Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gßγ Subunits.

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gßγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gßγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gßγ sink ßARK1-ct (C terminus of ß-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gßγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gßγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gßγ subunits and TRPM3. Since Gßγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gßγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gßγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gßγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

Gγ identity dictates efficacy of Gßγ signaling and macrophage migration.

G protein ßγ subunit (Gßγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gßγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gßγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gßγ and thereby differentially modulate Gßγ effector signaling in a Gγ-specific manner. Both Gßγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gßγ. We further show that the overall PM affinity of the Gßγ pool of a cell type is a strong predictor of its Gßγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gßγ behaviors not only recapitulated experimentally observed signaling of Gßγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gßγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gßγ.

PREX1 integrates G protein-coupled receptor and phosphoinositide 3-kinase signaling to promote glioblastoma invasion.

A defining feature of the brain cancer glioblastoma is its highly invasive nature. When glioblastoma cells are isolated from patients using serum free conditions, they accurately recapitulate this invasive behaviour in animal models. The Rac subclass of Rho GTPases has been shown to promote invasive behaviour in glioblastoma cells isolated in this manner. However the guanine nucleotide exchange factors responsible for activating Rac in this context have not been characterized previously. PREX1 is a Rac guanine nucleotide exchange factor that is synergistically activated by binding of G protein αγ subunits and the phosphoinositide 3-kinase pathway second messenger phosphatidylinositol 3,4,5 trisphosphate. This makes it of particular interest in glioblastoma, as the phosphoinositide 3-kinase pathway is aberrantly activated by mutation in almost all cases. We show that PREX1 is expressed in glioblastoma cells isolated under serum-free conditions and in patient biopsies. PREX1 promotes the motility and invasion of glioblastoma cells, promoting Rac-mediated activation of p21-associated kinases and atypical PKC, which have established roles in cell motility. Glioblastoma cell motility was inhibited by either inhibition of phosphoinositide 3-kinase or inhibition of G protein ßγ subunits. Motility was also inhibited by the generic dopamine receptor inhibitor haloperidol or a combination of the selective dopamine receptor D2 and D4 inhibitors L-741,626 and L-745,870. This establishes a role for dopamine receptor signaling via G protein ßγ subunits in glioblastoma invasion and shows that phosphoinositide 3-kinase mutations in glioblastoma require a context of basal G protein-coupled receptor activity in order to promote this invasion.

Inducible Inhibition of Gßγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on ßγ signaling at the Golgi, where ßγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous ßγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used ßγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit ßγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit ßγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by ßγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by ßγ inhibition, demonstrating that ßγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate ßγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for ßγ regulation of TGN to PM transport and a novel role for ßγ in mediating Golgi fragmentation.

Reversal of Ethanol-induced Intoxication by a Novel Modulator of Gßγ Protein Potentiation of the Glycine Receptor.

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of Gßγ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of Gßγ. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication.

On the selectivity of the Gαq inhibitor UBO-QIC: A comparison with the Gαi inhibitor pertussis toxin.

Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially ßγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gßγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gßγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the ßγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC.

Inhibition of G-protein ßγ signaling enhances T cell receptor-stimulated interleukin 2 transcription in CD4+ T helper cells.

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein ßγ (Gßγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gßγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gß1 and Gß2 mRNA accounted for >99% of Gß mRNA, and small interfering RNA (siRNA)-mediated silencing of Gß1 but not Gß2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gßγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gßγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gßγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gßγ in CD4+ T helper cells could have applications for autoimmune diseases.

G-protein ßγ subunit dimers modulate kidney repair after ischemia-reperfusion injury in rats.

Heterotrimeric G-proteins play a crucial role in the control of renal epithelial cell function during homeostasis and in response to injury. In this report, G-protein ßγ subunit (Gßγ) dimer activity was evaluated during the process of tubular repair after renal ischemia-reperfusion injury (IRI) in male Sprague Dawley rats. Rats were treated with a small molecule inhibitor of Gßγ activity, gallein (30 or 100 mg/kg), 1 hour after reperfusion and every 24 hours for 3 additional days. After IRI, renal dysfunction was prolonged after the high-dose gallein treatment in comparison with vehicle treatment during the 7-day recovery period. Renal tubular repair in the outer medulla 7 days after IRI was significantly (P < 0.001) attenuated after treatment with high-dose gallein (100 mg/kg) in comparison with low-dose gallein (30 mg/kg), or the vehicle and fluorescein control groups. Gallein treatment significantly reduced (P < 0.05) the number of proliferating cell nuclear antigen-positive tubular epithelial cells at 24 hours after the ischemia-reperfusion phase in vivo. In vitro application of gallein on normal rat kidney (NRK-52E) proximal tubule cells significantly reduced (P < 0.05) S-phase cell cycle entry compared with vehicle-treated cells as determined by 5'-bromo-2'-deoxyuridine incorporation. Taken together, these data suggest that Gßγ signaling contributes to the maintenance and repair of renal tubular epithelium and may be a novel therapeutic target for the development of drugs to treat acute kidney injury.

A chemical biology approach demonstrates G protein ßγ subunits are sufficient to mediate directional neutrophil chemotaxis.

Our laboratory has identified a number of small molecules that bind to G protein ßγ subunits (Gßγ) by competing for peptide binding to the Gßγ "hot spot." M119/Gallein were identified as inhibitors of Gßγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gßγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gßγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gßγ in vitro from Gαi1ß1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gßγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gßγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner.

Role for Gßγ G-proteins in protease regulation during remodeling of the murine femoral artery.

BACKGROUND: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. G-protein coupled receptors are involved in smooth muscle cell proliferation but the role of Gßγ in arterial intimal hyperplasia has not been well defined. The aim of this study is to characterize the expression of Gßγ G-proteins in the developing intimal hyperplasia in a murine model and the impact of disruption of Gßγ signaling on intimal hyperplasia development. METHODS: The murine femoral wire injury model was employed. Specimens were perfusion-fixed and sections were stained with H&E and Movat's stains such that morphometry could be performed using an Image-Pro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting for the Gßγ expression and for Western blotting and zymography to allow for the study of gelatinase and plasminogen activator expression and activation. Contralateral vessels were used as controls. Additional vessels were immersed in pluronic gel containing an adenovirus with the Gßγ inhibitor ßARK(CT). RESULTS: The injured femoral arteries developed intimal hyperplasia, while sham vessels did not produce such a response. Cell proliferation peaked at 3-5 d and cell migration at 7 d after injury. There was a marked time-dependent increase in Gßγ over the 28 d following injury. Inhibition of Gßγ with ßARK(CT) inhibited cell proliferation, cell migration and the development of intimal hyperplasia. Inhibition of Gßγ decreased peak uPA activity and expression without increasing early PAI-1 activity and expression. Inhibition of Gßγ reduced peak MMP-2 activity at d 1 but not at d 7 and also reduced peak MMP-9 activity at d 3. Protein expression for both MMP-2 and MMP-9 was also transiently decreased. There were no changes in TIMP-1 and TIMP-2 expression and activity. CONCLUSIONS: These data demonstrate a time-dependent increase in Gßγ G-protein expression following wire injury in the mouse. Inhibition of Gßγ alters cell proliferation and migration with associated changes in MMP-2, MMP-9, and uPA expression and activity.

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor.

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gßγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.

Rational design of a selective covalent modifier of G protein ßγ subunits.

G protein-coupled receptors transduce signals through heterotrimeric G protein Gα and Gßγ subunits, both of which interact with downstream effectors to regulate cell function. Gßγ signaling has been implicated in the pathophysiology of several diseases, suggesting that Gßγ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a protein-protein interaction "hot spot" on Gßγ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with Gßγ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies GßCys204, but also a second undefined site. The high potency of selenocystamine in Gßγ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on Gß. Using structural information about the "hot spot," we developed a strategy to selectively target redox reversible compounds to a specific site on Gßγ using peptide carriers such as SIGCAFKILGY(-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on Gß, likely GßCys204, and inhibits Gßγ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the Gßγ "hot spot" through rational incorporation into molecules that noncovalently bind to Gßγ.

Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition.

Direct interaction with the beta subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel alpha1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gbeta1gamma2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the alpha1 subunit, residues which are surface-exposed in the presence of the calcium channel beta subunit, contribute significantly to the functional inhibition by Gbeta1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gbeta1gamma2 subunits and with several isoforms of the auxiliary beta subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel.

Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex.

A large number of hormones and local agonists activating guanine-binding protein-coupled receptors (GPCR) play a major role in cancer progression. Here, we characterize the new imidazo-pyrazine derivative BIM-46174, which acts as a selective inhibitor of heterotrimeric G-protein complex. BIM-46174 prevents the heterotrimeric G-protein signaling linked to several GPCRs mediating (a) cyclic AMP generation (Galphas), (b) calcium release (Galphaq), and (c) cancer cell invasion by Wnt-2 frizzled receptors and high-affinity neurotensin receptors (Galphao/i and Galphaq). BIM-46174 inhibits the growth of a large panel of human cancer cell lines, including anticancer drug-resistant cells. Exposure of cancer cells to BIM-46174 leads to caspase-3-dependent apoptosis and poly(ADP-ribose) polymerase cleavage. National Cancer Institute COMPARE analysis for BIM-46174 supports its novel pharmacologic profile compared with 12,000 anticancer agents. The growth rate of human tumor xenografts in athymic mice is significantly reduced after administration of BIM-46174 combined with either cisplatin, farnesyltransferase inhibitor, or topoisomerase inhibitors. Our data validate the feasibility of targeting heterotrimeric G-protein functions downstream the GPCRs to improve anticancer chemotherapy.

G protein-coupled receptor internalization signaling is required for cardioprotection in ischemic preconditioning.

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gbetagamma-sequestering peptide, betaARKct (TG betaARKct mice), to test whether the protection of PC is Gbetagamma-dependent. To test the role of G(i) protein, we used wild-type mice pretreated with the G(i) inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG betaARKct mice. To determine the mechanism of Gbetagamma-induced protection in PC, we investigated one of the downstream targets of Gbetagamma, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG betaARKct hearts; therefore, we investigated other targets of Gbetagamma. Recent studies suggest a role for Gbetagamma in GPCR internalization. We found that betaARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kgamma, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kgamma blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.

Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol 3-kinase, protein kinase C, and c-Src.

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Regulation of endopeptidases EC3.4.24.15 and EC3.4.24.16 in vascular endothelial cells by cyclic strain: role of Gi protein signaling.

OBJECTIVE: Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs). METHODS AND RESULTS: BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3+/-0.4- and 1.9+/-0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi(alpha)- and Gbetagamma-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi(alpha2/3) inhibition and EP24.16 exhibiting greater sensitivity to Gi(alpha1/2) and Gbetagamma inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6+/-0.02- and 3.6+/-0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 +42%, EP24.16 +56%). CONCLUSIONS: Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.