Targeted nonviral gene-based inhibition of Gα(i/o)-mediated vagal signaling in the posterior left atrium decreases vagal-induced atrial fibrillation.

BACKGROUND: Pharmacologic and ablative therapies for atrial fibrillation (AF) have suboptimal efficacy. Newer gene-based approaches that target specific mechanisms underlying AF are likely to be more efficacious in treating AF. Parasympathetic signaling appears to be an important contributor to AF substrate. OBJECTIVE: The purpose of this study was to develop a nonviral gene-based strategy to selectively inhibit vagal signaling in the left atrium and thereby suppress vagal-induced AF. METHODS: In eight dogs, plasmid DNA vectors (minigenes) expressing Gα(i) C-terminal peptide (Gα(i)ctp) was injected in the posterior left atrium either alone or in combination with minigene expressing Gα(o)ctp, followed by electroporation. In five control dogs, minigene expressing scrambled peptide (Gα(R)ctp) was injected. Vagal- and carbachol-induced left atrial effective refractory periods (ERPs), AF inducibility, and Gα(i/o)ctp expression were assessed 3 days following minigene delivery. RESULTS: Vagal stimulation- and carbachol-induced effective refractory period shortening and AF inducibility were significantly attenuated in atria receiving a Gα(i2)ctp-expressing minigene and were nearly eliminated in atria receiving both Gα(i2)ctp- and Gα(o1)ctp-expressing minigenes. CONCLUSION: Inhibition of both G(i) and G(o) proteins is necessary to abrogate vagal-induced AF in the left atrium and can be achieved via constitutive expression of Gα(i/o)ctps expressed by nonviral plasmid vectors delivered to the posterior left atrium.

Different effects of the different natural CC chemokine receptor 2b ligands on beta-arrestin recruitment, Gαi signaling, and receptor internalization.

The chemokine receptor CCR2, which has been implicated in a variety of inflammatory, autoimmune, and cardiovascular conditions, binds several natural chemokine ligands. Here, we assessed the recruitment of ß-arrestin to CCR2 in response to these ligands using bioluminescence resonance energy transfer technology. Compared with CCL2, which was considered as a full agonist, other CCR2 ligands were partial agonists with reduced efficacy and potency. Agonist potencies were not a function of their affinity for CCR2. Efficacy of arrestin recruitment matched that of agonist-induced CCR2 internalization. Although the potency and efficacy rank orders of the ligands in arrestin recruitment were similar to those observed for Gα(i1) activation, arrestin recruitment was at least in part resistant to Gα(i/o)-inactivating pertussis toxin, suggesting partial independence from Gα(i/o). The degree of pertussis toxin resistance of arrestin recruitment was different between the chemokines. Moreover, qualitative differences between the arrestin responses to the different ligands were identified in the stability of the response: although CCL7-induced arrestin recruitment had a half-life of less than 15 min, CCL8 and CCL13 induced stable CCR2-arrestin interactions. Finally, the ligands stabilized different conformations of the CCR2 homodimer. Our results support the validity of models for receptor-ligand interactions in which different ligands stabilize different receptor conformations also for endogenous receptor ligands, with corresponding implications for drug development targeting CCR2.

Effects of chronic desipramine treatment on alpha2-adrenoceptors and mu-opioid receptors in the guinea pig cortex and hippocampus.

The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.

The time-course of agonist-induced solubilization of trimeric G(q)alpha/G(11)alpha proteins resolved by two-dimensional electrophoresis.

Prolonged agonist stimulation results in specific transfer of activated Galpha subunits of G(q)alpha/G(11)alpha family from particulate membrane fraction to soluble (cytosol) cell fraction isolated as 250,000 x g supernatant. In this study, we have used 2D electrophoresis for more defined resolution of Galpha subunits of G(q)alpha/G(11)alpha family and followed the time course of solubilization effect. The small signal of soluble G proteins was already detected in control, hormone-unexposed cells. Hormone stimulation resulted in a slow but continuous increase of both intensity and number of immunoreactive signals/spots of these G proteins (10, 30, 60, 120 and 240 min). At longer times of agonist exposure (>2 hours), a marked increase of G(q)alpha/G(11)alpha proteins was detected. The maximal level of soluble G(q)alpha/G(11)alpha proteins was reached after 16 hours of continuous agonist exposure. At this time interval, eight individual immunoreactive signals of G(q)alpha/G(11)alpha proteins could be resolved. The relative proportion among these spots was 15:42:10:11:7:7:2:5. Solubilization of this class of Galpha proteins was thus observed after prolonged agonist stimulation only, induced by ultra high concentration of hormone and in cells expressing a large number of GPCRs. Our data therefore rather indicate tight/persisting binding of G(q)alpha/G(11)alpha proteins to the membrane.

Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling.

Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Galphabetagamma heterotrimer or Galpha and Gbetagamma subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Galpha and Gbetagamma) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Galphabetagamma. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Galpha and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Galphabetagamma and the individual Galpha and Gbetagamma subunits.

Pharmacological characterization of mouse GPRC6A, an L-alpha-amino-acid receptor modulated by divalent cations.

BACKGROUND AND PURPOSE: GPRC6A is a novel member of family C of G protein-coupled receptors with so far unknown function. We have recently described both human and mouse GPRC6A as receptors for L-alpha-amino acids. To date, functional characterization of wild-type GPRC6A has been impaired by the lack of activity in quantitative functional assays. The aim of this study was thus to develop such an assay and extend the pharmacological characterization of GPRC6A. EXPERIMENTAL APPROACH: We have engineered a novel cell-based inositol phosphate turnover assay for wild-type mouse GPRC6A based on transient co-expression with the promiscuous Galpha(qG66D) protein, known to increase receptor signalling sensitivity. This assay allowed for measurements of L-alpha-amino acid potencies. Furthermore, in combination with an assay measuring inward currents at Ca(2+)-activated chloride channels in Xenopus oocytes, the divalent cation-sensing ability of the receptor was examined. KEY RESULTS: Using our novel assay, we demonstrate that the basic L-alpha-amino acids ornithine, lysine, and arginine are the most potent agonists at wild-type mouse GPRC6A. Using two different assay systems, we show that divalent cations do not activate the G(q) signalling pathway of mouse GPRC6A per se but positively modulate the amino-acid response. CONCLUSIONS AND IMPLICATIONS: This is the first reported assay for a wild-type GPRC6A successfully applied for quantitative pharmacological characterization of amino acid and divalent cation responses at mouse GPRC6A. The assay enables further search for GPRC6A ligands such as allosteric modulators, which may provide essential information about the physiological function of GPRC6A.

Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems.

Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.

Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex.

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.