Src Tyrosine Kinase Inhibitor PP2 Research Articles

Receptor-specific contributions of caveolae, PKC, and Src tyrosine kinase to serotonergic and adrenergic regulation of Kv channels and vasoconstriction

AimsCaveolae are invaginated, Ω-shaped membrane structures. They are now recognized as portals for signal transduction of multiple chemical and mechanical stimuli. Notably, the contribution of caveolae has been reported to be receptor-specific. However, details of how they differentially contribute to receptor signaling remain unclear. Main methodsUsing isometric tension measurements, patch-clamping, and western blotting, we examined the contribution of caveolae and their related signaling pathways to serotonergic (5-HT2A receptor-mediated) and adrenergic (α1-adrenoceptor-mediated) signaling in rat mesenteric arteries. Key findingsDisruption of caveolae by methyl-β-cyclodextrin effectively blocked vasoconstriction mediated by the 5-HT2A receptor (5-HT2AR), but not by the α1-adrenoceptor. Caveolar disruption selectively impaired 5-HT2AR-mediated voltage-dependent K+ channel (Kv) inhibition, but not α1-adrenoceptor-mediated Kv inhibition. In contrast, both serotonergic and α1-adrenergic effects on vasoconstriction, as well as Kv currents, were similarly blocked by the Src tyrosine kinase inhibitor PP2. However, inhibition of protein kinase C (PKC) by either GO6976 or chelerythrine selectively attenuated the effects mediated by the α1-adrenoceptor, but not by 5-HT2AR. Disruption of caveolae decreased 5-HT2AR-mediated Src phosphorylation, but not α1-adrenoceptor-mediated Src phosphorylation. Finally, the PKC inhibitor GO6976 blocked Src phosphorylation by the α1-adrenoceptor, but not by 5-HT2AR. Significance5-HT2AR-mediated Kv inhibition and vasoconstriction are dependent on caveolar integrity and Src tyrosine kinase, but not on PKC. In contrast, α1-adrenoceptor-mediated Kv inhibition and vasoconstriction are not dependent on caveolar integrity, but rather on PKC and Src tyrosine kinase. Caveolae-independent PKC is upstream of Src activation for α1-adrenoceptor-mediated Kv inhibition and vasoconstriction.

FUNDC1: a key mediator of adenosine A2BR activation-induced inhibition of cardiac mitophagy under ischemia/reperfusion conditions.

Mitophagy is an essential factor in mitochondrial quality control and myocardial ischaemia/reperfusion (I/R) injury protection. Because adenosine A2B receptor (A2BR) activation exerts a major role in reducing myocardial I/R injury, the effects of adenosine A2BR activation on cardiac mitophagy under reperfusion conditions were investigated. 110 adult Wistar rats (7-10 w), weighing 250-350 grams, were cultured in specific-pathogen-free (SPF) conditions before experiments. All hearts were removed and reperfused by Langendorff device. Six hearts with coronary flow (CF) values >28 or <10 mL/min were excluded. Others were arbitrarily divided into the following groups: sham operation group, I/R group, BAY60-6583 (BAY) (1-1,000 nM) + I/R group, PP2 + BAY + I/R group. After ischemia in rats, reperfusion was performed. H9c2 cells were placed in an imitated ischemic environment followed by Tyrode's solution to stimulate hypoxia/reoxygenation (H/R) injury. The mitochondrial fluorescence indicator MitoTracker Green and lysosomal fluorescence indicator LysoTracker Red were used to examine mitochondria and lysosomes, respectively. Colocalization of mitochondrial and autophagy marker proteins was determined by immunofluorescence. Autophagic flow currents were tested by Ad-mCherry-GFP-LC3B. Protein-protein interactions were predicted using a database and analyzed by co-immunoprecipitation. Autophagy marker protein, mitophagy marker protein, and mitophagy protein FUNDC1 were detected by immunoblotting. Compared with those in the I/R group, myocardial autophagy and mitophagy were suppressed by the selective adenosine A2BR agonist BAY, and this effect was inhibited by the selective Src tyrosine kinase inhibitor PP2, indicating that adenosine A2BR activation could inhibit myocardial autophagy and mitophagy by activating Src tyrosine kinase. In support, in H9c2 cells, the selective Src tyrosine kinase inhibitor PP2 inhibited the effect of BAY on TOM20 with LC3 or mitochondria with lysosomes colocalization and autophagy flow. Here, we showed that mitochondrial FUNDC1 co-precipitated with Src tyrosine kinase after BAY was added. Consistently, the immunofluorescence and western blotting results demonstrated that compared to that in the H/R group, the expression of mitochondrial FUNDC1 was reduced by BAY, but this effect was reversed by PP2. Adenosine A2BR activation may inhibit myocardial mitophagy by downregulating expression of the mitochondrial FUNDC1 by activating Src tyrosine kinase under I/R conditions and could increase the interaction between Src tyrosine kinase and FUNDC1.

Pivotal Pathogenic and Biomarker Role of Chlamydia Pneumoniae in Neurovascular Diseases.

Chlamydia Pneumoniae (C. Pn) is an obligatory intracellular bacterium that is associated with respiratory tract infections like pneumonia, pharyngitis and bronchitis. It has also been implicated in cerebrovascular (stroke) as well as cardiovascular diseases. The most possible pathway via which C. Pn elicits its pathogenesis could be via activation of Human Vascular Smooth Muscle Cells (VSMCs) proliferation resulting in the stimulation of Toll-Like Receptor-4 (TLR-4) and/or phospho-44/42(p44/p42) Mitogen-Activated Protein Kinases (MAPK). It is also established that tyrosine phosphorylation of IQ domain GTPase-activating protein 1 (IQGAP1) also contributed to C. Pn infection-triggered Vascular Endothelial Cell (VEC) movements via the SRC tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) resulting in angiogenesis. It is also proven that restricted inflammatory cell infiltrates as well as apoptosis have been linked to C. Pn or C. Pn-specific proteins in atherosclerotic plaques of patients with stroke. It is further an evidence that C. Pn enters the cerebral vasculature during the initial infection and worsen atherosclerosis either directly or indirectly. Chronic, persistent C. Pn infection is also capable of triggering the secretion of Chlamydial Heat Shock Protein 60 (cHSP60) in the vessel wall resulting in augmentation of inflammation. C. Pn also aids in the activation of explicit cell-intermediated immunity within plaques. Macrophages in the carotid plaques co-exist with CD4+ lymphocytes which are capable of triggering the release of pro-inflammatory cytokines resulting in the augmentation of atherogenic development during C. Pn infection. C. Pn actively participated in the modification of both histones H3 and H4 during chromatin analysis via the interleukin 8(IL-8) gene facilitator as well as conscription of nuclear factor kappa-B(NF-κB) or NF- κB/p65 complex and polymerase II (Pol II). This review, therefore, focuses on the crucial involvement of C. Pn in the pathogenesis of cerebrovascular events.

(475) - A Novel Nanoscale Formulation of Hydrophobic Src Tyrosine Kinase Inhibitor PP2 Prevents Pulmonary Ischemia Reperfusion Induced Acute Lung Injury

(475) - A Novel Nanoscale Formulation of Hydrophobic Src Tyrosine Kinase Inhibitor PP2 Prevents Pulmonary Ischemia Reperfusion Induced Acute Lung Injury

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr416) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.

MGluR1-mediated excitation of cerebellar GABAergic interneurons requires both G protein-dependent and Src-ERK1/2-dependent signaling pathways.

Stimulation of type I metabotropic glutamate receptors (mGluR1/5) in several neuronal types induces slow excitatory responses through activation of transient receptor potential canonical (TRPC) channels. GABAergic cerebellar molecular layer interneurons (MLIs) modulate firing patterns of Purkinje cells (PCs), which play a key role in cerebellar information processing. MLIs express mGluR1, and activation of mGluR1 induces an inward current, but its precise intracellular signaling pathways are unknown. We found that mGluR1 activation facilitated spontaneous firing of mouse cerebellar MLIs through an inward current mediated by TRPC1 channels. This mGluR1-mediated inward current depends on both G protein-dependent and -independent pathways. The nonselective protein tyrosine kinase inhibitors genistein and AG490 as well as the selective extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors PD98059 and SL327 suppressed the mGluR1-mediated current responses. Following G protein blockade, the residual mGluR1-mediated inward current was significantly reduced by the selective Src tyrosine kinase inhibitor PP2. In contrast to cerebellar PCs, GABAB receptor activation in MLIs did not alter the mGluR1-mediated inward current, suggesting that there is no cross-talk between mGluR1 and GABAB receptors in MLIs. Thus, activation of mGluR1 facilitates firing of MLIs through the TRPC1-mediated inward current, which depends on not only G protein-dependent but also Src–ERK1/2-dependent signaling pathways, and consequently depresses the excitability of cerebellar PCs.

Bisphenol AF-induced endogenous transcription is mediated by ERα and ERK1/2 activation in human breast cancer cells.

Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ERα and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ERα-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ERα-positive T47D and MCF7 cells as well as overexpression of ERα in ERα-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ERα and ERK1/2 activation in human breast cancer cells.

The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2 through the MAP kinases-NF-κB pathway

Rat primary cultures of spinal microglia were stimulated by UTP, a known P2Y2/4 receptor agonist, which resulted in the production and release of the C–C chemokine CCL2 (monocyte chemoattractant protein-1; MCP-1) measured by real-time PCR and ELISA, respectively. In an in vitro preparation of rat spinal microglia, with regard to the P2Y subtypes, the expression of P2Y1, 2, 6, 12, 13 and P2Y14, but not P2Y4, were detected by RT-PCR. The subtype of microglial P2Y receptor which could be involved in the production of CCL2 was also determined. The UTP-induced production of CCL2 was significantly blocked by pretreatment with reactive blue 2 and suramin, nonselective P2Y receptor antagonists, and MRS2578, a selective P2Y6 receptor antagonist. By contrast, knockdown of the P2Y2 receptor by RNA interference had no effect. The stimulatory effect of UTP was inhibited by phospholipase C (PLC) inhibitor U73122 and Src tyrosine kinase inhibitor PP2. A potential role of mitogen activated protein kinases was suggested since UTP-induced CCL2 production was significantly blocked by both U0126 and SB 202190, which are potent inhibitors of extracellular signal-regulated kinase (ERK) and p38, respectively. Moreover, UTP-stimulated phosphorylation of these kinases involved the activation of the P2Y6 receptor. Lastly, activation of nuclear factor-κB (NF-κB) by UTP is likely to be essential in the expression of CCL2. Together, these findings suggest that stimulation of spinal microglia P2Y6 receptors induce the production of CCL2 through either PLC-mediated ERK or p38 phosphorylation and the subsequent activation of NF-κB.

Lordosis facilitation by leptin in ovariectomized, estrogen-primed rats requires simultaneous or sequential activation of several protein kinase pathways

The present study tested the hypothesis that the Janus kinase 2, Src tyrosine kinases, and mitogen-activated protein kinase interact to regulate lordosis behavior induced by leptin in ovariectomized, estrogen-primed rats. The role of protein kinase A and protein kinase C in lordosis facilitation by leptin was also assessed. In experiment 1, the intracerebroventricular administration of leptin to ovariectomized, estradiol-primed rats significantly stimulated lordosis behavior at 1, 2 and 4h post-injection tests. In experiment 2, the Janus kinase 2 inhibitor AG490, the Src tyrosine kinase inhibitor PP2 and the mitogen-activated protein kinase inhibitor PD98059 were administered into the right lateral ventricle before leptin. The lordosis quotient and the lordosis score induced by leptin were significantly decreased by each of these kinase inhibitors. In experiment 3, we examined the effects of RpcAMPS and bisindolylmaleimide, protein kinase A and protein kinase C inhibitors on the lordosis elicited by leptin administration. Lordosis behavior induced by leptin was significantly decreased by both the protein kinase A and protein kinase C inhibitors at 1h post-leptin injection. The results confirm that multiple intracellular pathways participate in the expression of lordosis behavior in estrogen-primed rats elicited by leptin.

Roles of mitochondrial Src tyrosine kinase and zinc in nitric oxide-induced cardioprotection against ischemia/reperfusion injury

While nitric oxide (NO) induces cardioprotection by targeting the mitochondrial permeability transition pore (mPTP), the precise mitochondrial signaling events that mediate the action of NO remain unclear. The purpose of this study was to test whether NO induces cardioprotection against ischemia/reperfusion by inhibiting oxidative stress through mitochondrial zinc and Src tyrosine kinase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP) given before the onset of ischemia reduced cell death in rat cardiomyocytes subjected to simulated ischemia/reperfusion, and this was abolished by the zinc chelator N,N,N’,N’-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and the Src tyrosine kinase inhibitor PP2. SNAP also prevented loss of mitochondrial membrane potential (ΔΨm) at reperfusion, an effect that was blocked by TPEN and PP2. SNAP increased mitochondrion-free zinc upon reperfusion and enhanced mitochondrial Src phosphorylation in a zinc-dependent manner. SNAP inhibited both mitochondrial complex I activity and mitochondrial reactive oxygen species (ROS) generation at reperfusion through zinc and Src tyrosine kinase. Finally, the anti-infarct effect of SNAP was abrogated by TPEN and PP2 applied at reperfusion in isolated rat hearts. In conclusion, NO induces cardioprotection at reperfusion by targeting mitochondria through attenuation of oxidative stress resulted from the inhibition of complex I at reperfusion. Activation of mitochondrial Src tyrosine kinase by zinc may account for the inhibition of complex I.

Secreted Factors from Human Mast Cells Trigger Inflammatory Cytokine Production by Human Airway Smooth Muscle Cells

Background: A notable feature of allergic asthma is the infiltration of mast cells into smooth muscle in the human airway. Thus, mast cells and human airway smooth muscle (hASM) cells are likely to exhibit mutual functional modulation via direct cell-cell contact or through released factors. This study examined mast cell modulation of hASM cell cytokine release. Methods: The mast cell line HMCα was used to model mast cell function. hASM cells were either co-cultured directly with resting or IgE/antigen-stimulated HMCα cells or treated with HMCα-conditioned media to examine the impact on cytokine release. The activation pathways triggered in hASM cells by the mast cell-derived factors were examined through the use of selective inhibitors and by Western blotting. Results: HMCα cells, or their conditioned media, induced the expression of cytokines (IL-8 and IL-6) by hASM cells at both the mRNA and the protein level. Cytokine expression in hASM cells was greatly amplified when HMCα cells were IgE/antigen-activated. The effects of the conditioned media were not mediated by the chemokines MCP-1 and MIP-1α or by exosomes. While the mast cell-derived factor(s) increased p38^MAPK phosphorylation in hASM cells, cytokine production was not inhibited by the p38^MAPK inhibitor SB203580. hASM cell production of IL-8 induced by HMCα condition media but not IL-6 was, however, attenuated by the Src tyrosine kinase inhibitor PP2. Conclusions: Our study shows that the release of soluble mediators by activated mast cells can stimulate hASM cells to elicit production of proinflammatory cytokines that may then exacerbate airway inflammation in asthma.

Regulation of PI3K/Akt signaling by N-desmethylclozapine through activation of δ-opioid receptor

We have previously reported that N-desmethylclozapine (NDMC), a major clozapine metabolite, acts as a δ-opioid receptor agonist. Here, we show that in different cellular systems NDMC regulates protein kinase B/Akt (Akt) signaling through the activation of δ-opioid receptors. In Chinese hamster ovary cells transfected with the human δ-opioid receptor (CHO/DOR), NDMC induced a time- and concentration-dependent phosphorylation of Akt at Thr308 and glycogen synthase kinase-3β (GSK-3β) at Ser9 and these effects were fully blocked by the δ-opioid receptor antagonist naltrindole. NDMC-induced Akt and GSK-3β phosphorylations were completely prevented by pertussis toxin, the Src tyrosine kinase inhibitor PP2 and the selective insulin-like growth factor-I (IGF-I) receptor tyrosine kinase inhibitor tyrphostin AG 1024. NDMC stimulated IGF-I receptor β subunit tyrosine phosphorylation and this effect was prevented by either naltrindole or PP2. Blockade of phosphatidylinositol 3-kinase (PI3K) α, but not PI3Kγ, suppressed NDMC-induced Akt and GSK-3β phosphorylation, whereas inhibition of Akt curtailed the stimulation of GSK-3β phosphorylation. In rat nucleus accumbens, NDMC induced Akt and GSK-3β phosphorylation either in vitro or in vivo and these effects were prevented by naltrindole. NDMC also regulated Akt and GSK-3β phosphorylation through δ-opioid receptors in NG108-15 cells. In these cells NDMC counteracted oxidative stress-induced apoptosis and the effect was lost following PI3K inhibition. These data demonstrate that in different cell systems NDMC can stimulate Akt signaling by activating Gi/Go-coupled δ-opioid receptors, which, at least in CHO/DOR cells, regulate PI3Kα through Src-dependent transactivation of the IGF-I receptor, and indicate that through this mechanism NDMC can exert neuroprotective effects.

The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy

Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. For this purpose, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilizes the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-α production in the bronchoalveolar lavage fluid after LPS stimulation. The promising therapeutic efficacy and the formulation strategy developed in this work may help further translate PP2 and other hydrophobic therapeutic agents into clinical applications.

Induction of peroxiredoxin I gene expression by LPS involves the Src/PI3K/JNK signalling pathway

Peroxiredoxin I (Prx I) belongs to a family of proteins with thiol-dependent peroxidase activity and is involved in the cellular protection against oxidative stress, the modulation of intracellular signalling cascades as well as the regulation of cell proliferation and apoptosis. In RAW 264.7 mouse macrophage cells Prx I was up-regulated on the mRNA and protein level by lipopolysaccharide (LPS). Treatment of cells with LPS increased the phosphorylation of c-Jun-NH 2 terminal kinase (JNK) and protein kinase B (PKB). Both SP600125, an inhibitor of JNK, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), dose-dependently decreased LPS-induced Prx I mRNA expression. Furthermore, up-regulation of Prx I mRNA by LPS was diminished by the Src tyrosine kinase inhibitor PP2 and the iNOS inhibitor L-NMMA. LPS-dependent induction of Prx I is likely mediated by an activator protein-1 site within the Prx I promoter region binding JunB and c-Fos. In contrast, NFκB was not involved in the activation of Prx I transcription. Our results suggest that the up-regulation of Prx I gene expression by LPS is part of the cellular response to stress and may protect against oxidative stress-related injury in RAW 264.7 cells.

Contribution of Intracellular Ca2+ Concentration and Protein Dephosphorylation to the Induction of Dopamine Release from PC12 cells by the Green Odor Compound Hexanal

n-Hexanal (hexanal), a straight-chain six-carbon aldehyde, is mainly present in plants. Hexanal strongly affects the release of dopamine from rat striatal slices and rat pheochromocytoma (PC12) cells. In this study, we attempted to clarify the mechanism underlying the regulation of dopamine release by hexanal by using PC12 cells, which have the ability to synthesize, store, and release dopamine. The stimulation of PC12 cells with hexanal enhanced dopamine release in a time- and dose-dependent manner. Dopamine release was partially inhibited by pretreatment of the cells with BAPTA-AM, a cell-permeable Ca2+ chelator. In addition, the intracellular Ca2+ concentration was found to slowly increase after hexanal stimulation. Furthermore, the Src tyrosine kinase inhibitor PP2 partially inhibited hexanal-induced dopamine release. However, the levels of phosphorylated proteins decreased after hexanal stimulation. Hexanal stimulated the release of only a small amount of dopamine from reserpine-treated PC12 cells, in which the vesicular dopamine was depleted. These findings suggest that both an increase in the intracellular Ca2+ concentration and the dephosphorylation of phosphorylated proteins might be required for hexanal-stimulated release of dopamine, and that the dopamine released because of hexanal stimulation mainly comes from the dopamine vesicles. This study showed the cellular events that occurred in PC12 cells after stimulation of hexanal. Furthermore, it is important to examine the relationship between the cellular functions and the physiological effects of hexanal on dopamine release.

The β1-adrenergic receptor mediates extracellular signal-regulated kinase activation via Gαs

beta-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study, we investigated the molecular mechanism of beta1-adrenergic receptor (beta1AR)-mediated ERK activation in African green monkey kidney COS-7 cells. Treatment of cells with isoproterenol (ISO), a beta1AR selective agonist, induced phosphorylation of ERK1/2 in a dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gbetagamma inhibitor, betaAR kinase carboxyl terminal (betaARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA) with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Galphas subunit is involved in ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation. These observations suggest a mechanism of beta1AR-mediated ERK activity that involves the Galphas subunit, but not EGFR or Src tyrosine kinase.

Tyrosine phosphorylation controls nuclear localization and transcriptional activity of Ssdp1 in mammalian cells

The LIM-HD proteins interact with different cofactors, including Ssdp1 to regulate development in a diverse range of species. The single stranded DNA binding protein (Ssdp1) is a member of an evolutionarily conserved family of proteins that regulate critical transcriptional processes during embryonic development. Ssdp1 is localized predominantly in the cytoplasm of 293T cells but is translocated to the nucleus when co-transfected with Lck, a member of the Src family of non-receptor tyrosine kinases. The Src tyrosine kinase inhibitor PP2 blocked the nuclear translocation of Ssdp1. Western blot analysis showed that co-expression of Ssdp1 and Lck in 293T cells induces Ssdp1 phosphorylation. Mutation of the Ssdp1 N terminal tyrosine residues 23 and 25 markedly reduced both the phosphorylation and the nuclear localization of Ssdp1. Lck enhanced the transcriptional activity of Ssdp1 in the context of known components of a LIM-homeodomain (LIM-HD)/cofactor complex. We propose that phosphorylation involving N-terminal tyrosine residues of Ssdp1 is a means of regulating its nuclear localization and subsequent transcriptional activation of LIM-HD complexes.

Kir2.x inward rectifier potassium channels are differentially regulated by adrenergic α 1A receptors

Inhibition of I K1 currents by adrenergic α 1 receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native I K1 current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved.Adrenergic α 1A receptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native I K1 currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes.Activation of co-expressed adrenergic α 1A receptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of I K1 current by adrenergic α 1 receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of α 1A receptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein.In summary, on the molecular level the inhibitory regulation of I K1 currents by adrenergic α 1A receptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native I K1 current by adrenergic α 1 receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation.

Differential signaling mechanisms of HNP‐induced IL‐8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.

Sex-related differences in NMDA-evoked rat masseter muscle afferent discharge result from estrogen-mediated modulation of peripheral NMDA receptor activity

In the present study, the hypothesis that sex-related differences in glutamate-evoked rat masseter muscle afferent discharge may result from estrogen-related modulation of peripheral N-methyl-d-aspartate (NMDA) receptor activity and/or expression was tested by examining afferent fiber discharge in response to masseter injection of NMDA and the expression of NR2A/B subunits by masseter ganglion neurons in male and female rats. The results showed that injection of NMDA into the masseter muscle evoked discharges in putative mechanonociceptive afferent fibers and increased blood pressure that was concentration-dependent, however, a systemic action of NMDA appeared responsible for increased blood pressure. NMDA-evoked afferent discharge was significantly greater in female than in male rats, was positively correlated with plasma estrogen levels in females and was significantly greater in ovariectomized female rats treated with a high dose (5 μg/day) compared with a low dose (0.5 μg/day) of estrogen. Pre-treatment of high dose estrogen-treated–ovariectomized female rats with the Src tyrosine kinase inhibitor PP2 did not affect NMDA-evoked afferent discharge. NMDA-evoked afferent discharge was attenuated by the antagonists ketamine and ifenprodil, which is selective for NR2B containing NMDA receptors. Fewer masseter ganglion neurons expressed the NR2A (16%) subunit as compared with the NR2B subunit (38%), which was expressed at higher frequencies in intact female (46%) and high dose estrogen-treated ovariectomized female (60%) rats than in male (31%) rats. Taken together, these results suggest that sex-related differences in NMDA-evoked masseter afferent discharge are due, at least in part, to an estrogen-mediated increase in expression of peripheral NMDA receptors by masseter ganglion neurons in female rats.