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.     

Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells

The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G(i)-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCbeta activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCbeta. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.     

区域性血管床对局部注射植物雌激素三羟异黄酮的反应

在72只麻醉大鼠,分别采用后肢、肾脏和肠系膜动脉在体恒流灌注法,观察了向灌流环路中直接注射植物雌激素三羟异黄酮(genistein,GST)的血管效应,以所引起的灌流压增减反映血管的收缩和舒张。结果如下：(1)不同剂量的GST(0．4、0．8、1．2mg／k8)注射于股部灌注环路时,剂量依赖性地降低股动脉的灌流压。GST的这一效应可被L-硝基精氨酸甲酯(L-NAME)部分阻断,预先注射蛋白酪氨酸磷酸酶抑制剂正钒酸钠(50μg/kg),可部分抑制GST(0．8mg／kg)引起的效应;(2)向肾血管灌注环路中直接注射GST也可剂量依赖性地降低肾动脉的灌流压,预先注射正钒酸钠可完全抑制GST引起的效应,而L-NAME对此效应没有影响;(3)肠系膜血管灌流环路中注射GST可剂量依赖性地降低其灌流压,这一效应可被正钒酸钠部分抑制,而L-NAME对此无影响。根据上述结果得出的结论是：GST降低后肢、肾脏和肠系膜血管床的血管张力,其机制与酪氨酸激酶抑制有关,而在股动脉则与NO释放有部分关系。     

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.     

Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase

The aim of the present study was to investigate whether/how the recombinant human cardiac I_Ks could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I_Ks, and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I_Ks stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.     

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 muM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 muM) and the Src-family kinase inhibitor PP2 (10 muM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.     

Genistein and tyrphostin AG556 inhibit inwardly-rectifying Kir2.1 channels expressed in HEK 293 cells via protein tyrosine kinase inhibition

Previous studies reported the controversial effects that protein tyrosine kinase (PTK) inhibition could induce an up-regulation or down-regulation of Kir2.1 current. The present study investigates how the recombinant human Kir2.1 channels are regulated by PTKs using whole-cell patch voltage-clamp, immunoprecipitation and Western blot, and mutagenesis approaches. We found that hKir2.1 current was reversibly inhibited by the broad spectrum PTK inhibitor genistein and the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 in a concentration-dependent manner. The inhibition of hKir2.1 channels by genistein or AG556 was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of Kir2.1 channels was reduced by genistein or AG556, and the reduction was significantly antagonized by orthovanadate. The mutation of Y242 dramatically reduced the inhibitory response to AG556. The results obtained in this study demonstrate that hKir2.1 channels are down-regulated by PTK inhibition, suggesting that EGFR kinase participates in the modulation of human cardiac excitability.     

Budding yeast Cdc5 phosphorylates Net1 and assists Cdc14 release from the nucleolus

Lysophosphatidic acid (LPA) has been shown to be a potent mitogen for vascular smooth muscle cells. Src-dependent transactivation of receptor tyrosine kinases has been previously demonstrated to mediate LPA-induced activation of MAP kinase ERK1/2. Furthermore, generation of reactive oxygen species (ROS) by LPA is also known to contribute to MAP kinase activation. Rho family small G-proteins Rac and Cdc42, and their immediate downstream effector p21-activated kinase (PAK), have been demonstrated to mediate important effects on the cytoskeleton that are relevant for cell migration and proliferation. In the present report we evaluated stimulation of PAK by LPA in rat aortic vascular smooth muscle cells (VSMC) by PAK immunocomplex MBP in-gel kinase assay. LPA increased PAK activity 3-fold, peaking at 5 min and showing sustained activation up to 45 min. Inhibition of tyrosine kinases by pretreatment of VSMC with genistein or specific inhibition of Src by PP1 greatly diminished LPA-induced PAK activation, whereas specific inhibition of PDFG- and EGF receptor kinase by tyrphostin AG1296 and AG1478 had no effect. Furthermore, inhibition of Galpha(i) by pertussis toxin and inhibition of NADH/NADPH oxidase by diphenylene iodonium also diminished LPA-induced stimulation of PAK. This is the first study to demonstrate that LPA activates PAK. In VSMC, PAK activation by LPA is mediated by Galpha(i) and is dependent on Src, whereas EGF- or PDGF receptor transactivation are not involved. Furthermore, generation of ROS is required for LPA-induced activation of PAK.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

Paralytic peptide activates insect humoral immune response via epidermal growth factor receptor

Paralytic peptide (PP) activates innate immunity of silkworm Bombyx mori, inducing production of anti-microbial peptides (AMPs) and phagocytosis-related proteins; however the signal pathways of PP-dependent immune responses are not clear. In present study, we characterized BmE cells as a PP-responsive cell line by examining the expression of AMP genes and activation of p38 mitogen-activated protein kinase (p38 MAPK) under PP stimulation, and we also found PP directly binds to BmE cell membrane. Then we found that PP-dependent expression of AMP genes is suppressed by tyrosine kinase inhibitor (genistein) both in BmE cells and in fat body of silkworm larvae. Moreover, the specific tyrosine kinase epidermal growth factor receptor (EGFR) inhibitor (AG1478) attenuates PP-induced expression of AMP genes in BmE cells and fat body of silkworm and RNA interference (RNAi) to BmEGFR also suppresses PP-induced expression of AMP genes. Furthermore, the PP-induced p38 MAPK phosphorylation is inhibited by AG1478. Our results suggest that BmE cells can be used as a cell model to investigate the signal pathway of PP-dependent humoral immune response and receptor tyrosine kinase EGFR/p38 MAPK pathway is involved in the production of AMPs induced by PP.     

Interleukin-6 increases rat metalloproteinase-13 gene expression through Janus kinase-2-mediated inhibition of serine/threonine phosphatase-2A

Interleukin-6 (IL-6) increases metalloproteinase-13 (MMP-13) gene expression by increasing phosphorylated c-Jun and by inhibiting serine/threonine phosphatase-2A (PP2A) activity. We investigated the mechanisms by which IL-6 induces c-Jun phosphorylation and PP2A inactivation in Rat-1 fibroblasts. We show that IL-6 increased MMP-13 mRNA, phosphorylated c-Jun, and activator protein 1 (AP1) binding activity without increasing c-Jun-N-terminal kinase (JNK) activity. These effects did not seem to be mediated by ERK, p38 MAP kinase, phosphatidylinositol-3-kinase, calmoduline-dependent protein kinase, protein kinase C (PKC) or protein kinase A since inhibition with specific inhibitors did not abrogate these effects. IL-6 increases PP2A catalytic subunit tyrosine phosphorylation. Inhibition of the tyrosine kinase Jak2, with the specific inhibitor AG490, abrogated this effect. Likewise, this Jak2 inhibitor blocked the effects of IL-6 on c-Jun phosphorylation, AP1 binding activity and metalloproteinase-13 gene expression. We conclude that IL-6 increases MMP-13 gene expression by activation of Jak2, resulting in tyrosine phosphorylation of the catalytic subunit of PP2A, which in turn decreases PP2A activity and prolongs c-Jun phosphorylation.     

Transactivation of epidermal growth factor receptor by enhanced levels of endogenous angiotensin II contributes to the overexpression of Giα proteins in vascular smooth muscle cells from SHR

We earlier showed that the increased expression of Gi proteins exhibited by vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) was attributed to the enhanced levels of endogenous endothelin. Since the levels of angiotensin II (Ang II) are also enhanced in VSMC from SHR, the present study was undertaken to examine the role of enhanced levels of endogenous Ang II in the overexpression of Giα proteins in VSMC from SHR and to further explore the underlying mechanisms responsible for this increase. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR compared to WKY was attenuated by the captopril, losartan and AG1478, inhibitors of angiotensin converting enzyme, AT₁ receptor and epidermal growth factor receptor (EGFR) respectively as well as by the siRNAs of AT1, cSrc and EGFR. The enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent functions) and of inhibitory responses of hormones on adenylyl cyclase activity (receptor-dependent functions) in VSMC from SHR was also attenuated by losartan. Furthermore, the enhanced phosphorylation of EGFR in VSMC from SHR was also restored to control levels by captopril, losartan, PP2, a c-Src inhibitor and N-acetyl-L-cysteine (NAC), superoxide anion (O₂⁻) scavenger, whereas enhanced ERK1/2 phosphorylation was attenuated by captopril and losartan. Furthermore, NAC also restored the enhanced phosphorylation of c-Src in SHR to control levels. These results suggest that the enhanced levels of endogenous Ang II in VSMC from SHR, transactivate EGFR, which through MAP kinase signaling, enhance the expression of Giα proteins and associated adenylyl cyclase signaling.     

Contractile Force Is Enhanced in Aortas from Pendrin Null Mice Due to Stimulation of Angiotensin II-Dependent Signaling

Pendrin is a Cl⁻/HCO₃ ⁻ exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca²⁺ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation.     

ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC as well as EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse embryonic stem cells

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [(3)H] thymidine incorporation in a time- (>4 h) and dose- (>10(-9) M) dependent manner. The ANG II-induced increase in [(3)H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT(1)) receptor but not by ANG II type 2 (AT(2)) receptor, and AT(1) receptor was expressed. ANG II increased inositol phosphates formation and [Ca(2+)](i), and translocated PKC alpha, delta, and zeta to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [(3)H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [(3)H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [(3)H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT(1) receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21(cip1/waf1) and p27(kip1), CDK inhibitory proteins. These proteins were blocked by the inhibition of AT(1) receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells.     

Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells.

Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.     

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.