Uneven cellular expression of recombinant alpha2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition

Two populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (alpha2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Stimulation of the ATP, Mg-dependent protein phosphatase by p-nitrophenyl phosphate

The phosphorylase phosphatase activity of the ATP,Mg-dependent protein phosphatase is stimulated by p-nitrophenyl phosphate (pNPP). All the active forms of this type of enzyme show this property, which seems to be unrelated to any pNPP-hydrolyzing activity. The increase in activity is due to an increase in Vm, the Km being unchanged. The possibility that pNPP acts as a deinhibitor is excluded. pNPP acts as a competitive inhibitor on the phosphorylase phosphatase activity of the different polycation-stimulated protein phosphatases. Stimulation by pNPP can be used as a differential criterion in a specific assay of the active forms of the ATP,Mg-dependent phosphatase.     

Effects of the antipsychotic drug haloperidol on the somastostatinergic system in SH-SY5Y neuroblastoma cells

Antipsychotics are established drugs in schizophrenia treatment which, however, are not free of side effects. Lipid rafts are critical for normal brain function. Several G protein-coupled receptors, such as somatostatin (SRIF) receptors, have been shown to localize to lipid rafts. The aim of this study was to investigate whether haloperidol treatment affects the composition and functionality of lipid rafts in SH-SY5Y neuroblastoma cells. Haloperidol inhibited cholesterol biosynthesis, leading to a marked reduction in cell cholesterol content and to an accumulation of sterol intermediates, particularly cholesta-8,14-dien-3β-ol. These changes were accompanied by a loss of flotillin-1 and Fyn from the lipid rafts. We next studied the functionality of the SRIF receptor. Treatment with haloperidol reduced the inhibitory effect of SRIF on adenylyl cyclase (AC) activity. On the other side, haloperidol decreased basal AC activity but increased forskolin-stimulated AC activity. Addition of free cholesterol to the culture medium abrogated the effects of haloperidol on lipid raft composition and SRIF signaling whereas the AC response to forskolin remained elevated. The results show that haloperidol, by affecting cholesterol homeostasis, ultimately alters SRIF signaling and AC activity, which might have physiological consequences.     

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.     

Novel short isoforms of adenylyl cyclase as negative regulators of cAMP production

Here, we cloned a new family of four adenylyl cyclase (AC) splice variants from interleukin-1β (IL-1β)-transdifferentiated vascular smooth muscle cells (VSMCs) encoding short forms of AC8 that we have named “AC8E-H”. Using biosensor imaging and biochemical approaches, we showed that AC8E-H isoforms have no cyclase activity and act as dominant-negative regulators by forming heterodimers with other full-length ACs, impeding the traffic of functional units towards the plasma membrane. The existence of these dominant-negative isoforms may account for an unsuspected additional degree of cAMP signaling regulation. It also reconciles the induction of an AC in transdifferentiated VSMCs with the vasoprotective influence of cAMP. The generation of alternative splice variants of ACs may constitute a generalized strategy of adaptation to the cell's environment whose scope had so far been ignored in physiological and/or pathological contexts.     

Insulin desensitized beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase in SK-N-MC cells.

Receptor cross-talk is an emerging field which investigates cross-regulation between distinct classes of receptors. In the present work, we investigated the influence of activating the insulin receptor, a tyrosine kinase receptor, on beta-agonist activation of adenylyl cyclase, which is mediated by a G protein-linked receptor. Treatment of SK-N-MC neuroepithelioma cells with insulin generated a marked attenuation of beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase. This effect required nanomolar concentrations of insulin, occurred within minutes of exposure of these cells to insulin, and did not result from down-regulation of beta-adrenergic receptors. Insulin alone reduced the maximal isoproterenol-mediated stimulation of adenylyl cyclase by 50%, while the co-addition of the phosphatase inhibitor sodium vanadate increased the magnitude of insulin inhibition to 90%. Insulin provides an additional avenue for heterologous desensitization of beta-adrenergic receptors and their transmembranal signalling pathway.     

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.     

Cannabinoid Receptor Activation Differentially Regulates the Various Adenylyl Cyclase Isozymes

Abstract: Two cannabinoid receptors belonging to the superfamily of G protein-coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the G_i/o subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to α_s, α_i/o, and βγ subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active α_s, thyroid-stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.     

Beta-adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation

Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.     

Role of Sulfhydryl Groups in Functioning of Adenylyl Cyclase Signaling System

This review considers the literature data and author's own results on the role of SH-groups in functioning of the hormone-sensitive adenylyl cyclase system (ACS). It has been shown that the state of SH-groups affects crucially all main stages of the hormonal signal transudation: the ligand-binding properties of receptor and its coupling to G-proteins, interaction of G-proteins with adenylyl cyclase (AC) and its catalytic activity. It is noted that for the receptors, coupled to AC by a stimulating mode, the central aspect of the SH-dependent regulation of ACS is shifted to the receptor, while for the receptors coupled to AC by an inhibiting mode, it coincides with G-protein of the inhibiting type, which is sensitive to the SH-group state. Based on the performed comparative analysis of primary structures of signalling proteins—ACS components and of literature data, there are revealed the cysteine residues determining the functional activity of these proteins in the process of the hormonal signal transudation. The conclusion is made that the SH-group state (the ratio of free SH-groups and disulfide bonds) is the main factor determining the ACS reactivity to hormonal effects and selectivity of process of the signal transudation.     

Opioid tolerance/dependence in neuroblastoma x glioma (NG108-15) hybrid cells is associated with a reduction in spontaneous stimulatory receptor activity

Chronic opioid regulation of stimulatory receptor activity was investigated in neuroblastoma x glioma (NG108-15) hybrid cells stably transfected to express the human beta(2)-adrenoceptor (beta(2)-AR). Expressed beta(2)-ARs are functionally coupled to G proteins and display ligand-independent signalling activity, as demonstrated by the ability of an inverse agonist to attenuate basal adenylyl cyclase (AC) activity. Despite the relative increase in basal AC activity due to the development of tolerance/dependence, chronic morphine treatment was found to completely abolish spontaneous beta(2)-AR activity by reducing basal receptor/G protein precoupling. A similar chronic opioid effect was observed in transiently transfected COS-7 cells. These results indicate that during the state of opioid tolerance/dependence basal levels of AC activity are no longer under the control of spontaneously active stimulatory receptors.     

Adenine nucleotides decrease the apparent Km of endogenous natriuretic peptide receptors for GTP

Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent K(m) of both receptors for GTP but had little effect on the V(max). The EC(50) for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wild-type receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent K(m) for GTP.     

Tumor necrosis factor alpha stimulates adenylyl cyclase activity in human myometrial cells

Cytokines such as tumor necrosis factor alpha (TNFalpha) have been implicated in amniotic fluid infections and preterm and term labor. The underlying mechanisms are incompletely understood. In some smooth muscle cells, TNFalpha affects function of the beta-adrenergic/adenylyl cyclase pathway. The present study was performed to examine the effects of chronic TNFalpha exposure on adenylyl cyclase activity in cell cultures of human myometrium. Chronic TNFalpha exposure led to a dose- and time-dependent increase in basal-, GTP-, NaF-, and forskolin-stimulated adenylyl cyclase (AC) activity. The increase in AC activity was not mediated by changes in the expression of the heterotrimeric G proteins G(s)alpha or G(i)alpha as determined by immunoblotting. In addition, increases in AC activity occurred in the presence of indomethacin, indicating that these changes were not provoked by TNFalpha-induced changes in prostaglandin production. The present results suggest that TNFalpha-induced increases in AC activity in human myometrial cells obtained from the lower uterine segment occur at the level of G-protein/AC interaction or at the level of the AC enzyme itself.     

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell. The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector. Unlike the baker yeast Saccharomyces cerevisiae, S. pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides. The optimal pH for the S. pombe adenylyl cyclase activity is 6.0. The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.     

Regulation of adenylyl cyclase signaling system by insulin, biogenic amines and glucagon at their separate and combined action in muscle membranes of mollusc Anodonta cygnea

In the smooth muscles of mollusc Anodonta cygnea the regulatory action of hormones on adenylyl cyclase signaling system (ACSS) are realized through the receptors of serpentine type (biogenic amines, isoproterenol, glucagon) and receptor tyrosine kinase (insulin) type. Intracellular mechanisms of their interaction are interconnected. Application of hormones, their antagonists and pertussis toxin in combination with insulin and biogenic amines or glucagon on adenylyl cyclase (AC) activity allows revealing the possible sites of cross-linking in the mechanisms of their action. Combined influence of insulin and serotonin or glucagon leads to decreased stimulation of adenylyl cyclase (AC) by these hormones, whereas combined application of insulin and isoproterenol suppresses AC-stimulating effect of insulin, but AC-inhibiting effect of isoproterenol is maintained in the presence and absence of non-hydrolysable analog of GTP—guanylyl imido diphosphate (GIDP). The specific blockage of AC-stimulating effect of serotonin by cyproheptadine—antagonist of serotonin receptors, did not change AC stimulation by insulin. Beta-adrenoblockers (propranolol and alprenolol) prevent inhibition of AC activity by isoproterenol, but did not change AC stimulation by insulin. Pertussis toxin blocked AC-inhibiting effect of isoproterenol and weakened AC-stimulating action of insulin. Thus, in the muscles of Anodonta cygnea negative interaction between ACS have been revealed, which are realized under combined influence of insulin and serotonin or glucagon, most probably, at the level of receptor of serpentine type (serotonin, glucagon), whereas under action of insulin and isoproterenol at the level of G_i protein and AC interaction.     

Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D₂ receptors. [³H]-Sulpiride binding to 7315c cell homogenates was specific and saturable, and K _i values for compounds to compete for these sites were highly correlated with values for the same compounds at D₂ receptors in brain. Dopamine maximally inhibited ∼65% of forskolin-stimulated cyclase activity in cell membranes. Some D₂ agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that G_i1, G_i2, G_i3, G_o, G_q, and G_s are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C-terminal regions of Gα_i1 and Gα_i2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C-terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D₂ receptor regulates cyclase inhibition predominantly via G_i1 and/or G_i2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D₂ receptors in the absence of other dopamine receptor subtypes.     

Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Increases Functional Coupling Between a G Protein (GS) and Adenylyl Cyclase

Abstract: It has been reported that antidepressant treatment in rats results in a significant increase of G_s-mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down-regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl-5′-imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21-day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant-induced β-receptor desensitization occurred earlier than the enhancement of Gpp(NH)p-activated adenylyl cyclase, and extensive desensitization of β receptors by isoproterenol treatment did not enhance the Gpp(NH)p-stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p-stimulated adenylyl cyclase activity is not correlated with desensitization of β-adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially G_s) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (α_s, α_i, α_o, and β) nor their association with the plasma membrane was changed after antidepressant treatment. Thus, these results are consistent with the hypothesis that chronic antidepressant treatment acts directly at the postsynaptic membrane to increase the coupling between G_s and adenylyl cyclase.