Inhibition of L-Type Cav1.2 Ca2+ Channels by 2,(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one(LY294002) and 2-[1-(3-Dimethyl-aminopropyl)-5
Institut fe Pharmakologie und Toxikologie, Technische Universitt Mechen, Mechen, Germany
Abstract
Phosphatidylinositol 3-kinase (PI3-K) is involved in physiological processes of cellular proliferation and inflammation and, as postulated recently, in the regulation of L-type Ca2+ channels. The latter conclusion arose in part from the inhibitory action of the compound 2,(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), which has been established as a selective PI3-K inhibitor (IC50 = 1.4 e). Herein we show, however, that LY294002 and an inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide (G6983), act as direct Ca2+-channel inhibitors, with IC50 values of approximately 20 and 10 e, respectively. Because both drugs are commonly used at concentrations of approximately 10 e or higher, the interpretation of such experiments is questionable with respect to a regulatory action of PI3-K or PKC on L-type Ca2+ channels.
L-type Ca2+-channel activity is crucial for heartbeat and smooth muscle tonus. Recent evidence has shown that, in both tissues, L-type Ca2+ channels are modulated by the phosphatidylinositol 3-kinase (PI3-K) pathway, which is mainly known to be involved in cell proliferation and inflammation (Wymann et al., 2003). In smooth muscle, PI3-K was reported to trigger activation of L-type Ca2+ channels after stimulation of acetylcholine and angiotensin-II receptors (Quignard et al., 2001; Callaghan et al., 2004; Le Blanc et al., 2004). In heart muscle, PI3-K has been proposed to regulate excitation-contraction coupling (McDowell et al., 2004). However, most of the evidence either for coupling of cholinergic stimulation to Ca2+-channel activity or for regulating excitation-contraction coupling via PI3-K comes from the inhibitory effect of the compound LY294002, which has been introduced as a selective PI3-K inhibitor (Vlahos et al., 1994). In the present study, we show that LY2934002 and the protein kinase C (PKC) inhibitor G6983 directly inhibit L-type Ca2+-channel activity. Therefore, a cautionary interpretation is recommended when these drugs are used to confirm an influence of the PI3-K and PKC pathways on biological effects.
Materials and Methods
Generation of Cav1.2-Deficient Mice. All experiments complied with the animal protection laws of Germany. The generation of control (CTR) mice and mice deficient in the smooth muscle Cav1.2 calcium channel (smooth muscle 1c-subunit calcium-channel knockout, SMACKO) has been described previously (Moosmang et al., 2003).
Cell Transfection and Culture. HEK 293 cells were stably transfected with the 1b (CaV1.2b) and the 2a subunit of the smooth muscle L-type calcium channel (GenBank accession numbers X55763 [GenBank] and X64298 [GenBank] , respectively) or with only a carboxy-terminaleCtruncated version of the 1b subunit, as described previously (Seisenberger et al., 1995).
Western Blot. Western blot analysis was performed on transfected HEK 293 cells and on brain tissue from mice using an antibody against the p110 subunit of PI-3K (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as described previously (Rong et al., 2003).
Tension Recordings. Muscles strips from murine urinary bladder were prepared as described previously (Wegener et al., 2004). Tension was recorded isometrically at 36 ± 1°C using the myograph 601 (DMT A/S, Aarhus, Denmark).
Current Recordings. L-type Ca2+-channel current was measured in transfected HEK 293 cells by the whole-cell configuration of the patch-clamp technique. Experiments were performed at room temperature using Ba2+ as the charge carrier. The bath solution contained 104 mM NaCl, 20 mM tetraethylammonium chloride, 5.4 mM CsCl, 5 mM BaCl2, 1 mM MgCl2, 1 mM NaH2PO4, 10 mM glucose, and 5 mM HEPES, pH 7.4 (NaOH). The pipette solution contained 112 mM CsCl, 1 mM MgSO4, 3 mM Na2ATP, 10 mM EGTA, and 5 mM HEPES; pH was adjusted to 7.4 with CsOH. The holding potential was eC80 mV. Trains of test pulses were to 0 or +10 mV for 100 ms with 0.2 Hz. Cumulative dose-inhibition curves were measured using two to three different drug concentrations per cell. Drugs were freshly diluted from the stock solution into the bath solution on each experimental day. IC50 values were calculated by fitting the averaged dose-inhibition curves to the Hill equation.
Chemicals. All chemicals used were at least of reagent grade and were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. LY294002, wortmannin, G6983, and G6976 were obtained from Calbiochem (San Diego, CA).
Evaluation of Results. Data are presented as original recordings or expressed as means ± S.E.M. Effects of the drugs were obtained in quasieCsteady-state conditions. Concentration-response curves were fitted using Prism 4.0 software (GraphPad Software Inc., San Diego, CA). Correlation coefficients were >0.95.
Results and Discussion
L-type Cav1.2 calcium-channel activity mediates contraction induced by stimulation of muscarinic receptors in urinary bladder smooth muscle from mice (Wegener et al., 2004). We tested the possibility that the PI3-K pathway is involved in this cascade as described for vascular smooth muscle (Quignard et al., 2001; Callaghan et al., 2004). Therefore, we examined the effects of LY294002, an inhibitor of PI3-K (Vlahos et al., 1994), and of G6983, an inhibitor of PKC isoforms including the PKC isoform (Gschwendt et al., 1996). Both LY294002 (20 e) and G6983 (10 e) inhibited carbachol-induced contractions of bladder muscles from control mice to approximately 50% but not from SMACKO mice (Fig. 1, A and B). The protein kinase C inhibitor G6976 (10 e), which does not act on the PKC isoform (Gschwendt et al., 1996), was without effect (data not shown). These results implied that the PI3-K/PKC pathway is involved in the coupling of muscarinic receptors to CaV1.2 Ca2+ channels, leading to contraction in urinary bladder muscle. However, wortmannin, another inhibitor of PI3-K (IC50 = 5 nM) (Arcaro and Wymann, 1993), did not inhibit carbachol-induced contractions at 100 nM (Fig. 1C). Higher concentrations of wortmannin (10 e) reduced contractions (data not shown), probably because of inhibition of myosin light-chain kinase (IC50 = 200 nM) (Burdyga and Wray, 1999) but not Ca2+ current (Fig. 4C). In addition, in control experiments in which contraction was induced by K+ depolarization, both LY294002 and G6983 reduced contractions, with IC50 values of approximately 18 and 9 e, respectively (Fig. 2). Identical results were obtained with muscle rings from aorta (data not shown). Atropine (1 e), an unselective muscarinic antagonist, did not affect K+-induced contractions in bladder muscle, confirming that muscarinic receptors are not involved in this response. These results suggested a more direct inhibitory action of the drugs on L-type Ca2+ channels because contraction induced by depolarization is completely blocked by dihydropyridines and, thus, primarily mediated by this channel type. Therefore, we tested the effects of both drugs on HEK 293 cells expressing functional L-type Ca2+ channels. Because there exist contrary reports whether or not HEK 293 cells contain endogenous PI3-K activity (Naga Prasad et al., 2001; Brock et al., 2003), Western blot analysis on our transfected HEK cells was performed; no p110 protein of the PI3-K isoform, which is believed to influence Ca2+ channels (Viard et al., 2004), could be detected (Fig. 3). LY294002 and G6983, but not wortmannin, inhibited voltage-activated Ca2+-channel currents in these cells being transfected with 1b plus 2a subunit with IC50 values of 12 and 20 e, respectively (Fig. 4). A similar IC50 value for LY294002 was measured for cells only expressing the 1b subunit (data not shown). No frequency-(0.1 versus 1 Hz) or voltage-dependent effects (eC80 versus eC40 mV holding potential) of the drugs were observed. However, current inactivation was accelerated by LY294002 and by G6983, indicating, at least partially, an interaction with the open channel (Fig. 4); time constants were 0.07 ± 0.005 s (n = 5) and 0.27 ± 0.09 s (n = 3) in the presence of LY294002 (30 e) and G6983 (30 e), respectively.
In summary, the present study shows that both LY294002 and G6983 exhibit Ca2+ antagonistic properties in the concentration range usually applied to selectively block PI3-K (Quignard et al., 2001; Northcott et al., 2002; Wang et al., 2002; Callaghan et al., 2004; McDowell et al., 2004) and PKC (Tao et al., 2003), respectively. Therefore, evidence of a functional coupling of the PI3-K signaling pathway to L-type Ca2+-channel activity is hampered by the inhibitory effects of the compounds LY294002 and G6983 on L-type Ca2+ channels. The side effects of the putative PI3-K inhibitor LY294002, together with its recently described effect on K+ currents (El-Kholy et al., 2003; Sun et al., 2004), limits the usefulness of this drug in the analysis of PI3-K function.
Acknowledgements
We thank S. Paparisto for excellent technical assistance.
This study was supported by Deutsche Forschungsgemeinschaft und Fond der Chemischen Industrie.
doi:10.1124/mol.104.006049.
References
Arcaro A and Wymann MP (1993) Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J 296: 297eC301.
Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, and Nurnberg B (2003) Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma. J Cell Biol 160: 89eC99.
Burdyga TV and Wray S (1999) The relationship between the action potential, intracellular calcium and force in intact phasic, guinea-pig uretic smooth muscle. J Physiol 520: 867eC883.
Callaghan B, Koh SD, and Keef KD (2004) Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol-3-kinase, protein kinase C and c-Src. Circ Res 94: 626eC633.
El-Kholy W, Macdonald PE, Lin JH, Wang J, Fox JM, Light PE, Wang Q, Tsushima RG, and Wheeler MB (2003) The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks KV currents via a direct mechanism. FASEB J 17: 720eC722.
Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, and Johannes FJ (1996) Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase C isoenzymes. FEBS Lett 392: 77eC80.
Le Blanc C, Mironneau C, Barbot C, Henaff M, Bondeva T, Wetzker R, and Macrez N (2004) Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate. Circ Res 95: 300eC307.
McDowell SA, McCall E, Matter WF, Estridge TB, and Vlahos CJ (2004) Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes. Am J Physiol 286: H796eCH805.
Moosmang S, Schulla V, Welling A, Feil R, Feil S, Wegener JW, Hofmann F, and Klugbauer N (2003) Dominant role of smooth muscle L-type calcium channel Cav1.2 for blood pressure regulation. EMBO (Eur Mol Biol Organ) J 22: 6027eC6034.
Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, and Rockman HA (2001) Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by -adrenergic receptor kinase 1. A role in receptor sequestration. J Biol Chem 276: 18953eC18959.
Northcott CA, Poy MN, Najjar SM, and Watts SW (2002) Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats. Circ Res 91: 360eC369.
Quignard JF, Mironneau J, Carricaburu V, Fournier B, Babich A, Nurnberg B, Mironneau C, and Macrez N (2001) Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes. J Biol Chem 276: 32545eC32551.
Rong R, Ahn JY, Huang H, Nagata E, Kalman D, Kapp JA, Tu J, Worley PF, Snyder SH, and Ye K (2003) PI3 kinase enhancer-Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis. Nat Neurosci 6: 1153eC1161.
Seisenberger C, Welling A, Schuster A, and Hofmann F (1995) Two stable cell lines for screening of calcium channel blockers. Naunyn Schmiedeberg's Arch Pharmacol 352: 662eC669.
Sun H, Oudit GY, Ramirez RJ, Costantini D, and Backx PH (2004) The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents. Cardiovasc Res 62: 509eC520.
Tao FC, Shah S, Pradhan AA, Tolloczko B, and Martin JG (2003) Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats. Am J Physiol 284: L90eCL99.
Viard P, Butcher AJ, Halet G, Davies A, Nurnberg B, Heblich F, and Dolphin AC (2004) PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane. Nat Neurosci 7: 939eC946.
Vlahos CJ, Matter WF, Hui KY, and Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269: 5241eC5248.
Wang YG, Dedkova EN, Steinberg SF, Blatter LA, and Lipsius SL (2002) Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes. J Gen Physiol 119: 69eC82.
Wegener JW, Schulla V, Lee TS, Koller A, Feil S, Feil R, Kleppisch T, Klugbauer N, Moosmang S, Welling A, et al. (2004) An essential role of Cav1.2 L-type calcium channel for urinary bladder function. FASEB J 18: 1159eC1161.
Wymann MP, Zvelebil M, and Laffargue M (2003) Phosphoinositide 3-kinase signaling—which way to target Trends Pharmacol Sci 24: 366eC376., 百拇医药(A. Welling, F. Hofmann, a)
Abstract
Phosphatidylinositol 3-kinase (PI3-K) is involved in physiological processes of cellular proliferation and inflammation and, as postulated recently, in the regulation of L-type Ca2+ channels. The latter conclusion arose in part from the inhibitory action of the compound 2,(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), which has been established as a selective PI3-K inhibitor (IC50 = 1.4 e). Herein we show, however, that LY294002 and an inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide (G6983), act as direct Ca2+-channel inhibitors, with IC50 values of approximately 20 and 10 e, respectively. Because both drugs are commonly used at concentrations of approximately 10 e or higher, the interpretation of such experiments is questionable with respect to a regulatory action of PI3-K or PKC on L-type Ca2+ channels.
L-type Ca2+-channel activity is crucial for heartbeat and smooth muscle tonus. Recent evidence has shown that, in both tissues, L-type Ca2+ channels are modulated by the phosphatidylinositol 3-kinase (PI3-K) pathway, which is mainly known to be involved in cell proliferation and inflammation (Wymann et al., 2003). In smooth muscle, PI3-K was reported to trigger activation of L-type Ca2+ channels after stimulation of acetylcholine and angiotensin-II receptors (Quignard et al., 2001; Callaghan et al., 2004; Le Blanc et al., 2004). In heart muscle, PI3-K has been proposed to regulate excitation-contraction coupling (McDowell et al., 2004). However, most of the evidence either for coupling of cholinergic stimulation to Ca2+-channel activity or for regulating excitation-contraction coupling via PI3-K comes from the inhibitory effect of the compound LY294002, which has been introduced as a selective PI3-K inhibitor (Vlahos et al., 1994). In the present study, we show that LY2934002 and the protein kinase C (PKC) inhibitor G6983 directly inhibit L-type Ca2+-channel activity. Therefore, a cautionary interpretation is recommended when these drugs are used to confirm an influence of the PI3-K and PKC pathways on biological effects.
Materials and Methods
Generation of Cav1.2-Deficient Mice. All experiments complied with the animal protection laws of Germany. The generation of control (CTR) mice and mice deficient in the smooth muscle Cav1.2 calcium channel (smooth muscle 1c-subunit calcium-channel knockout, SMACKO) has been described previously (Moosmang et al., 2003).
Cell Transfection and Culture. HEK 293 cells were stably transfected with the 1b (CaV1.2b) and the 2a subunit of the smooth muscle L-type calcium channel (GenBank accession numbers X55763 [GenBank] and X64298 [GenBank] , respectively) or with only a carboxy-terminaleCtruncated version of the 1b subunit, as described previously (Seisenberger et al., 1995).
Western Blot. Western blot analysis was performed on transfected HEK 293 cells and on brain tissue from mice using an antibody against the p110 subunit of PI-3K (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as described previously (Rong et al., 2003).
Tension Recordings. Muscles strips from murine urinary bladder were prepared as described previously (Wegener et al., 2004). Tension was recorded isometrically at 36 ± 1°C using the myograph 601 (DMT A/S, Aarhus, Denmark).
Current Recordings. L-type Ca2+-channel current was measured in transfected HEK 293 cells by the whole-cell configuration of the patch-clamp technique. Experiments were performed at room temperature using Ba2+ as the charge carrier. The bath solution contained 104 mM NaCl, 20 mM tetraethylammonium chloride, 5.4 mM CsCl, 5 mM BaCl2, 1 mM MgCl2, 1 mM NaH2PO4, 10 mM glucose, and 5 mM HEPES, pH 7.4 (NaOH). The pipette solution contained 112 mM CsCl, 1 mM MgSO4, 3 mM Na2ATP, 10 mM EGTA, and 5 mM HEPES; pH was adjusted to 7.4 with CsOH. The holding potential was eC80 mV. Trains of test pulses were to 0 or +10 mV for 100 ms with 0.2 Hz. Cumulative dose-inhibition curves were measured using two to three different drug concentrations per cell. Drugs were freshly diluted from the stock solution into the bath solution on each experimental day. IC50 values were calculated by fitting the averaged dose-inhibition curves to the Hill equation.
Chemicals. All chemicals used were at least of reagent grade and were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. LY294002, wortmannin, G6983, and G6976 were obtained from Calbiochem (San Diego, CA).
Evaluation of Results. Data are presented as original recordings or expressed as means ± S.E.M. Effects of the drugs were obtained in quasieCsteady-state conditions. Concentration-response curves were fitted using Prism 4.0 software (GraphPad Software Inc., San Diego, CA). Correlation coefficients were >0.95.
Results and Discussion
L-type Cav1.2 calcium-channel activity mediates contraction induced by stimulation of muscarinic receptors in urinary bladder smooth muscle from mice (Wegener et al., 2004). We tested the possibility that the PI3-K pathway is involved in this cascade as described for vascular smooth muscle (Quignard et al., 2001; Callaghan et al., 2004). Therefore, we examined the effects of LY294002, an inhibitor of PI3-K (Vlahos et al., 1994), and of G6983, an inhibitor of PKC isoforms including the PKC isoform (Gschwendt et al., 1996). Both LY294002 (20 e) and G6983 (10 e) inhibited carbachol-induced contractions of bladder muscles from control mice to approximately 50% but not from SMACKO mice (Fig. 1, A and B). The protein kinase C inhibitor G6976 (10 e), which does not act on the PKC isoform (Gschwendt et al., 1996), was without effect (data not shown). These results implied that the PI3-K/PKC pathway is involved in the coupling of muscarinic receptors to CaV1.2 Ca2+ channels, leading to contraction in urinary bladder muscle. However, wortmannin, another inhibitor of PI3-K (IC50 = 5 nM) (Arcaro and Wymann, 1993), did not inhibit carbachol-induced contractions at 100 nM (Fig. 1C). Higher concentrations of wortmannin (10 e) reduced contractions (data not shown), probably because of inhibition of myosin light-chain kinase (IC50 = 200 nM) (Burdyga and Wray, 1999) but not Ca2+ current (Fig. 4C). In addition, in control experiments in which contraction was induced by K+ depolarization, both LY294002 and G6983 reduced contractions, with IC50 values of approximately 18 and 9 e, respectively (Fig. 2). Identical results were obtained with muscle rings from aorta (data not shown). Atropine (1 e), an unselective muscarinic antagonist, did not affect K+-induced contractions in bladder muscle, confirming that muscarinic receptors are not involved in this response. These results suggested a more direct inhibitory action of the drugs on L-type Ca2+ channels because contraction induced by depolarization is completely blocked by dihydropyridines and, thus, primarily mediated by this channel type. Therefore, we tested the effects of both drugs on HEK 293 cells expressing functional L-type Ca2+ channels. Because there exist contrary reports whether or not HEK 293 cells contain endogenous PI3-K activity (Naga Prasad et al., 2001; Brock et al., 2003), Western blot analysis on our transfected HEK cells was performed; no p110 protein of the PI3-K isoform, which is believed to influence Ca2+ channels (Viard et al., 2004), could be detected (Fig. 3). LY294002 and G6983, but not wortmannin, inhibited voltage-activated Ca2+-channel currents in these cells being transfected with 1b plus 2a subunit with IC50 values of 12 and 20 e, respectively (Fig. 4). A similar IC50 value for LY294002 was measured for cells only expressing the 1b subunit (data not shown). No frequency-(0.1 versus 1 Hz) or voltage-dependent effects (eC80 versus eC40 mV holding potential) of the drugs were observed. However, current inactivation was accelerated by LY294002 and by G6983, indicating, at least partially, an interaction with the open channel (Fig. 4); time constants were 0.07 ± 0.005 s (n = 5) and 0.27 ± 0.09 s (n = 3) in the presence of LY294002 (30 e) and G6983 (30 e), respectively.
In summary, the present study shows that both LY294002 and G6983 exhibit Ca2+ antagonistic properties in the concentration range usually applied to selectively block PI3-K (Quignard et al., 2001; Northcott et al., 2002; Wang et al., 2002; Callaghan et al., 2004; McDowell et al., 2004) and PKC (Tao et al., 2003), respectively. Therefore, evidence of a functional coupling of the PI3-K signaling pathway to L-type Ca2+-channel activity is hampered by the inhibitory effects of the compounds LY294002 and G6983 on L-type Ca2+ channels. The side effects of the putative PI3-K inhibitor LY294002, together with its recently described effect on K+ currents (El-Kholy et al., 2003; Sun et al., 2004), limits the usefulness of this drug in the analysis of PI3-K function.
Acknowledgements
We thank S. Paparisto for excellent technical assistance.
This study was supported by Deutsche Forschungsgemeinschaft und Fond der Chemischen Industrie.
doi:10.1124/mol.104.006049.
References
Arcaro A and Wymann MP (1993) Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J 296: 297eC301.
Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, and Nurnberg B (2003) Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma. J Cell Biol 160: 89eC99.
Burdyga TV and Wray S (1999) The relationship between the action potential, intracellular calcium and force in intact phasic, guinea-pig uretic smooth muscle. J Physiol 520: 867eC883.
Callaghan B, Koh SD, and Keef KD (2004) Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol-3-kinase, protein kinase C and c-Src. Circ Res 94: 626eC633.
El-Kholy W, Macdonald PE, Lin JH, Wang J, Fox JM, Light PE, Wang Q, Tsushima RG, and Wheeler MB (2003) The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks KV currents via a direct mechanism. FASEB J 17: 720eC722.
Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, and Johannes FJ (1996) Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase C isoenzymes. FEBS Lett 392: 77eC80.
Le Blanc C, Mironneau C, Barbot C, Henaff M, Bondeva T, Wetzker R, and Macrez N (2004) Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate. Circ Res 95: 300eC307.
McDowell SA, McCall E, Matter WF, Estridge TB, and Vlahos CJ (2004) Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes. Am J Physiol 286: H796eCH805.
Moosmang S, Schulla V, Welling A, Feil R, Feil S, Wegener JW, Hofmann F, and Klugbauer N (2003) Dominant role of smooth muscle L-type calcium channel Cav1.2 for blood pressure regulation. EMBO (Eur Mol Biol Organ) J 22: 6027eC6034.
Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, and Rockman HA (2001) Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by -adrenergic receptor kinase 1. A role in receptor sequestration. J Biol Chem 276: 18953eC18959.
Northcott CA, Poy MN, Najjar SM, and Watts SW (2002) Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats. Circ Res 91: 360eC369.
Quignard JF, Mironneau J, Carricaburu V, Fournier B, Babich A, Nurnberg B, Mironneau C, and Macrez N (2001) Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes. J Biol Chem 276: 32545eC32551.
Rong R, Ahn JY, Huang H, Nagata E, Kalman D, Kapp JA, Tu J, Worley PF, Snyder SH, and Ye K (2003) PI3 kinase enhancer-Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis. Nat Neurosci 6: 1153eC1161.
Seisenberger C, Welling A, Schuster A, and Hofmann F (1995) Two stable cell lines for screening of calcium channel blockers. Naunyn Schmiedeberg's Arch Pharmacol 352: 662eC669.
Sun H, Oudit GY, Ramirez RJ, Costantini D, and Backx PH (2004) The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents. Cardiovasc Res 62: 509eC520.
Tao FC, Shah S, Pradhan AA, Tolloczko B, and Martin JG (2003) Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats. Am J Physiol 284: L90eCL99.
Viard P, Butcher AJ, Halet G, Davies A, Nurnberg B, Heblich F, and Dolphin AC (2004) PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane. Nat Neurosci 7: 939eC946.
Vlahos CJ, Matter WF, Hui KY, and Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269: 5241eC5248.
Wang YG, Dedkova EN, Steinberg SF, Blatter LA, and Lipsius SL (2002) Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes. J Gen Physiol 119: 69eC82.
Wegener JW, Schulla V, Lee TS, Koller A, Feil S, Feil R, Kleppisch T, Klugbauer N, Moosmang S, Welling A, et al. (2004) An essential role of Cav1.2 L-type calcium channel for urinary bladder function. FASEB J 18: 1159eC1161.
Wymann MP, Zvelebil M, and Laffargue M (2003) Phosphoinositide 3-kinase signaling—which way to target Trends Pharmacol Sci 24: 366eC376., 百拇医药(A. Welling, F. Hofmann, a)