Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes.

Previously we demonstrated in a cell-free ovarian follicular plasma membrane model that agonist-dependent desensitization of the luteinizing hormone/choriogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addition of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selectively inhibited by synthetic N-terminal ARF6 peptides. We therefore sought direct evidence that activation of the LH/CG R promotes activation of ARF1 and/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyzed ADP-ribosylation of G alpha(s), results show that LH/CG R activation stimulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N-terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated ARF activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6. These results suggest that ARF6 is the predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R does not appear to signal through the C-terminal regions of G alpha(i) or G alpha(q) or through the second or third intracellular loops or the N terminus of the cytoplasmic tail of the LH/CG R. Although exogenous recombinant ARNO promotes only a small increase in ARF6 activation in the presence of activated LH/CG R, hCG-stimulated ARF6 activation is reduced to basal levels by catalytically inactive ARF nucleotide binding-site opener. These results provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.

Chronic treatment of C6 glioma cells with antidepressant drugs results in a redistribution of Gsalpha.

Previous studies have demonstrated that chronic treatment of C6 glioma cells with the antidepressants desipramine and fluoxetine increases the Triton X-100 solubility of the G protein Gsalpha (Toki et al., 1999). The antidepressants also caused a 50% decrease in the amount of Gsalpha localized to caveolae-enriched membrane domains. In this study, laser scanning confocal microscopy reveals that Gsalpha is localized to the plasma membrane as well as the cytosol in both treated and control cells. However, striking differences are seen in the distribution of Gsalpha in the long cellular processes after chronic treatment with these antidepressant drugs. Control cells display Gsalpha along the entire process with an especially high concentration of that G protein at the distal ends. Desipramine- or fluoxetine-treated cells show a more centralized clustering of Gsalpha in the Golgi region of the cell and a drastic reduction of Gsalpha in the cellular processes. There is no change in the distribution of Goalpha after desipramine treatment and the antipsychotic drug chlorpromazine does not alter Gsalpha. These results suggest that antidepressant-induced changes in the association of Gsalpha with the plasma membrane may translate into altered cellular localization of this signal transducing protein. Thus, modification of the coupling between Gs-coupled receptors and adenylyl cyclase may underlie both antidepressant therapy and depressive illnesses. This report also suggests that modification of the membrane domain occupied by Gsalpha might represent a mechanism for chronic antidepressant effects.

Differences in Ca(2+) signaling underlie age-specific effects of secretagogues on colonic Cl(-) transport.

Taurodeoxycholic acid (TDC) stimulates Cl(-) transport in adult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates that stimuli like neurotensin (NT) are also age specific and identifies the age-dependent signaling step. Bile acid actions are segment and bile acid specific. Thus although TDC and taurochenodeoxycholate stimulate Cl(-) transport in AD distal but not proximal colon, taurocholate has no effect in either segment. TDC increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in AD, but not in WN and NB, colonocytes. In AD cells, TDC (5 min) action on Cl(-) transport needs intra- but not extracellular Ca(2+). NT, histamine, and bethanechol increase Cl(-) transport and [Ca(2+)](i) in AD, but not WN, distal colonocytes. However, A-23187 increased [Ca(2+)](i) and Cl(-) transport in all age groups, suggesting that Ca(2+)-sensitive Cl(-) transport is present from birth. Study of the proximal steps in Ca(2+) signaling revealed that NT, but not TDC, activates a GTP-binding protein, Galpha(q), in AD and WN cells. In addition, although WN and AD colonocytes had similar levels of phosphatidylinositol 4,5-bisphosphate, NT and TDC increased 1,4,5-inositol trisphosphate content only in AD cells. Nonresponsiveness of WN cells to Ca(2+)-dependent stimuli, therefore, is due to the absence of measurable phospholipase C activity. Thus delays in Ca(2+) signaling afford a crucial protective mechanism to meet the changing demands of the developing colon.

Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the beta-adrenergic receptor: a potential mechanism of G protein activation.

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.

Bacterial peptidoglycan binds to tubulin.

A search for cellular binding proteins for peptidoglycan (PGN), a CD14- and TLR2-dependent macrophage activator from Gram-positive bacteria, using PGN-affinity chromatography and N-terminal micro-sequencing, revealed that tubulin was a major PGN-binding protein in mouse macrophages. Tubulin also co-eluted with PGN from anti-PGN vancomycin affinity column and bound to PGN coupled to agarose. Tubulin-PGN binding was preferential under the conditions that promote tubulin polymerization, required macromolecular PGN, was competitively inhibited by soluble PGN and tubulin, did not require microtubule-associated proteins, and had an affinity of 100-150 nM. By contrast, binding of tubulin to lipopolysaccharide (LPS) had 2-3 times lower affinity, faster kinetics of binding, and showed positive cooperativity. PGN enhanced tubulin polymerization in the presence of 4 M glycerol, but in the absence of glycerol, both PGN and LPS decreased microtubule polymerization. These results indicate that tubulin is a major PGN-binding protein and that PGN modulates tubulin polymerization.

Muscarinic receptor activation promotes the membrane association of tubulin for the regulation of Gq-mediated phospholipase Cbeta(1) signaling.

The microtubule protein tubulin regulates adenylyl cyclase and phospholipase Cbeta(1) (PLCbeta(1)) signaling via transactivation of the G-protein subunits Galphas, Galphai1, and Galphaq. Because most tubulin is not membrane associated, this study investigates whether tubulin translocates to the membrane in response to an agonist so that it might regulate G-protein signaling. This was studied in SK-N-SH neuroblastoma cells, which possess a muscarinic receptor-regulated PLCbeta(1)-signaling pathway. Tubulin, at nanomolar concentrations, transactivated Galphaq by the direct transfer of a GTP analog and potentiated carbachol-activated PLCbeta(1). A specific and time-dependent association of tubulin with plasma membranes was observed when SK-N-SH cells were treated with carbachol. The same phenomenon was observed with membranes from Sf9 cells, expressing a recombinant PLCbeta(1) cascade. The time course of this event was concordant both with transactivation of Galphaq by the direct transfer of [(32)P]P(3)(4-azidoanilido)-P(1)-5'-GTP from tubulin as well as with the activation of PLCbeta(1). In SK-N-SH cells, carbachol induced a rapid and transient translocation of tubulin to the plasma membrane, microtubule reorganization, and a change in cell shape as demonstrated by confocal immunofluorescence microscopy. These observations presented a spatial and temporal resolution of the sequence of events underlying receptor-evoked involvement of tubulin in G-protein-mediated signaling. It is suggested that G-protein-coupled receptors might modulate cytoskeletal dynamics, intracellular traffic, and cellular architecture.

Treatment of C6 glioma cells and rats with antidepressant drugs increases the detergent extraction of G(s alpha) from plasma membrane.

Results from previous studies suggested that chronic treatment of rats or C6 glioma cells with antidepressants augments the coupling between Gs and adenylyl cyclase. As these effects on C6 glioma cells are seen in the absence of presynaptic input, several antidepressant drugs may have a direct "postsynaptic" effect on their target cells. It was hypothesized that the target of antidepressant action was some membrane protein that may regulate coupling between G proteins and adenylyl cyclase. To test this, C6 glioma cells were treated with amitriptyline, desipramine, iprindole, or fluoxetine for 3 days. Chlorpromazine served as a control for these treatments. Membrane proteins were extracted sequentially with Triton X-100 and Triton X-114 from C6 glioma cells. Triton X-100 extracted more G(s alpha) in membranes prepared from antidepressant-treated C6 glioma cells than from control groups. In addition, cell fractionation studies revealed that the amount of G(s alpha) in caveolin-enriched domains was reduced after antidepressant treatment and that adenylyl cyclase comigrated with G(s alpha) in the gradients. These data suggest that some postsynaptic component that increases availability of Gs to activate effector molecules, such as adenylyl cyclase, might be a target of antidepressant treatment.

Stimulation of cAMP synthesis by Gi-coupled receptors upon ablation of distinct Galphai protein expression. Gi subtype specificity of the 5-HT1A receptor.

The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level.

G protein alpha subunits activate tubulin GTPase and modulate microtubule polymerization dynamics.

G proteins serve many functions involving the transfer of signals from cell surface receptors to intracellular effector molecules. Considerable evidence suggests that there is an interaction between G proteins and the cytoskeleton. In this report, G protein alpha subunits Gi1alpha, Gsalpha, and Goalpha are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. Gialpha inhibited polymerization of tubulin-GTP into microtubules by 80-90% in the absence of exogenous GTP. Addition of exogenous GTP, but not guanylylimidodiphosphate, which is resistant to hydrolysis, overcame the inhibition. Analysis of the dynamics of individual microtubules by video microscopy demonstrated that Gi1alpha increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, Galpha may play a role in modulating microtubule dynamic instability, providing a mechanism for the modification of the cytoskeleton by extracellular signals.

Expression of G protein alpha subunits in normal rat colon and in azoxymethane-induced colonic neoplasms.

BACKGROUND & AIMS: Heterotrimeric G proteins are important in growth-regulating signal transduction. The aim of this study was to characterize the relative expression of G protein alpha subunits in rat colonocytes, colonocyte antipodal plasma membranes, and colonic neoplasms. METHODS: Antipodal plasma membranes were prepared from isolated colonocytes. Azoxymethane was administered to rats to induce colonic neoplasms. K-ras mutations in the neoplasms were determined by oligonucleotide hybridization and confirmed by primer mediated-restriction fragment length polymorphism. Colonocyte and tumor homogenates or membranes were probed for Galpha subunits by Western blotting with isoform-specific antibodies. RESULTS: The expressions of Galphai2, alphai3, and alphaq/11 were significantly enriched in the basolateral compared with brush border fraction of colonic antipodal plasma membranes. In neoplasms without K-ras mutations, the expression of Galphai2 increased 4-fold, Galphas(long) increased 2.5-fold, and Galphai3 increased 1.5-2-fold. Expression did not differ among tumor grades. K-ras mutations were associated with lowered expression of G proteins, especially Galphao. CONCLUSIONS: In colonocytes, Galpha subunits are localized primarily in basolateral plasma membranes. The increased expressions of Galphai2 and, to a lesser degree, Galphai3 and Galphas(long) in tumors was independent of tumor grade but was modulated by the presence of K-ras mutations.

Luteinizing hormone/choriogonadotropin receptor-mediated activation of heterotrimeric guanine nucleotide binding proteins in ovarian follicular membranes.

The LH/CG receptor signals to adenylyl cyclase via the stimulatory heterotrimeric GTP binding regulatory protein, Gs, and to phospholipase C and potentially to other effectors, such as ion channels, via a G protein or proteins that have not been identified in gonadal cells. To identify G proteins activated in a physiological membrane environment upon LH/CG receptor activation, we used the ability of activated G proteins to bind GTP and incubated ovarian follicular membranes with the photoaffinity GTP analog, P3-(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). Results showed that human CG (hCG) stimulated the binding of [32P]AAGTP to a 45-kDa protein(s) in follicular membranes that comigrated with immunoreactive G alphas, G alphaq/11, and G alpha13. When G alpha proteins were immunoprecipitated from Triton X-100 solubilized membrane extracts after photoaffinity labeling with [32P]AAGTP, a time-dependent increase in hCG-dependent [32P]AAGTP binding to G alphas, G alphaq/11, and G alphai was detected. hCG-dependent [32P]AAGTP binding to G alpha13 was also detected. These results demonstrate that agonist-dependent LH/CG receptor activation promotes the activation of Gs, Gi, Gq/11, and G13 in porcine ovarian follicular membranes. These results further suggest that G alphas remains coupled to the agonist-bound LH/CG receptor during at least the initial 10 min after agonist-dependent receptor activation.

Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460.

Gastrin-releasing peptide (GRP) causes multiple effects in humans by activating a specific heptaspanning receptor. Within the gastrointestinal tract, GRP receptors (GRP-R) are not normally expressed by mucosal epithelial cells except for those lining the gastric antrum. In contrast, recent studies have shown that up to 40% of resected colon cancers aberrantly express this receptor. This is important because the GRP-R can cause the proliferation of many, but not all, tissues in which it is expressed. Since GRP and other agonists are not known to exist in the colonic lumen, it has not been clear how or even if GRP-R expression in colon cancer contributes to cell proliferation. To evaluate the functional consequence of GRP-R expression on colonic epithelium, we transfected the recently isolated nonmalignant human colon epithelial cell line NCM460 with the cDNA for this receptor. All NCM460 cell lines expressing varying numbers of GRP-R bound selected agonists and antagonists indistinguishably from receptors expressed by other human tissues. Furthermore GRP-R-expressing transfected cell lines, but not wild-type NCM460 cells, proliferated independently of serum or other growth factors. Further evaluation revealed that GRP-R in these cells tonically stimulated G alpha q/11, resulting in increased phospholipase C activation. Since transfected cells do not secrete GRP, nor is their growth influenced by exposure to receptor-specific antagonists, these data indicate that GRP-R ectopically expressed by NCM460 cells are constitutively active. This report provides the first evidence of mutation-independent heptaspanning receptor constitutive activation resulting in cell proliferation, and identifies a potential mechanism whereby the GRP-R may act as an oncogene in human colon cancer.

Luteinizing hormone/choriogonadotropin-dependent, cholera toxin-catalyzed adenosine 5'-diphosphate (ADP)-ribosylation of the long and short forms of Gs alpha and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha*.

Although it is well established that activated LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, Gs) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and Gs or any other G protein(s). We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G alpha proteins were associated with the LH/CG receptor in ovarian follicular membranes. Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G alpha protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by [32P]NAD. Results demonstrated the time- and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time- and hCG-dependent manner. Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45- and 48/50 kDa proteins as the short and long forms of Gs alpha and the 40-kDa protein as Gi alpha. A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of Gs alpha and Gi. These results suggest that a portion of Gi along with the long and short forms of Gs alpha are associated physically with the LH/CG receptor in ovarian follicular membranes.

Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase Cbeta1 signaling.

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein alpha subunits, Galphas or Galphai1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Galpha conformation. To study the possibility of tubulin involvement in Galphaq-mediated phospholipase Cbeta1 (PLCbeta1) signaling, the m1 muscarinic receptor, Galphaq, and PLCbeta1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCbeta1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCbeta1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Galphaq and PLCbeta1, accompanied by guanine nucleotide transfer from tubulin to Galphaq, is suggested as a mechanism for the enzyme activation. The PLCbeta1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCbeta1 by tubulin, since high tubulin concentrations might prevent the access of PLCbeta1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Galphaq for tubulin. Involvement of tubulin in the articulation between PLCbeta1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.

Transgenic mice overexpressing the beta 1-adrenergic receptor in adipose tissue are resistant to obesity.

The ratio of alpha- to beta-receptors is thought to regulate the lipolytic index of adipose depots. To determine whether increasing the activity of the beta 1-adrenergic receptor (AR) in adipose tissue would affect the lipolytic rate or the development of this tissue, we used the enhancer-promoter region of the adipocyte lipid-binding protein (aP2) gene to direct expression of the human beta 1 AR cDNA to adipose tissue. Expression of the transgene was seen only in brown and white adipose tissue. Adipocytes from transgenic mice were more responsive to beta AR agonists than were adipocytes from nontransgenic mice, both in terms of cAMP production and lipolytic rates. Transgenic animals were partially resistant to diet-induced obesity. They had smaller adipose tissue depots than their nontransgenic littermates, reflecting decreased lipid accumulation in their adipocytes. In addition to increasing the lipolytic rate, overexpression of the beta 1 AR induced the abundant appearance of brown fat cells in subcutaneous white adipose tissue. These results demonstrate that the beta 1 AR is involved in both stimulation of lipolysis and the proliferation of brown fat cells in the context of the whole organism. Moreover, it appears that it is the overall beta AR activity, rather than the particular subtype, that controls these phenomena.

G protein beta1gamma2 subunits promote microtubule assembly.

alpha and betagamma subunits of G proteins are thought to transduce signals from cell surface receptors to intracellular effector molecules. Galpha and Gbetagamma have also been implicated in cell growth and differentiation, perhaps due to their association with cytoskeletal components. In this report Gbetagamma is shown to modulate the cytoskeleton by regulation of microtubule assembly. Specificity among betagamma species exists, as beta1gamma2 stimulates microtubule assembly, and beta1gamma1 is without any effect. Furthermore, a mutant beta1gamma2, beta1gamma2(C68S), which does not undergo prenylation and subsequent carboxyl-terminal processing on the gamma subunit, does not stimulate the formation of microtubules. beta immunoreactivity was detected exclusively in the microtubule fraction after assembly in the presence of beta1gamma2, suggesting a preferential association with microtubules rather than soluble tubulin. Crude microtubule fractions from ovine brain contain Gbetagamma, and electron microscopy reveals a specific association with microtubules. The decoration of microtubules by Gbetagamma appears to be strikingly similar to the periodic pattern observed for microtubule-associated proteins, suggesting a similar site of activation of microtubule assembly by both agents. It is suggested that reformation of the cytoskeleton represents an additional cellular process mediated by Gbetagamma.

Synaptic membrane G proteins are complexed with tubulin in situ.

The G proteins G S and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express G s alpha but not G i alpha 1, responded to the addition of tubulin with a stable activation of adenyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both G s and G i 1, responded to exogenous tubulin with a stable inhibition of adenyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and G i 1 and G s from detergent extracts of these membranes. Nearly 90% of the G s alpha from Triton X-114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein-cytoskeleton link that participates in the modulation of cellular signal transduction.

G protein-mediated signal transduction as a target of antidepressant and antibipolar drug action: evidence from model systems.

While the molecular locus of antidepressant and antibipolar drug action has not yet been established, it has become increasingly likely that the targets of such drugs lie distal to neurotransmitter receptors along the signal transduction pathway. These targets are likely to involve G protein-mediated signal transduction systems such as adenylyl cyclase and phospholipase C. This article will demonstrate model systems that have been developed in an attempt to determine how and where these drugs work. It appears that these model systems not only represent good experimental paradigms for studying the mechanisms of antibipolar and antimanic drugs, but are useful as simplified, yet realistic venues in which to test the speed and efficacy of suspected therapeutic agents.

Chronic antidepressant treatment facilitates G protein activation of adenylyl cyclase without altering G protein content.

It has been suggested that the molecular basis of antidepressant action involves postreceptor components. Results from our studies have suggested that a G protein (Gs) is one of those targets and that chronic antidepressant treatment facilitates the activation of adenylyl cyclase by Gs alpha. This report represents an attempt to define which aspects of G protein function are altered by chronic antidepressant treatment. Rats were treated for 21 days with amitriptyline, desipramine, ABT 200 (a pyrollidine with putative antidepressant effects) or electroconvulsive shock, and membranes were prepared from the cerebral cortexes. Each of these treatments caused an increase in membrane adenylyl cyclase assayed in the presence of guanyl-5'-imidodiphosphate (> or = 1 microM). Results of acute antidepressant treatments were no different than those of control treatment. Chronic treatment with amphetamine, which inhibits neurotransmitter reuptake without displaying antidepressant effect, was also ineffective in increasing Gs alpha stimulation of adenylyl cyclase. Chronic antidepressant treatment did not change the content of G protein, as no change at the level of Gs alpha, Gi alpha, Go alpha or G beta protein was detected by immunoblotting. Although there was no change in the amount of G proteins, antidepressant treatment increased the number of active Gs alpha/adenylyl cyclase complexes immunoprecipitated by an anti-Gs alpha antibody. It is suggested that chronic antidepressant treatment alters certain membrane components such that a greater proportion of Gs alpha is activated, Gs alpha enjoys a more fruitful interaction with adenylyl cyclase, or both.