A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg(2+)-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg(2+)-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.     

Opposite effects of pressurized steady versus pulsatile perfusion on vascular endothelial cell cytosolic pH: role of tyrosine kinase and mitogen-activated protein kinase signaling

Endothelial cytosolic pH (pH(i)) modulates ion channel function, vascular tone, and cell proliferation. Steady shear induces rapid acidification in bicarbonate buffer. However, in vivo shear is typically pulsatile, potentially altering this response. We tested effects and mechanisms of pH(i) modulation by flow pulsatility, comparing pressurized steady versus pulse-flow responses in bovine aortic endothelial cells cultured within glass capillary tubes. Cells were loaded with the fluorescent pH(i) indicator carboxy seminaphthorhodafluor-1 and perfused with physiological pulsatile pressure and flow generated by a custom servo-control system. Raising mean pressure from 0 to 90 mm Hg at 0.5 mL/min steady flow in bicarbonate buffer induced sustained acidification (-0.33+/-0.09 pH units, P<0.01). A subsequent increase in steady flow resulted in further acidification. In contrast, if mean pressure and flow were unchanged but perfusion made pulsatile, pH(i) rose +0.3+/-0.03 (P<0. 0001) over 30 to 60 minutes. HCO(3)(-) removal and use of acid/base exchange inhibitors 5-(N-ethyl-N-isopropyl)amiloride or diisothiocyanato stilbene disulfonic acid identified both extracellular Na(+)-independent Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers as activated by static pressure, whereas pulsatility activated extracellular Na(+)-dependent Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers to raise pH(i). Pulse-perfusion alkalinization occurred with or without flow reversal and increased 1.6-fold in Ca(2+)-free buffer. Inhibition of c-Src tyrosine kinase (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo [3,4-d]pyrimidine; PP2) or MEK-1 (mitogen-activated protein kinase [MAP]/extracellular signal-regulated kinase [ERK]-1) (PD98059, blocking ERK1/2) blocked or reversed the pulsatile-flow pH(i) change to acidification. In contrast, PP2 had no effect on steady flow acidification, whereas MEK-1 inhibition converted it to alkalinization. Thus, pulsatile and steady flow trigger opposite effects on endothelial pH(i) by differential activation of acid/base exchangers linked to c-Src and MAP kinase phosphorylation, but not to Ca(2+). These data highlight specific signaling responses triggered by phasic shear profiles.     

Shp2 negatively regulates growth in cardiomyocytes by controlling focal adhesion kinase/Src and mTOR pathways

The aim of this study was to investigate whether Shp2 (Src homology region 2, phosphatase 2) controls focal adhesion kinase (FAK) activity and its trophic actions in cardiomyocytes. We show that low phosphorylation levels of FAK in nonstretched neonatal rat ventricular myocytes (NRVMs) coincided with a relatively high basal association of FAK with Shp2 and Shp2 phosphatase activity. Cyclic stretch (15% above initial length) enhanced FAK phosphorylation at Tyr397 and reduced FAK/Shp2 association and phosphatase activity in anti-Shp2 precipitates. Recombinant Shp2 C-terminal protein tyrosine phosphatase domain (Shp2-PTP) interacted with nonphosphorylated recombinant FAK and dephosphorylated FAK immunoprecipitated from NRVMs. Depletion of Shp2 by specific small interfering RNA increased the phosphorylation of FAK Tyr397, Src Tyr418, AKT Ser473, TSC2 Thr1462, and S6 kinase Thr389 and induced hypertrophy of nonstretched NRVMs. Inhibition of FAK/Src activity by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2, and S6 kinase, as well as the hypertrophy of NRVMs induced by Shp2 depletion. Inhibition of mTOR (mammalian target of rapamycin) with rapamycin blunted the hypertrophy in NRVMs depleted of Shp2. NRVMs treated with PP2 or depleted of FAK by specific small interfering RNA were defective in FAK, Src, extracellular signal-regulated kinase, AKT, TSC2, and S6 kinase phosphorylation, as well as in the hypertrophic response to prolonged stretch. The stretch-induced hypertrophy of NRVMs was also prevented by rapamycin. These findings demonstrate that basal Shp2 tyrosine phosphatase activity controls the size of cardiomyocytes by downregulating a pathway that involves FAK/Src and mTOR signaling pathways.     

5-hydroxytryptamine 2B receptor regulates cell-cycle progression: cross-talk with tyrosine kinase pathways

In this paper, we present evidence that activation of 5-hydroxytryptamine 2B (5-HT2B) receptors by serotonin (5-HT) leads to cell-cycle progression through retinoblastoma protein hyperphosphorylation and through activation of both cyclin D1/cdk4 and cyclin E/cdk2 kinases by a mechanism that depends on induction of cyclin D1 and cyclin E protein levels. The induction of cyclin D1 expression, but not that of cyclin E, is under mitogen-activated protein kinase (MAPK) control, indicating an independent regulation of these two cyclins in the 5-HT2B receptor mitogenesis. Moreover, by using the specific platelet-derived growth factor receptor (PDGFR) inhibitor AG 1296 or by overexpressing a kinase-mutant PDGFR, we show that PDGFR kinase activity is essential for 5-HT2B-triggered MAPK/cyclin D1, but not cyclin E, signaling pathways. 5-HT2B receptor activation also increases activity of the Src family kinase, c-Src, Fyn, and c-Yes. Strikingly, c-Src, but not Fyn or c-Yes, is the crucial molecule between the G(q) protein-coupled 5-HT2B receptor and the cell-cycle regulators. Inhibition of c-Src activity by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) or depletion of c-Src is sufficient to abolish the 5-HT-induced (i) PDGFR tyrosine kinase phosphorylation and MAPK activation, (ii) cyclin D1 and cyclin E expression levels, and (iii) thymidine incorporation. This paper elucidates a model of 5-HT2B receptor mitogenesis in which c-Src acts alone to control cyclin E induction and in concert with the receptor tyrosine kinase PDGFR to induce cyclin D1 expression via the MAPK/ERK pathway.     

Transactivation of fetal liver kinase-1/kinase-insert domain-containing receptor by lysophosphatidylcholine induces vascular endothelial cell proliferation

Lysophosphatidylcholine (LPC), a major lipid component of oxidized low-density lipoprotein, is a bioactive lipid molecule involved in numerous biological processes including the progression of atherosclerosis. Recently orphan G protein-coupled receptors were identified as high-affinity receptors for LPC. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, LPC-stimulated transactivation of receptor tyrosine kinase has not yet been reported. Here we observed for the first time that LPC treatment of human umbilical vein endothelial cells (HUVECs) induces tyrosyl phosphorylation of vascular endothelial growth factor receptor 2 [fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)]. Flk-1/KDR transactivation by LPC was inhibited by vascular endothelial growth factor receptor tyrosine kinase inhibitors, SU1498 and 4-[(4'-chloro-2'-fluoro) phenylamino]6,7-dimethoxyquinazoline (VTKi) in immunoprecipitation. Furthermore, we examined the effects of the Src family kinases inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), on LPC-induced Flk-1/KDR transactivation. Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation because herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from Western blots, ERK1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also activated by LPC, and this was inhibited by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in HUVECs. LPC-induced stimulation of HUVEC proliferation was shown to be secondary to transactivation because it was suppressed by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in dimethylthiazoldiphenyltetra-zoliumbromide assay. These findings suggest that LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the progression of atherosclerosis.     

Distinct pathways regulate expression of cardiac electrical and mechanical junction proteins in response to stretch

To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by approximately 2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-beta1 and anti-beta3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.     

Role of protein kinase C- or RhoA-induced Ca(2+) sensitization in stretch-induced myogenic tone

OBJECTIVE: It has been suggested that Ca(2+) sensitization mechanisms might contribute to myogenic tone. However, specific mechanisms have yet to be fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced Ca(2+) sensitization in myogenic tone of the rabbit basilar vessel. METHODS: Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 Ca(2+) signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. RESULTS: Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) only in the presence of extracellular Ca(2+). Stretch evoked greater contraction than high K(+) at a given [Ca(2+)](i). The stretch-induced increase in [Ca(2+)](i) and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent Ca(2+) channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing [Ca(2+)](i). Immunoblotting using isoform-specific antibodies showed the presence of PKCalpha and PKCepsilon in the rabbit basilar artery. PKCalpha, but not PKCepsilon, and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. CONCLUSIONS: Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of PKCalpha and/or RhoA may be key mechanisms for the Ca(2+) sensitization associated myogenic tone in basilar vessels.     

Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)

Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC(50) in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.     

Ca(2+)-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.     

Ca-sensitive tyrosine kinase Pyk2/CAK β-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.     

Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts

OBJECTIVE: Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex. METHODS AND RESULTS: In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 muM followed by an 8-minute washout before 30 min of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cgamma1/protein kinase Cepsilon (PLC-gamma1/PKCepsilon) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-gamma1/PKCepsilon pathway, but also cardiac protection. This protection was also blocked by a PKCepsilon translocation inhibitor peptide (PKCepsilon TIP). CONCLUSION: Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-gamma1 and PKCepsilon.     

Zinc induces a Src family kinase-mediated up-regulation of NMDA receptor activity and excitotoxicity

Zinc is coreleased with glutamate from excitatory nerve terminals throughout the central nervous system and acutely inhibits N-methyl-d-aspartate (NMDA) receptor activation. Here we report that cultured murine cortical neurons briefly exposed to sublethal concentrations of zinc developed increased intracellular free Na(+), phosphorylation of Src kinase at tyrosine 220, and tyrosine phosphorylation of NMDA receptor 2A/2B subunits, in a fashion sensitive to the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, PP2. Functionally, this zinc exposure produced a delayed increase in NMDA receptor current in perforated patch but not conventional whole-cell recordings, as well as an increase in NMDA receptor-mediated cell death. These observations suggest that the effect of synaptically released zinc on neuronal NMDA receptors may be biphasic: acute block, followed by Src family kinase-mediated up-regulation of NMDA receptor activity and cytotoxicity.     

Differential D1 and D5 receptor regulation and degradation of the angiotensin type 1 receptor

Renal sodium transport is increased by the angiotensin type 1 receptor (AT(1)R), which is counterregulated by dopamine via unknown mechanisms involving either the dopamine type 1 (D(1)R) or dopamine type 5 receptor (D(5)R) that belong to the D(1)-like receptor family of dopamine receptors. We hypothesize that the D(1)R and D(5)R differentially regulate AT(1)R protein expression and signaling, which may have important implications in the pathogenesis of essential hypertension. D(1)R and D(5)R share the same agonists and antagonists; therefore, the selective effects of either D(1)R or D(5)R stimulation on AT(1)R expression in human renal proximal tubule cells were determined using antisense oligonucleotides selective to either D(1)R or D(5)R. We also determined the role of receptor tyrosine kinase and the proteosome on the D(1)R/D(5)R-mediated effects on AT(1)R expression and internalization. In renal proximal tubule cells, D(5)R (not D(1)R) decreased AT(1)R expression (half-life: 0.47+/-0.18 hours) and AT(1)R-mediated extracellular signal-regulated kinase 1/2 phosphorylation (232+/-18.9 U with angiotensin II [10(-7) mol/L] versus 81+/-8.9 U with angiotensin II [10(-7) mol/L] and fenoldopam [D(1)R/D(5)R agonist; 10(-6) mol/L; P<0.05; n=6). The fenoldopam-induced decrease in AT(1)R expression was reversed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3,4-d) pyrimidine (c-Src tyrosine-kinase inhibitor) and clasto-lactacystin beta-lactone (proteasome inhibitor), demonstrating that the fenoldopam-mediated decrease in total cell AT(1)R expression is a result of a c-Src- and proteasome-dependent process. D(5)R stimulation decreases AT(1)R expression and is c-Src and proteasome dependent. The discovery of differential regulation by D(1)R and D(5)R opens new avenues for the development of agonists selective to either receptor subtype as targeted antihypertensive agents that can decrease AT(1)R-mediated antinatriuresis.     

Agonist-stimulated endothelial nitric oxide synthase activation and vascular relaxation. Role of eNOS phosphorylation at Tyr83

Tyr83 in endothelial nitric oxide synthase (eNOS) has been identified previously as a site of Src kinase-mediated phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) that is phosphorylated in response to oxidant stress. In the present study, we have used a phospho-specific antibody to show that Tyr83 in eNOS is also phosphorylated in both BAECs and intact blood vessel segments in response to treatment with a variety of different eNOS-activating agonists, including thapsigargin, vascular endothelial growth factor, bradykinin, ATP, sphingosine-1-phosphate, estrogen, angiopoietin, and acetylcholine. Agonist stimulation of eNOS Tyr83 phosphorylation as well as agonist stimulation of endothelial NO release in BAECs is blocked by Src kinase inhibition by either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) or by dominant negative Src. Mutation of Tyr83 to a nonphosphorylatable Phe blocks agonist stimulation of NO release from eNOS-reconstituted eNOS knockdown endothelial cells. Mutation of Tyr83 also attenuates agonist-induced relaxation of eNOS-reconstituted aortic rings from eNOS knockout mice. Phosphorylation of eNOS at Tyr83 thus appears to be a common covalent modification that is induced, not only by oxidant stress but also by other physiologically relevant extracellular signals known to be important in regulation of eNOS activity in vivo. Moreover, our results demonstrate an important role for Src-mediated phosphorylation of eNOS at Tyr83 in agonist stimulation of eNOS activation and vascular relaxation.     

Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells

The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.     

Angiotensin II and myosin light-chain phosphorylation contribute to the stretch-induced slow force response in human atrial myocardium

AIMS: Stretch is an important regulator of atrial function. The functional effects of stretch on human atrium, however, are poorly understood. Thus, we characterized the stretch-induced force response in human atrium and evaluated the underlying cellular mechanisms. METHODS AND RESULTS: Isometric twitch force of human atrial trabeculae (n = 252) was recorded (37 degrees C, 1 Hz stimulation) following stretch from 88 (L88) to 98% (L98) of optimal length. [Na(+)](i) and pH(i) were measured using SBFI and BCECF epifluorescence, respectively. Stretch induced a biphasic force increase: an immediate increase [first-phase, Frank-Starling mechanism (FSM)] to approximately 190% of force at L88 followed by an additional slower increase [5-10 min; slow force response (SFR)] to approximately 120% of the FSM. FSM and SFR were unaffected by gender, age, ejection fraction, and pre-medication with major cardiovascular drugs. There was a positive correlation between the amplitude of the FSM and the SFR. [Na(+)](i) rose by approximately 1 mmol/L and pH(i) remained unchanged during the SFR. Inhibition of Na(+)/H(+)-exchange (3 microM HOE642), Na(+)/Ca(2+)-exchange (5 microM KB-R7943), or stretch-activated channels (0.5 microM GsMtx-4 and 80 microM streptomycin) did not reduce the SFR. Inhibition of angiotensin-II (AngII) receptors (5 microM saralasin and 0.5 microM PD123319) or pre-application of 0.5 microM AngII, however, reduced the SFR by approximately 40-60%. Moreover, stretch increased phosphorylation of myosin light chain 2 (MLC2a) and inhibition of MLC kinase (10 microM ML-7 and 5 microM wortmannin) decreased the SFR by approximately 40-85%. CONCLUSION: Stretch elicits a SFR in human atrium. The atrial SFR is mediated by stretch-induced release and autocrine/paracrine actions of AngII and increased myofilament Ca(2+) responsiveness via phosphorylation of MLC2a by MLC kinase.     

Implication of Gbetagamma proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes

Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca(2+) (EGTA, 0.5 mM) and chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetoxymethyl ester (BAPTA) (5 microM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca(2+) is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Galphas and Gbeta, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gbeta subunits by preincubation of cells with a Gbeta antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gbetagamma-c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.     

Role of EGF Receptor and Pyk2 in endothelin-1-induced ERK activation in rat cardiomyocytes

G protein-coupled receptor (GPCR)-evoked signal transduction pathways leading to the activation of extracellular signal-regulated kinases (ERK) are quite different among cell types. In cardiomyocytes, much attention has been focused on the activation of protein kinase C (PKC) or mobilization of intracellular Ca(2+) ([Ca(2+)](i)), however, the contributions of tyrosine kinases are controversial. In the present study, we characterized the signaling pathways involving tyrosine kinases, Pyk2 and epidermal growth factor receptor (EGFR), and their contribution to ERK activation in cultured cardiomyocytes. We initially investigated the potential involvement of [Ca(2+)](i) and PKC on the activation of these kinases in endothelin-stimulated cardiomyocytes. Interestingly, activation of Pyk2 was abrogated by chelating [Ca(2+)](i) or by downregulation of PKC, whereas transactivation of EGFR was solely dependent on PKC. By using a compound that selectively interferes with EGFR (AG1478), c-Src (PP1), or disrupts actin cytoskeleton (cytochalasin D), we demonstrated that cytochalasin D completely inhibited the activation of Pyk2, but not that of EGFR, whereas AG1478 did not inhibit the activation of Pyk2, indicating that transactivation of EGFR and signaling pathways involving Pyk2 were distinct pathways. Furthermore, activation of ERK and Shc, and c- fos gene expression were significantly inhibited by AG1478 but not by cytochalasin D or PP1. Overexpression of deletion mutant of EGFR attenuated the activation of ERK. These facts demonstrated the existence of two distinct tyrosine kinase pathways requiring Pyk2 or EGFR downstream from GPCR in cardiomyocytes. EGFR was Ca(2+)-independently activated and predominantly contributed to Shc/ERK/c- fos activation, while Pyk2 or c-Src contributed less to it.     

Insulin-induced oxidative stress up-regulates heme oxygenase-1 via diverse signaling cascades in the C2 skeletal myoblast cell line

Impaired insulin sensitivity (insulin resistance) is a common denominator in many metabolic disorders, exerting pleiotropic effects on skeletal muscle, liver, and adipose tissue function. Heme oxygenase-1 (HOX-1), the rate-limiting enzyme in heme catabolism, has recently been shown to confer an antidiabetic effect while regulating cellular redox-buffering capacity. Therefore, in the present study, we probed into the mechanisms underlying the effect of insulin on HOX-1 in C2 skeletal myoblasts. Hence, insulin was found to suppress C2 myoblasts viability via stimulation of oxidative stress, with HOX-1 counteracting this action. Insulin induced HOX-1 expression in a time- and dose-dependent manner, an effect attenuated by selective inhibitors of ERK1/2 (PD98059), Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and c-Jun terminal kinases 1 and 2 (SP600125) pathways. Furthermore, nuclear factor-κB role in insulin-induced HOX-1 up-regulation was verified, with ERK1/2, Src, and c-Jun terminal kinases 1 and 2 mediating p65-nuclear factor-κB subunit phosphorylation. Overall, our novel findings highlight for the first time the transduction mechanisms mediating HOX-1 induction in insulin-treated C2 myoblasts. This effect was established to be cell type specific because insulin failed to promote HOX-1 expression in HepG2 hepatoma cells. Deciphering the signaling networks involved in insulin-stimulated HOX-1 up-regulation is of prominent significance because it may potentially contribute to elucidation of the mechanisms involved in associated metabolic pathologies.