Side-by-side comparison of the effects of Gq- and Gi-DREADD-mediated astrocyte modulation on intracellular calcium dynamics and synaptic plasticity in the hippocampal CA1.

Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca2+. In addition, Ca2+-dependent and/or Ca2+-independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca2+ signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca2+ dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca2+ events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca2+-dependent and Ca2+-independent mechanisms, respectively.

Neuronal-driven glioma growth requires Gαi1 and Gαi3.

Neuroligin-3 (NLGN3) is necessary and sufficient to promote glioma cell growth. The recruitment of Gαi1/3 to the ligand-activated receptor tyrosine kinases (RTKs) is essential for mediating oncogenic signaling. Methods: Various genetic strategies were utilized to examine the requirement of Gαi1/3 in NLGN3-driven glioma cell growth. Results: NLGN3-induced Akt-mTORC1 and Erk activation was inhibited by decreasing Gαi1/3 expression. In contrast ectopic Gαi1/3 overexpression enhanced NLGN3-induced signaling. In glioma cells, NLGN3-induced cell growth, proliferation and migration were attenuated by Gαi1/3 depletion with shRNA, but facilitated with Gαi1/3 overexpression. Significantly, Gαi1/3 silencing inhibited orthotopic growth of patient-derived glioma xenografts in mouse brain, whereas forced Gαi1/3-overexpression in primary glioma xenografts significantly enhanced growth. The growth of brain-metastatic human lung cancer cells in mouse brain was largely inhibited with Gαi1/3 silencing. It was however expedited with ectopic Gαi1/3 overexpression. In human glioma Gαi3 upregulation was detected, correlating with poor prognosis. Conclusion: Gαi1/3 mediation of NLGN3-induced signaling is essential for neuronal-driven glioma growth.

Astrocytes in the Ventromedial Hypothalamus Involve Chronic Stress-Induced Anxiety and Bone Loss in Mice.

Chronic stress is one of the main risk factors of bone loss. While the neurons and neural circuits of the ventromedial hypothalamus (VMH) mediate bone loss induced by chronic stress, the detailed intrinsic mechanisms within the VMH nucleus still need to be explored. Astrocytes in brain regions play important roles in the regulation of metabolism and anxiety-like behavior through interactions with surrounding neurons. However, whether astrocytes in the VMH affect neuronal activity and therefore regulate chronic stress-induced anxiety and bone loss remain elusive. In this study, we found that VMH astrocytes were activated during chronic stress-induced anxiety and bone loss. Pharmacogenetic activation of the Gi and Gq pathways in VMH astrocytes reduced and increased the levels of anxiety and bone loss, respectively. Furthermore, activation of VMH astrocytes by optogenetics induced depolarization in neighboring steroidogenic factor-1 (SF-1) neurons, which was diminished by administration of N-methyl-D-aspartic acid (NMDA) receptor blocker but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker. These results suggest that there may be a functional "glial-neuron microcircuit" in VMH nuclei that mediates anxiety and bone loss induced by chronic stress. This study not only advances our understanding of glial cell function but also provides a potential intervention target for chronic stress-induced anxiety and bone loss therapy.

PAK Membrane Translocation and Phosphorylation Regulate Platelet Aggregation Downstream of Gi and G12/13 Pathways.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.

Increased Gi protein signaling potentiates the negative chronotropic effect of adenosine in the SHR right atrium.

Hypertension is a risk factor for cardiovascular diseases, which have been associated with dysfunction of sympathetic and purinergic neurotransmission. Therefore, herein, we evaluated whether modifications of adenosine receptor signaling may contribute to the cardiac dysfunction observed in hypertension. Isolated right atria from spontaneously hypertensive (SHR) or normotensive Wistar rats (NWR) were used to investigate the influence of adenosine receptor signaling cascade in the cardiac chronotropism. Our results showed that adenosine, the endogenous agonist of adenosine receptors, and CPA, a selective agonist of A1 receptor, decreased the atrial chronotropism of NWR and SHR in a concentration- and time-dependent manner, culminating in cardiac arrest (0 bpm). Interestingly, a 3-fold lower concentration of adenosine was required to induce the negative chronotropic effect in SHR atria. Pre-incubation of tissues from both strains with DPCPX, a selective A1 receptor antagonist, inhibited the negative chronotropic effect of CPA, while simultaneous inhibition of A2 and A3 receptors, with ZM241385 and MRS1523, did not change the adenosine chronotropic effects. Moreover, 1 µg/ml pertussis toxin, which inactivates the Gαi protein subunit, reduced by 80% the negative chronotropic effects of adenosine in the NWR atrium, with minor effects in SHR tissue. These data indicate that the negative chronotropic effect of adenosine in right atrium depends exclusively on the activation of A1 receptors. Moreover, the distinct responsiveness of NWR and SHR atria to pertussis toxin reveals that the enhanced negative chronotropic response of SHR right atrium is probably due to an increased activity of Gαi protein-mediated.

GABABR-Induced EGFR Transactivation Promotes Migration of Human Prostate Cancer Cells.

G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) act in concert to regulate cell growth, proliferation, survival, and migration. Metabotropic GABAB receptor (GABABR) is the GPCR for the main inhibitory neurotransmitter GABA in the central nervous system. Increased expression of GABABR has been detected in human cancer tissues and cancer cell lines, but the role of GABABR in these cells is controversial and the underlying mechanism remains poorly understood. Here, we investigated whether GABABR hijacks RTK signaling to modulate the fates of human prostate cancer cells. RTK array analysis revealed that the GABABR-specific agonist baclofen selectively induced the transactivation of EGFR in PC-3 cells. EGFR transactivation resulted in the activation of ERK1/2 by a mechanism that is dependent on Gi/o protein and that requires matrix metalloproteinase-mediated proligand shedding. Positive allosteric modulators (PAMs) of GABABR, such as CGP7930, rac-BHFF, and GS39783, can function as PAM agonists to induce EGFR transactivation and subsequent ERK1/2 activation. Moreover, both baclofen and CGP7930 promoted cell migration and invasion through EGFR signaling. In summary, our observations demonstrated that GABABR transactivated EGFR in a ligand-dependent mechanism to promote prostate cancer cell migration and invasion, thus providing new insights into developing a novel strategy for prostate cancer treatment by targeting neurotransmitter signaling.

Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCß pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gß subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.

Cell-based assay of nongenomic actions of progestins revealed inhibitory G protein coupling to membrane progestin receptor α (mPRα).

Previously, we established cell lines stably producing goldfish membrane progestin receptor α (goldfish mPRα) proteins, which mediate steroidal nongenomic actions. In this study, we transfected these cell lines (MDA-MD-231) with cDNAs encoding a recombinant luciferase gene (GloSensor). These cells can be used for monitoring the effects of ligands that bind to mPR by means of luminescence, the intensity of which reflects intracellular cyclic adenosine monophosphate (cAMP) levels. Luminescence intensity of the cells increased significantly when cells were treated with forskolin, strong activator of adenylyl cyclase. Then, we established a strategy to measure changes in luminescence that correlated with the actions of the ligands. The actions of ligands were measurable by the prevention of stimulation caused by forskolin after ligand stimulation. The studies using these cell lines indicated that cAMP concentrations were decreased specifically by the mPR ligands 17α,20ß-dihydroxy-4-pregnen-3-one, diethylstilbestrol and progesterone. Furthermore, pertussis toxin inhibited the decrease in cAMP levels caused by mPR ligands. These results support evidence from previous results that mPRα is coupled to an inhibitory G protein.

Cardioprotective trafficking of caveolin to mitochondria is Gi-protein dependent.

BACKGROUND: Caveolae are a nexus for protective signaling. Trafficking of caveolin to mitochondria is essential for adaptation to cellular stress though the trafficking mechanisms remain unknown. The authors hypothesized that G protein-coupled receptor/inhibitory G protein (Gi) activation leads to caveolin trafficking to mitochondria. METHODS: Mice were exposed to isoflurane or oxygen vehicle (30 min, ± 36 h pertussis toxin pretreatment, an irreversible Gi inhibitor). Caveolin trafficking, cardioprotective "survival kinase" signaling, mitochondrial function, and ultrastructure were assessed. RESULTS: Isoflurane increased cardiac caveolae (n = 8 per group; data presented as mean ± SD for Ctrl versus isoflurane; [caveolin-1: 1.78 ± 0.12 vs. 3.53 ± 0.77; P < 0.05]; [caveolin-3: 1.68 ± 0.29 vs. 2.67 ± 0.46; P < 0.05]) and mitochondrial caveolin levels (n = 16 per group; [caveolin-1: 0.87 ± 0.18 vs. 1.89 ± .19; P < 0.05]; [caveolin-3: 1.10 ± 0.29 vs. 2.26 ± 0.28; P < 0.05]), and caveolin-enriched mitochondria exhibited improved respiratory function (n = 4 per group; [state 3/complex I: 10.67 ± 1.54 vs. 37.6 ± 7.34; P < 0.05]; [state 3/complex II: 37.19 ± 4.61 vs. 71.48 ± 15.28; P < 0.05]). Isoflurane increased phosphorylation of survival kinases (n = 8 per group; [protein kinase B: 0.63 ± 0.20 vs. 1.47 ± 0.18; P < 0.05]; [glycogen synthase kinase 3ß: 1.23 ± 0.20 vs. 2.35 ± 0.20; P < 0.05]). The beneficial effects were blocked by pertussis toxin. CONCLUSIONS: Gi proteins are involved in trafficking caveolin to mitochondria to enhance stress resistance. Agents that target Gi activation and caveolin trafficking may be viable cardioprotective agents.

A real-time method for measuring cAMP production modulated by Gαi/o-coupled metabotropic glutamate receptors.

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Atypical G(αi) signal transduction.

G protein signaling is an extremely complex event that is involved in almost every cellular process. As such, G protein-coupled receptors are the most commonly found type of transmembrane receptors used by cells to initiate intracellular signaling events. However, the widely accepted model of cyclical GDP-GTP exchange in response to ligand binding to 7TMRs, followed by dissociation of the G protein subunits and activation of intracellular signaling cascades, has repeatedly been challenged in recent years. Some of the exceptions that have been brought forth include signaling by a non-dissociated, rearranged heterotrimer and the existence of "reverse-mode", active G proteins that interact with active receptors. Here, we focus on G(αi/o), one of the common G(α) classes, and outline a major exception to the classical model, that of G protein coupling to RTKs. We then describe a novel concept in G(αi/o) signaling, namely that the pathways induced by agonist binding circumvent the typical signaling pathways responsive to decreases in the second messenger cAMP, via adenylyl cyclase inhibition.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

Impaired periamygdaloid-cortex prodynorphin is characteristic of opiate addiction and depression.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

Cognate antigen directs CD8+ T cell migration to vascularized transplants.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Potentiation of sulfonylurea action by an EPAC-selective cAMP analog in INS-1 cells: comparison of tolbutamide and gliclazide and a potential role for EPAC activation of a 2-APB-sensitive Ca2+ influx.

Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 µM tolbutamide and 20 µM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 µM) or the L-type Ca(2+) channel blocker nicardipine (2 µM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 µM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 µM but not 1 µM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 µM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 µM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 µM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 µM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx.

Defective macrophage migration in Gαi2- but not Gαi3-deficient mice.

Various heterotrimeric G(i) proteins are considered to be involved in cell migration and effector function of immune cells. The underlying mechanisms, how they control the activation of myeloid effector cells, are not well understood. To elucidate isoform-redundant and -specific roles for Gα(i) proteins in these processes, we analyzed mice genetically deficient in Gα(i2) or Gα(i3). First, we show an altered distribution of tissue macrophages and blood monocytes in the absence of Gα(i2) but not Gα(i3). Gα(i2)-deficient but not wild-type or Gα(i3)-deficient mice exhibited reduced recruitment of macrophages in experimental models of thioglycollate-induced peritonitis and LPS-triggered lung injury. In contrast, genetic ablation of Gα(i2) had no effect on Gα(i)-dependent peritoneal cytokine production in vitro and the phagocytosis-promoting function of the Gα(i)-coupled C5a anaphylatoxin receptor by liver macrophages in vivo. Interestingly, actin rearrangement and CCL2- and C5a anaphylatoxin receptor-induced chemotaxis but not macrophage CCR2 and C5a anaphylatoxin receptor expression were reduced in the specific absence of Gα(i2). Furthermore, knockdown of Gα(i2) caused decreased cell migration and motility of RAW 264.7 cells, which was rescued by transfection of Gα(i2) but not Gα(i3). These results indicate that Gα(i2), albeit redundant to Gα(i3) in some macrophage activation processes, clearly exhibits a Gα(i) isoform-specific role in the regulation of macrophage migration.

Chronic receptor-mediated activation of Gi/o proteins alters basal t-tubular and sarcolemmal L-type Ca²âº channel activity through phosphatases in heart failure.

L-type Ca(2+) channels (LTCCs) play an essential role in the excitation-contraction coupling of ventricular myocytes. We previously found that t-tubular (TT) LTCC current density was halved by the activation of protein phosphatase (PP)1 and/or PP2A, whereas surface sarcolemmal (SS) LTCC current density was increased by the inhibition of PP1 and/or PP2A activity in failing ventricular myocytes of mice chronically treated with isoproterenol (ISO mice). In the present study, we examined the possible involvement of inhibitory heterotrimeric G proteins (G(i/o)) in these abnormalities by chronically administrating pertussis toxin (PTX) to ISO mice (ISO + PTX mice). Compared with ISO mice, ISO + PTX mice exhibited significantly higher fractional shortening of the left ventricle. The expression level of Gα(i2) proteins was not altered by the treatment of mice with ISO and/or PTX. ISO + PTX myocytes had normal TT and SS LTCC current densities because they had higher and lower availability and/or open probability of TT and SS LTCCs than ISO myocytes, respectively. A selective PKA inhibitor, H-89, did not affect LTCC current densities in ISO + PTX myocytes. A selective PP2A inhibitor, fostriecin, did not affect SS or TT current density in control or ISO + PTX myocytes but significantly increased TT but not SS LTCC current density in ISO myocytes. These results indicate that chronic receptor-mediated activation of G(i/o) in vivo decreases basal TT LTCC activity by activating PP2A and increases basal SS LTCC activity by inhibiting PP1 without modulating PKA in heart failure.

Stress and glucocorticoids impair memory retrieval via ß2-adrenergic, Gi/o-coupled suppression of cAMP signaling.

Acute stress impairs the retrieval of hippocampus-dependent memory, and this effect is mimicked by exogenous administration of stress-responsive glucocorticoid hormones. It has been proposed that glucocorticoids affect memory by promoting the release and/or blocking the reuptake of norepinephrine (NE), a stress-responsive neurotransmitter. It has also been proposed that this enhanced NE signaling impairs memory retrieval by stimulating ß(1)-adrenergic receptors and elevating levels of cAMP. In contrast, other evidence indicates that NE, ß(1), and cAMP signaling is transiently required for the retrieval of hippocampus-dependent memory. To resolve this discrepancy, wild-type rats and mice with and without gene-targeted mutations were stressed or treated with glucocorticoids and/or adrenergic receptor drugs before testing memory for inhibitory avoidance or fear conditioning. Here we report that glucocorticoids do not require NE to impair retrieval. However, stress- and glucocorticoid-induced impairments of retrieval depend on the activation of ß(2) (but not ß(1))-adrenergic receptors. Offering an explanation for the opposing functions of these two receptors, the impairing effects of stress, glucocorticoids and ß(2) agonists on retrieval are blocked by pertussis toxin, which inactivates signaling by G(i/o)-coupled receptors. In hippocampal slices, ß(2) signaling decreases cAMP levels and greatly reduces the increase in cAMP mediated by ß(1) signaling. Finally, augmenting cAMP signaling in the hippocampus prevents the impairment of retrieval by systemic ß(2) agonists or glucocorticoids. These results demonstrate that the ß(2) receptor can be a critical effector of acute stress, and that ß(1) and ß(2) receptors can have quite distinct roles in CNS signaling and cognition.