Expression profiling of plac1 in murine cancer cell lines

AIM: Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2% to 42.5%. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization.

Delivery of Exogenous miR-124 to Glioblastoma Multiform Cells by Wharton's Jelly Mesenchymal Stem Cells Decreases Cell Proliferation and Migration, and Confers Chemosensitivity.

MicroRNAs (miRs) are potential therapeutic targets in glioblastoma multiforme (GBM), but the difficulties associated with their delivery to tumor target cells have hampered their widespread use. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers, including GBM and exert anti-tumor effects. In this study, it is shown that Wharton's jelly-MSCs (WJ-MSCs) have the ability to deliver exogenous miRs to GBM cells and the functional impact of this delivery is characterized. It is found that the labeled miR-124, as an example for miR of interest, can be successfully delivered with WJ-MSCs to U87 GBM cells via dependent or exosome-independent processes. It is demonstrated that the delivered exogenous miR-124 significantly decreases the luciferase activity of the target gene CDK6. In addition, the delivered miR-124 enhances the chemosensitivity of GBM cells to temozolomide and decreases the migration of GBM cells. These results suggest that the use of exogenous miRNA delivery with the derived exosomes from WJ-MSCs may provide a novel approach for miRNA replacement therapy in GBM cancers.

Stable silencing of IGF1R using Lentiviral-mediated shRNA in HEK293T cells.

Insulin-like growth factors are among the peptide mitogens that regulate cell proliferation and differentiation as well as mediator of antiapoptotic signals. The imbalance between the expression and activities of these molecules may lead to malignancy in cells. Evidences have suggested the insulin-like growth factor 1 receptor (IGF-1R) signaling pathway as a therapeutic target in the management and treatment of cancer. In this present study, we have generated silencing stable clones of HEK cells using six different pGIPZ (lentiviral vector) shRNAs targeted to human IGF-1R gene and a pGIPZ non-silencing shRNAmir lentiviral vector (as negative control). The recombinant lentiviral vectors were separately transduced into human embryonic kidney 293 T (HEK293T) cell lines. The knockdown of IGF-1R was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and the relative IGF-1R mRNA levels were expressed as a ratio of IGF-1R to ß-actin by REST software. The results showed significant reduction in the expression of IGF-1R mRNAs in cells transduced with all six pGIPZ-IGF-1R recombinant lentivirals compared to non-silencing negative control. No significant difference was observed among the six cassettes. Results indicated that recombinant lentiviral vectors provided an efficient and stable knockdown of IGF-1R providing useful tool for IGF-1R pathway studies.

Reduction of autophagy markers mediated protective effects of JNK inhibitor and bucladesine on memory deficit induced by Aß in rats.

Autophagy, the process of self-degradation of cellular components, has an important role in neurodegenerative diseases, such as Alzheimer's disease. In this study, we investigated the effects of SP600125 as c-Jun N-terminal kinase (JNK) inhibitor and bucladesine as a cyclic adenosine 3',5'-monophosphate (cAMP) analog on spatial memory and expression of autophagic factors in Aß-injected rats. Male Wistar rats were used. Rats were randomly allocated into five groups as following: amyloid beta (Aß)-only group, Aß + SP600125 (30 µg/1 µ/side, n = 7) and/or bucladesine (100 µM/1 µl/side, n = 7), and the normal control (vehicle only) group. The treatments were administered bilaterally to the CA1 sub-region of the hippocampus stereotaxically. Spatial reference memory was performed using Morris Water Maze 21 days later. The expression of authophagy markers (beclin1, Atg7, Atg12, and LC3 II/LC3 I) in the hippocampus was evaluated using western blotting. Compared to the vehicle group, Aß administration reduced spatial reference learning (P < 0.001) and memory (P < 0.01) and upregulated the expression of beclin1, Atg7, Atg12, and LC3 II/I (P < 0.0001). Compare to Aß-only group, the administration of SP600125 and/or bucladesine improved spatial reference learning (P < 0.001) and memory (P < 0.01). Compared to the Aß-only group, the treatment with SP600125 and/or bucladesine also reduced beclin1, Atg7, Atg12, and LC3 II/I (P < 0.0001) which was similar to amount of normal rats. In summary, it seems that the improvement of spatial memory by SP600125 and/or bucladesine in Aß-injected rats is in relation with normalizing of autophagy to the physiologic level, possibly through neuroprotection and/or neuroplasticity.

uPAR peptide antagonist alters regulation of MAP kinases and Bcl-2 family members in favor of apoptosis in MDA-MB-231 cell line.

Urokinase plasminogen activator receptor (uPAR) and its ligands play a major role in many tumors by mediating extracellular matrix degradation and signaling cascades leading to tumor growth, invasion and metastasis. Recently we introduced uPAR decapeptide antagonist with cytotoxic effect on MDA-MB-231 cell line. In this study we assessed the alteration in uPAR downstream signaling following treatment with the peptide antagonist. In this regard, extracellular-signal-regulated kinase (ERK) and p38 from mitogen-activated protein kinase family and Bcl-2, Bim and Bax from Bcl-2 protein family were investigated. Our data revealed that the peptide caused p38 activation and low ERK activation. On the other hand, the peptide induced down-regulation of Bcl-2 and up-regulation of Bim without Bax modulation. Changes in target protein expression/activation explain the apoptotic property of the peptide and highlight its potential to be used as a therapeutic agent in cancerous cells expressing high levels of uPAR.

Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function.

The vasopressin V2 receptor belongs to the large family of the G-protein coupled receptors and is responsible for the antidiuretic effect of the neurohypophyseal hormone arginine vasopressin (AVP). Based on bioinformatic studies it seems that Ala300 and Asp297 of the V2 vasopressin receptor (V2R) are involved in receptor binding. Ala300Glu mutation resulted in lower energy while Asp297Tyr mutation resulted in higher energy in AVP-V2R docked complex rather than the wild type. Therefore we hypothesized that the Ala300Glu mutation results in stronger and Asp297Tyr mutation leads to weaker ligand-receptor binding. Site directed mutagenesis of Asp297Tyr and Ala300Glu was performed using nested polymerase chain reaction. After restriction enzyme digestion, the inserts were ligated into the pcDNA3 vector and Escherichia coli XL1-Blue competent cells were transformed using commercial kit and electroporation methods. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using ESCORT™ IV transfection reagent, the adenylyl cyclase activity assay was performed for functional studies. The cell surface expression of V2R was analyzed by indirect ELISA method. Based on the obtained results, the Ala300Glu mutation of V2R led to reduced levels of cAMP production without a marked effect on the receptor expression and the receptor binding. Effect of Asp297Tyr mutation on cell surface expression of V2R was the same as the wild type receptor. Pretreatment with 1 nM vasopressin showed an increased level of Asp297Tyr mutant receptor internalization as compared to the wild type receptor, while the effect of 100 nM vasopressin was similar in the mutant and wild type receptors. These data suggest that alterations in Asp297 but not Ala300 would affect the hormone receptor binding.

Gabapentin Enhances Neurogenesis in E14 Rat Embryonic Neocortex Stem Cells.

Many anticonvulsant drugs have been studied for their non conventional therapeutic effects on neurodegerative diseases but merely a few demonstrated potential neurogenic characteristic. Gabapentin as a well-known mood stabilizer was studied for its potential capability to promote neurogenesis in embryonic rat cortical stem cells. Rat E14 cortical stem cells were exposed to gabapentin during differentiation for 7 days and subjected to immunocytochemistry. The phenotypic changes were evaluated in the ultimately survived and differentiated cells. Gabapentin (16 µg/ml) exposure significantly increased the number and percentage of MAP2 immunopositive neurons with no significant alterations in nestin or GFAP immunopositivity in neural or glial progenitors. The enhanced number of neurons by therapeutic doses of gabapentin via augmentation of the neuronal differentiation in neural stem cells may participate to the therapeutic properties of gabapentin in the treatment of mood disorder.

Optimization of transfection methods for Huh-7 and Vero cells: comparative study.

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI and Lipofectamine 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 +/- 2.36 and 73.9 +/- 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI was respectively 14.2 +/- 0.69 and 28 +/- 1.11% for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.

Optimization of transfection methods for Huh-7 and Vero cells: A comparative study.

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.

Cyclooxygenase 2 plays a role in Emdogain-induced proliferation.

BACKGROUND AND OBJECTIVE: Enamel matrix proteins are involved in the development and regeneration of root cementum and in its attachment to dentin; however, the mechanisms through which this occurs have yet to be elucidated. The present study was therefore carried out to evaluate the mitogenic and proliferative responses of human periodontal fibroblast (HPLF) cells to Emdogain (EMD), and the potential role of cyclooxygenase 2 (COX-2) in this process. MATERIAL AND METHODS: We investigated the effects of EMD on 5-bromo-2'-deoxyuridine (BrdU) incorporation, colchicine freezing of mitosis, XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and Trypan Blue dye exclusion, with or without celecoxibe, a selective cyclooxygenase-2 (COX-2) inhibitor; we also evaluated the expression of COX-2 mRNA and COX-2 protein in response to EMD. RESULTS: EMD significantly enhanced mitosis in, and proliferation of, human periodontal ligament fibroblasts in a dose-dependent manner; however, there was a small increase of DNA synthesis only in response to a high dose of EMD (200 µg/mL). EMD (100 and 200 µg/mL) elicited an increase in COX-2 expression (p ≤ 0.05). Celecoxibe (20 µm) diminished the EMD-induced mitosis and proliferation of HPLF cells (p ≤ 0.05). CONCLUSION: Celecoxibe hampered EMD-induced mitosis and proliferation, which, in association with EMD-increased COX-2 expression, indicates that COX-2 may be involved in the proliferative response of HPLF cells to EMD.

The expression of p38, ERK1 and Bax proteins has increased during the treatment of newly diagnosed acute promyelocytic leukemia with arsenic trioxide.

BACKGROUND: Promising reports exist regarding the use of arsenic trioxide (ATO) as first-line treatment in acute promyelocytic leukemia (APL). Although the in vitro effect of ATO is extensively studied, the in vivo mechanism(s) of ATO action is mostly unknown. PATIENTS AND METHODS: Newly diagnosed APL patients were involved and received ATO (0.15 mg.kg/day) for 28 days as induction followed by consolidation therapy. Bone marrow (BM) aspirates were obtained on days 0, 14 and 28 of treatment for further molecular studies. Clinical findings and white blood cell counts were recorded as well. RESULTS: Complete remission was observed in 17 (85%) patients with the median duration of 28 days (18-38) and cumulative dosage of median 280 mg (180-350). Hyperleukocytosis and APL differentiation syndrome (63%), gastrointestinal disorders (30%), liver enzyme elevation and night sweating (50%) were the most prevalent side-effects. The expression of Bax, ERK1 and p38 proteins and caspase-3 activity increased significantly in promyelocytes of BM aspirates at days 14 and 28 of induction therapy. CONCLUSION(S): These findings point toward the role of p38 and Bax in the induction of apoptosis, which was confirmed by increase in caspase-3 activity. However, the increase in ERK1 expression with regard to leukocytosis could translate to a proliferative/differentiation effect.

Primary WWOX phosphorylation and JNK activation during etoposide induces cytotoxicity in HEK293 cells.

BACKGROUND AND THE PURPOSE OF THE STUDY: Etoposide is an antineoplastic agent used in multiple cancers. It is known that etoposide induce cell death via interaction with topoisomerase II; however, the etopoisde cellular response is poorly understood. Upon etoposide induced DNA damage, many stress signaling pathways including JNK are activated. In response to DNA damage, it has been shown that WWOX, a recently introduced tumor suppressor, can be activated. In this study the activation of WWOX and JNK and their interaction following etoposide treatment were evaluated. MATERIALS AND METHODS: HEK293 cells treated with etoposide were lysed in a time course manner. The whole cell lysates were used to evaluate JNK and WWOX activation pattern using Phospho specific antibodies on western blots. The viability of cells treated with etoposide, JNK specific inhibitor and their combination was examined using MTT assay. RESULTS: Findings of this study indicate that WWOX and JNK are activated in a simultaneous way in response to DNA damage. Moreover, JNK inhibition enhances etoposide induced cytotoxicity in HEK293. CONCLUSION: Taken together, our results indicate that etoposide induces cytotoxicity and WWOX phosphorylation and the cytotoxicty is augmented by blocking JNK pathway.

The effect of full length and mature NAG-1 protein overexpression on cytotoxicity of celecoxib, tamoxifen and doxorubicin in HT1080.

BACKGROUND AND THE PURPOSE OF THE STUDY: Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) is involved in inflammation, apoptosis/survival and tumorigenesis as well as resistance to chemotherapy. NAG-1 protein is synthesized as pro-peptide, cleaved and secreted as mature protein. Regulation of NAG-1 is not completely discovered and increased level of NAG-1 has been reported in many cancers. The expression of NAG-1 in cancer cells could affect the progression of tumor growth. In addition the secretion of full length and mature forms of NAG-1 can influence cell proliferation in other cells. In this study the role of full length and mature forms of NAG-1 on viability of HT-1080 and MCF-7 cells were evaluated, and the cytotoxicity of celecoxib, indomethacin, tamoxifen and doxorubicin in HT1080 cells stably expressing NAG-1 were also tested. METHODS: Full length and mature NAG-1 was cloned from cDNA library of HCT116 cells and stably transfected in HT1080 cells. Cells were treated with different concentrations of indomethacin, celecoxib, tamoxifen and doxorubicin and viability was assessed by MTT assay. The effect of conditioned medium of NAG-1 expressing cells on proliferation of MCF-7 and HT1080 cells were also tested. RESULTS: The growth curves of HT1080 cells expressing full length and mature NAG-1 were not different. The viability of HT1080 cells expressing NAG-1 in the presence of indomethacin, doxorubicin and tamoxifen compared to untransfected cells was higher. The proliferation of HT1080 and MCF-7 cells were inhibited by conditioned medium of NAG-1 expressing cells in 24 and 48 hrs. MAJOR CONCLUSION: NAG-1 expression enhances drug resistance to tamoxifen, indomethacin and doxorubicin in HT1080. In addition, condition medium of NAG-1 expression cells inhibits proliferation in MCF-7 and HT1080 cells. Thus, NAG-1 expression can induce drug resistance in NAG-1 expressing cells and inhibition of viability in non expressing cells. Thus, NAG-1 is suggested as a marker for effective cancer chemotherapy and tumor progression.

Neurodegenerative, with expression ATF-2 by p38 in cortical neurons.

DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal cell death evoked by the DNA-damaging agent, camptothecin. The topisomerase-1 inhibitor, camptothecin, has been shown to induce cortical neuronal cell death in a reproducible and synchronistic manner. Primary embryonic neuronal cell culture cortical neurons were prepared. In the study, the survival % of neurons in camptothecin P38 group, after 6 hours (85%), 24 hours (64%) and 48 hours (50%), compared to camptothecin ATF-2 and P38 group after 4 hours (97 and 95%), have been significantly lower, and the expression % of neurons in camptothecin P38 group , after 6 hours (20%), 24 hours (40%) and 48 hours (55%), compared to camptothecin ATF-2 and P38 group after 4 hours (5 and 3%) have been significant lower (p<0.05). The expression % of neurons in camptothecin P38 group, after 24 hours (40%), compared to camptothecin ATF-2 group after 24hours (30%), have been significant lower (p<0.05). This study revealed that camptothecin induces P38 expression and P38 in embryonic cortical neurons to determine the importance of the P38 pathway in neuronal death following DNA damage, and P38 is induce phosphorylation of ATF-2 in embryonic cortical neurons following DNA damage.

ATP depletion as a consequence of hypoxia enhances tamoxifen antiproliferative effects in T47D breast carcinoma cells.

Tamoxifen causes a mitochondrial transmembrane potential dysfunction and ATP depletion, which may play a role in tamoxifen cytotoxicity. Administration of oligomycin-2 deoxy glucose (2DG) enhanced tamoxifen antiproliferative effects, which may be due to exacerbated ATP depletion following tamoxifen and oligomycin-2DG coadministration. Sodium nitroprusside (SNP) did not significantly change tamoxifen responsiveness at 0.1, 0.5, and 1 mM; however, 2 mM SNP hampered tamoxifen effects on cell proliferation and cell cycle. Oligomycin-2DG neither changed iNOS expression nor altered its attenuated expression due to tamoxifen exposure, suggesting that ATP depletion-mediated sensitivity to tamoxifen seems to be apart from iNOS.

Effect of chronic lithium administration on endothelium-dependent relaxation in rat aorta.

1. The effects of chronic lithium administration on the relaxant responses of rat thoracic aortic rings in the presence of indomethacin (a cyclo-oxygenase inhibitor) and/or NG-nitro-L-arginine (L-NOARG; a nitric oxide synthase inhibitor) to acetylcholine (ACh) or sodium nitroprusside were investigated in the present study. 2. Acetylcholine produced a concentration-dependent relaxation in vessels precontracted by phenylephrine (PE), while in lithium-treated rats the maximal relaxation was significantly increased. 3. Indomethacin (20 mumol/L) significantly potentiated the ACh-induced relaxation in lithium-treated and control rats. 4. NG-Nitro-L-arginine (1 mumol/L) decreased the ACh-induced relaxation in both control and lithium-treated rats. In contrast, indomethacin (20 mumol/L) reversed the inhibitory effect of L-NOARG. 5. Sodium nitroprusside produced similar concentration-dependent relaxations of vessels from both control and lithium-treated rats, which was not affected by indomethacin. In endothelium-denuded rings, indomethacin (20 mumol/L) caused a rightward shift in the concentration-contraction curve to PE. 6. These data support evidence for a possible increase in endothelium-dependent relaxation induced by ACh during long-term administration of lithium in rat aortic rings.

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells.

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.

Constitutive G(i2)-dependent activation of adenylyl cyclase type II by the 5-HT1A receptor. Inhibition by anxiolytic partial agonists.

The 5-HT1A receptor is implicated in depression and anxiety. This receptor couples to G(i) proteins to inhibit adenylyl cyclase (AC) activity but can stimulate AC in tissues (e.g. hippocampus) that express ACII. The role of ACII in receptor-mediated stimulation of cAMP formation was examined in HEK-293 cells transfected with the 5-HT1A receptor, which mediated inhibition of basal and G(s)-induced cAMP formation in the absence of ACII. In cells cotransfected with 5-HT1A receptor and ACII plasmids, 5-HT1A agonists induced a 1. 5-fold increase in cAMP level. Cotransfection of 5-HT1A receptor, ACII, and Galpha(i2), but not Galpha(i1), Galpha(i3), or Galpha(o), resulted in an agonist-independent 6-fold increase in the basal cAMP level, suggesting that G(i2) preferentially coupled the receptor to ACII. The 5-HT1B receptor also constitutively activated ACII. Constitutive activity of the 5-HT1A receptor was blocked by pertussis toxin and the Gbetagamma antagonist, betaCT, suggesting an important role for Gbetagamma-mediated activation of ACII. The Thr-149 --> Ala mutation in the second intracellular domain of the 5-HT1A receptor disrupted Gbetagamma-selective activation of ACII. Spontaneous 5-HT1A receptor activity was partially attenuated by 5-HT1A receptor partial agonists with anxiolytic activity (e.g. buspirone and flesinoxan) but was not altered by full agonists or antagonists. Thus, anxiolytic activity may involve inhibition of spontaneous 5-HT1A receptor activity.

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.

Distinct roles for Galphai2, Galphai3, and Gbeta gamma in modulation offorskolin- or Gs-mediated cAMP accumulation and calcium mobilization by dopamine D2S receptors.

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.