166 2. The conformations of the former two structures differ quite dramatically from that of 2. The most notable differences lie in the relative orientations of the pendant phenyl rings in the two series, as well as the fact that the ethylamine fragment in 2 approximates a gauche conformation, while the comparable orientation in 5a and 5c more nearly approaches an antiperiplanar conformation. These findings will be used to refine further the model of the dopamine D1 agonist receptor that we have previously developed.
Assuntos
Agonistas de Dopamina/síntese química , Fenantridinas/química , Fenantridinas/síntese química , Receptores de Dopamina D1/agonistas , Animais , Simulação por Computador , Corpo Estriado/metabolismo , Agonistas de Dopamina/química , Agonistas de Dopamina/farmacologia , Indicadores e Reagentes , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fenantridinas/farmacologia , Ratos , Receptores de Dopamina D1/metabolismo , Software , Espiperona/metabolismo , Relação Estrutura-AtividadeRESUMO
Prolonged stimulation of Gi-coupled receptors often sensitizes adenylate cyclase to subsequent activation by forskolin or Gs-coupled receptors. To identify mechanisms of heterologous sensitization, we characterized sensitization of cAMP accumulation that was induced by activation of recombinant dopamine D2 receptors expressed in C6 glioma and human embryonic kidney (HEK)293 cells. Pretreatment with dopamine or other agonists for 2 hr induced heterologous sensitization that was blocked by the D2 antagonist spiperone but not by the beta-adrenergic receptor antagonist propranolol. Sensitization was evident after 15 min of treatment with dopamine and persisted for at least 2 hr after washout. The EC50 value for sensitization by dopamine in HEK-D2L cells was 100 nM(r) approximately 80-fold higher than the IC50 value for dopamine inhibition of cAMP accumulation. The D2 receptor agonists quinpirole, 7-hydroxy-dipropylamin-otetralin, and pergolide also induced sensitization, whereas the high affinity ergot agonists bromocriptine and lisuride did not. Stimulation of either D2L or D2S receptors sensitized cAMP accumulation to similar extents, but stimulation of D3 receptors did not. In C6-D21 cells, sensitization of isoproterenol-stimulated activity was manifested as a > 100% increase in maximal response, with no change in potency. In contrast, the potency for forskolin-stimulated activity was increased 4-fold, with no apparent change in maximal response. Overnight treatment with pertussis toxin (25 ng/ml) had little effect on isoproterenol or forskolin activation of adenylate cyclase per se but prevented D2 receptor-mediated sensitization in both C6-D2L and HEK-D2L cells, indicating an involvement of one or more of the pertussis toxin-sensitive G proteins, Gi/Gzero. D2 receptor stimulation also sensitized type I and type II adenylate cyclases, each expressed in HEK293 cells together with D2L dopamine receptors. Rapid D2 receptor-mediated heterologous sensitization may be the result of enhanced interaction of G6 with adenylate cyclase and may represent a novel mechanism for modulation of neural activity by D2 receptors.
Assuntos
Adenilil Ciclases/fisiologia , Isoenzimas/fisiologia , Receptores de Dopamina D2/fisiologia , Adenilil Ciclases/metabolismo , Adenilil Ciclases/farmacologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Humanos , Isoproterenol/farmacologia , Cinética , Receptores de Dopamina D2/agonistas , Proteínas Recombinantes/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Sensibilidade e Especificidade , Espiperona/farmacologia , Estimulação Química , Tetra-Hidronaftalenos/farmacologiaRESUMO
Compute-aided conformational analysis was used to characterize the agonist pharmacophore for D1 dopamine receptor recognition and activation. Dihydrexidine (DHX), a high-affinity full agonist with limited conformational flexibility, served as a structural template that aided in determining a molecular geometry that would be common for other more flexible, biologically active agonists. The intrinsic activity of the drugs at D1 receptors was assessed by their ability to stimulate adenylate cyclase activity in rat striatal homogenates (the accepted measure of D1 receptor activation). In addition, affinity data on 12 agonists including six purported full agonists (dopamine, dihydrexidine, SKF89626, SKF82958, A70108, and A77636), as well as six less efficacious structural analogs, were obtained from D1 dopamine radioreceptor-binding assays. The active analog approach to pharmacophore building was applied as implemented in the SYBYL software package. Conformational analysis and molecular mechanics calculations were used to determine the lowest energy conformation of the active analogs (i.e., full agonists), as well as the conformations of each compound that displayed a common pharmacophoric geometry. It is hypothesized that DHX and other full agonists may share a D1 pharmacophore made up of two hydroxy groups, the nitrogen atom (ca. 7 A from the oxygen of m-hydroxyl) and the accessory ring system characterized by the angle between its plane and that of the catechol ring (except for dopamine and A77636). For all full agonists (DHX, SKF89626, SKF82958, A70108, A77636, and dopamine), the energy difference between the lowest energy conformer and those that displayed a common pharmacophore geometry was relatively small (< 5 kcal/mol). The pharmacophoric conformations of the full agonists were also used to infer the shape of the receptor binding site. Based on the union of the van der Waals density maps of the active analogs, the excluded receptor volume was calculated. Various inactive analogs (partial agonists with D1 K0.5 > 300 nM) subsequently were used to define the receptor essential volume (i.e., sterically intolerable receptor regions). These volumes, together with the pharmacophore results, were integrated into a three-dimensional model estimating the D1 receptor active site topography.
Assuntos
Agonistas de Dopamina/química , Agonistas de Dopamina/farmacologia , Receptores de Dopamina D1/química , Adenilil Ciclases/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Simulação por Computador , Dopamina/farmacologia , Agonistas de Dopamina/metabolismo , Ligantes , Conformação Molecular , Estrutura Molecular , Fenantridinas/química , Fenantridinas/farmacologia , Ratos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Software , Relação Estrutura-AtividadeRESUMO
The present work reports the synthesis and preliminary pharmacological characterization of 8,9-dihydroxy-2,3,7,11b-tetrahydro-1H-naph[1,2,3-de] isoquinoline (4, dinapsoline). This molecule was designed to conserve the essential elements contained in our D1 agonist pharmacophore model (i.e., position and orientation of the nitrogen, hydroxyls, and phenyl rings). It involved taking the backbone of dihydrexidine [3; (+/-)-trans-10, 11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a] phenanthridine], the first high-affinity full D1 agonist, and tethering the two phenyl rings of dihydrexidine through a methylene bridge and removing the C(7)-C(8) ethano bridge. Preliminary molecular modeling studies demonstrated that these modifications conserved the essential elements of the hypothesized pharmacopore. Dinapsoline 4 had almost identical affinity (KI = 5.9 nM) to 3 at rat striatal D1 receptors and had a shallow competition curve (nH = 0.66) that suggested agonist properties. Consistent with this, in both rat striatum and C-6-mD1 cells, dinapsoline 4 was a full agonist with an EC50 of ca. 30 nM in stimulating synthesis of cAMP via D1 receptors. The design and synthesis of dinapsoline 4 provide a powerful test of the model of the D1 pharmacophore we have developed and provide another chemical series that can be useful probes for the study of D1 receptors. An interesting property of 3 is that it also has relatively high D2 affinity (K0.5 = 50 nM) despite having an accessory phenyl ring usually though to convey D1 selectivity. Dinapsoline 4 was found to have even higher affinity for the D2 receptor (K0.5 = 31 nM) than 3. Because of the high affinity of 4 for D2 receptors, it and its analogs can be powerful tools for exploring the mechanisms of "functional selectivity" (i.e., that 3 is an agonist at some D2 receptors, but an antagonist at others). Together, these data suggest that 4 and its derivatives may be powerful tools in the study of dopamine receptor function and also have potential clinical utility in Parkinson's disease and other conditions where perturbation of dopamine receptors is useful.
Assuntos
Agonistas de Dopamina/farmacologia , Isoquinolinas/farmacologia , Naftóis/farmacologia , Adenilil Ciclases/metabolismo , Animais , Benzazepinas/metabolismo , Benzazepinas/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Corpo Estriado/metabolismo , AMP Cíclico/metabolismo , Agonistas de Dopamina/química , Agonistas de Dopamina/metabolismo , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Isoquinolinas/química , Macaca mulatta , Masculino , Naftóis/química , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Células Tumorais CultivadasRESUMO
A critical assumption in the unilateral 6-hydroxydopamine (6-OHDA) model is that interactions between the intact and denervated hemispheres do not influence the response to insult. The present study examined this issue by assessing the effects of unilateral substantia nigra 6-OHDA lesions in rats that previously had received corpus callosum transections, a treatment designed to minimize interhemispheric influences. Quantitative autoradiography in the caudate-putamen ipsilateral to the lesion revealed that corpus callosum transection did not alter the increase in D2-like receptors ([125I]-epidepride-labeled sites) that is induced by unilateral 6-OHDA lesion. There were no effects of either 6-OHDA lesion or transection on D1 receptor density ([125I]-SCH23982 autoradiography). As a functional endpoint, dopamine-stimulated cAMP efflux was measured in superfused striatal slices. In this paradigm, the net effect of dopamine (DA) represents a combination of D1 receptor-mediated stimulation and D2 receptor-mediated inhibition. 6-OHDA lesion increased cAMP efflux induced by exposure to 100 microM DA alone; corpus callosum transection did not alter this effect. An interaction between 6-OHDA lesion and transection status was revealed, however, by comparison of results obtained with DA alone vs. DA plus the D2 antagonist sulpiride (to block the D2 inhibitory effects of 100 microM DA). This comparison revealed two important effects of 6-OHDA lesion in rats with an intact corpus callosum: 1) a moderate decrease in dopamine D1 receptor-mediated stimulation; and 2) a dramatic decrease in the ability of D2 receptors to inhibit this stimulation. Corpus callosum transection prevented these effects of 6-OHDA. These results provide a biochemical demonstration of D1:D2 receptor uncoupling in unilateral 6-OHDA lesioned rats, and suggest that interhemispheric influences (e.g., contralateral cortico-striatal glutamatergic projections) may contribute to lesion-induced alterations in D1:D2 receptor interactions.
Assuntos
Corpo Caloso/fisiologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Substância Negra/fisiologia , Animais , Núcleo Caudado/metabolismo , AMP Cíclico/metabolismo , Denervação , Dopamina/metabolismo , Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Masculino , Oxidopamina/farmacologia , Putamen/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Sulpirida/farmacologiaRESUMO
The intrinsic activities of selected dopamine D1 receptor agonists were compared in three distinct molecular expression systems, C-6, Ltk, and GH4 cells transfected with primate D1A receptors. The influence of the cell expression system on intrinsic activity varied markedly among agonists. Dihydrexidine (DHX), a potent full agonist with dramatic antiparkinsonian actions, displayed intrinsic activity similar to dopamine in all three cell lines. In contrast, SKF82958 and SKF38393 (full and partial agonists, respectively, in rat striatum) had intrinsic activities equal to dopamine in GH4 cells that expressed a high density of D1 receptors, yet were of lower intrinsic activity in C-6 cells having 15-fold fewer receptors. The idea that spare receptors are one important determinant of observed intrinsic activity was explored directly by "receptor titration," in which ca. 90% of D1 receptors in Ltk cells were inactivated using EEDQ, an irreversible antagonist. Whereas EEDQ pretreatment decreased the potency of all agonists, it changed the intrinsic activity of some, but not all, drugs. A 40% decrease was seen with the partial agonist SKF38393, and, surprisingly, a 30% decrease was seen with the purported full agonist SKF82958. Conversely, the intrinsic activity of DHX and A68930 were unaffected by the EEDQ treatment. The data demonstrate that significant and biologically meaningful differences in intrinsic efficacy (e.g., DHX vs. SKF82958) may be obscured in test systems that have sufficient receptor reserve (e.g., the striatum). Such differences in intrinsic efficacy may be an important predictor of the clinical utility of D1 agonists.