Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

Regulation of aryl hydrocarbon receptor signal transduction by protein tyrosine kinases

The involvement of protein tyrosine kinases (PTKs) in aryl hydrocarbon receptor (AhR)-mediated signalling by omeprazole and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in hepatoma cells. Both omeprazole- and TCDD-dependent AhR signalling was attenuated by inhibition of c-src kinase, either by using pyrazolopyrimidine 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4 ]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitors or by expression of dominant-negative c-src. These results indicate that the overall AhR function is modulated by c-src kinase activity. In contrast, a selective inhibition of omeprazole-mediated AhR signalling was revealed by tyrosine kinase inhibitors, tyrphostins AG17 and AG879. Furthermore, omeprazole-dependent AhR activation was abolished by mutation of Tyr320 to Phe, suggesting that this residue is a putative phosphorylation site. TCDD-dependent AhR signalling was neither affected by tyrphostins nor by this mutation. Our results are consistent with activation of the AhR by omeprazole in a ligand-independent manner, via a signal transduction pathway that involves protein tyrosine kinases, and are different from the mechanism exerted by high-affinity ligands.     

Tyrosine phosphorylation of HPK1 by activated Src promotes ischemic brain injury in rat hippocampal CA1 region

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that HPK1 is involved in c-Jun NH2-terminal kinase (JNK) signaling pathway by sequential activation of MLK3-MKK7-JNK3 after cerebral ischemia. Here, we used 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) and MK801 to investigate the events upstream of HPK1 in ischemic brain injury. Immunoprecipitation and immunoblot results showed that PP2 and MK801 significantly decreased the activation of Src, HPK1, MLK3, JNK3 and c-Jun, respectively, during ischemia/reperfusion. Histology and TUNEL staining showed PP2 or MK801 protects against neuron death after brain ischemia. We speculate that this unique signaling pathway through the tyrosine phosphorylation of HPK1 promotes ischemic brain injury by activated Src via N-methyl-d-aspartate receptor and, ultimately, the activation of the MLK3-MKK7-JNK3 pathway after cerebral ischemia.     

Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

Src kinase mediates the regulation of phospholipase C-gamma activity by glycosphingolipids

Glucosylceramide-based glycosphingolipids have been previously demonstrated to regulate negatively the formation of inositol 1,4,5-trisphosphate by phospholipase C-gamma1. In the present study, the depletion of endogenous glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells induced autophosphorylation of Src kinase at tyrosine residue 418 within the catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529 within the carboxyl-terminal regulatory region. Phosphotransferase activities of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src kinase activity and phosphorylations at Src Tyr-418 and epidermal growth factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in response to 100 nm D-t-EtDO-P4 compared with control cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529 residues of c-Src were reversed by treatment of 4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine (PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were maintained. Compared with untreated cells, tyrosine phosphorylation of phospholipase C-gamma1 was enhanced by EGF stimulation in glucosylceramide-depleted cells, associated with enhanced tyrosine phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 in control cells, whereas in glucosylceramide-depleted cells, suppression of Src kinase activity by PP2 toward EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 was less significant. Thus the activation of Src kinase by depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a critical up-stream event in the activation of phospholipase C-gamma1.     

c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin enhances pancreatic adenocarcinoma cell adhesion to extracellular matrix components

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member with a diversity of extracellular ligands that is implicated in the initiation and progression of a variety of malignancies. We sought to characterize the effects of CEACAM6 crosslinking on pancreatic adenocarcinoma cellular interaction with the extracellular matrix (ECM) components fibronectin and vitronectin. Antibody-mediated CEACAM6 crosslinking was performed and the ability of BxPC3 cells, which inherently overexpress CEACAM6, to adhere to fibronectin and vitronectin was quantified. The roles of the archetypal fibronectin (alpha5beta1 integrin) and vitronectin (alphavbeta3 integrin) receptors were determined. The effects of c-Src inhibition were investigated using the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and c-Src specific RNA interference. CEACAM6 crosslinking initiates c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin, leading to increased ECM component adhesion. CEACAM6-mediated signaling events may contribute to the invasive and metastatic potential of pancreatic adenocarcinoma cells by promoting their interaction with ECM components.     

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.     

Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531

We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).     

Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2

Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H(2)O(2)). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H(2)O(2) to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.     

Src tyrosine kinase inhibitor PP2 markedly enhances Ras-independent activation of Raf-1 protein kinase by phorbol myristate acetate and H2O2

Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026-1033). In this study we have demonstrated that PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H2O2 to activate Raf-1 kinase, whereas it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H2O2 treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H2O2.     

Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3-L1 cells.

Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.     

Interleukin-1beta, Src- and non-Src tyrosine kinases, and nitric oxide synthase induction in rat aorta in vitro

We studied the potential roles for endogenous interleukin-1beta (IL-1beta) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1beta augmented, whereas the IL-1beta receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1beta mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3' kinase inhibition did not block iNOS induction, whereas nuclear factor kappaB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1beta mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1beta. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-1beta mRNA but blocked both the IL-1beta-mediated and spontaneous induction of iNOS. We conclude that 1) the upregulation of tissue IL-1beta, via a signaling pathway involving a Src family kinase, plays a key role in rat vascular iNOS induction and 2) non-Src tyrosine kinases play roles downstream from IL-1beta for iNOS induction.     

Role of Yes kinase during early zebrafish development

We have identified the Yes kinase in zebrafish eggs and investigated its role in development of the zebrafish embryo. In situ hybridization as well as immunofluorescence techniques demonstrated that Yes kinase is maternally expressed and is localized to the cortical region of the unfertilized egg. Fertilization resulted in concentration of Yes kinase to the blastodisc where it continued to be localized to the blastoderm cells through cleavage, gastrulation, and later development. Yes kinase activity was found to decrease abruptly at fertilization, then increase progressively during epiboly, and was maintained at high levels throughout gastrulation. The role of Yes kinase in development was tested by treating embryos with chemical protein tyrosine kinase (PTK) inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) and by injection of antisense morpholinos. Both treatments resulted in the arrest of development at the beginning of the epiboly. Co-immunoprecipitation studies demonstrated that Yes kinase participates in a stable complex with focal adhesion kinase (FAK), which is phosphorylated in vitro. These results demonstrate that Yes kinase plays an important role in epiboly and indicate that Yes kinase participates in signaling by focal adhesion kinase during early development.     

Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.     

Synthesis,biological evaluation and docking studies of new pyrrolo[2,3-d] pyrimidine derivatives as Src family-selective tyrosine kinase inhibitors

In this study, the synthesis and potential enzyme interactions of new Pyrrolo[2,3-d]pyrimidine derivatives along with their inhibitory activity against SFK enzymes such as Fyn, Lyn, Hck, and c-Src were reported. The results indicated that compounds were slightly active of tested SFK enzymes in comparison with PP2 for Fyn, A-419259 for Lyn and CGP77675 for c-Src. Compound N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-4-(3,4-dimethoxyphenyl)butanamide (5) was identified as a non-selective slight inhibitor against Fyn, Lyn and c-Src. However, compounds did not show any inhibitory effects on Hck. Docking studies were performed to analyze the binding mode of compounds against SFKs. The best interaction was obtained between compound 5 and the active site of Fyn and c-Src enzymes in comparison with reference compounds PP2 and CGP77675, respectively.     

Antitumor activity of new pyrazolo[3,4-d]pyrimidine SRC kinase inhibitors in Burkitt lymphoma cell lines and its enhancement by WEE1 inhibition

Burkitt lymphoma (BL) is a highly aggressive B cell neoplasm. Although intensive polychemotherapy regimens have proven very effective, they are associated with significant toxicities. Therefore, more rational therapies that selectively target the molecular abnormalities of BL are needed. Recent data suggest that the tyrosine kinase SRC could represent a therapeutic target for BL. We found that new pyrazolo[3,4-d]pyrimidine SRC inhibitors exerted a significant cytotoxic effect and induced apoptosis on two BL cell lines, as determined by MTS assays, cytofluorimetric analyses and caspase 3 assay. Notably, our SRC inhibitors proved to be more effective than the well-known SRC inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine] in BL cells. Moreover, our small molecules induced a G₂/M arrest in BL cells through a possible new mechanism, whereby SRC inhibition hinders an AKT-WEE1-cyclin-dependent kinase 1 (CDK1) axis, leading to inhibition of CDK1, the main trigger of entry into mitosis. By using a small-molecule inhibitor of WEE1, a crucial CDK1 negative regulator, we were able to shift the balance toward apoptosis rather than growth arrest and enhance the efficacy of the SRC inhibitors, suggesting a possible use of these selective drugs in combination for a safe and efficient treatment of BL.     

Peroxynitrite enhances astrocytic volume-sensitive excitatory amino acid release via a src tyrosine kinase-dependent mechanism

Volume-regulated anion channels (VRACs) are critically important for cell volume homeostasis, and under pathological conditions contribute to neuronal damage via excitatory amino (EAA) release. The precise mechanisms by which brain VRACs are activated and/or modulated remain elusive. In the present work we explored the possible involvement of nitric oxide (NO) and NO-related reactive species in the regulation of VRAC activity and EAA release, using primary astrocyte cultures. The NO donors sodium nitroprusside and spermine NONOate did not affect volume-activated d-[3H]aspartate release. In contrast, the peroxynitrite (ONOO-) donor 3-morpholinosydnomine hydrochloride (SIN-1) increased volume-dependent EAA release by approx. 80-110% under identical conditions. Inhibition of ONOO- formation with superoxide dismutase completely abolished the effects of SIN-1. Both the volume- and SIN-1-induced EAA release were sensitive to the VRAC blockers NPPB and ATP. Further pharmacological analysis ruled out the involvement of cGMP-dependent reactions and modification of sulfhydryl groups in the SIN-1-inducedmodulation of EAA release. The src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2), but not its inactive analog PP3, abolished the effects of SIN-1. A broader spectrum tyrosine kinase inhibitor tyrphostin A51, also completely eliminated the SIN-1-induced EAA release. Our data suggest that ONOO- up-regulates VRAC activity via a src tyrosine kinase-dependent mechanism. This modulation may contribute to EAA-mediated neuronal damage in ischemia and other pathological conditions favoring cell swelling and ONOO- production.