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Role of TRP channels in endothelial pathophysiology—evidence for vascular TRPs as a potential target for drug therapy
International Congress Series 1262 (2004) 137 – 140
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Role of TRP channels in endothelial pathophysiology—evidence for vascular TRPs as a potential target for drug therapy Annarita Graziani a, Michael Poteser b, Christian Rosker a, Klaus Groschner a,* a
Department of Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria b Department of Hygiene and Preventive Medicine, Yamagata University, Japan
Abstract. Proteins related to the Drosophila transient receptor potential (TRP) gene product are key players in Ca2 + homeostasis of mammalian cells. TRP proteins form cation channels, which function as sensors for a variety of stimuli including oxidative stress. The canonical TRP proteins TRPC3 and TRPC4 are likely to play a role in endothelial physiology and pathophysiology. Here, we report that overexpression of TRPC3 or TRPC4 generates a redox-sensitive cation conductance in human embryonic kidney cells (HEK293). Redox activation of these TRPC species appears to be due to oxidant-induced promotion of phospholipase C activity and/or sensitisation of TRPC proteins to regulation by phospholipase C. Our results suggest TRPC species as attractive, novel targets for cardiovascular therapy. D 2003 Elsevier B.V. All rights reserved. Keywords: Endothelial cells; Oxidative stress; Nonselective cation channels; Transient receptor potential
1. Introduction The function of vascular endothelium is governed by the activity of nonselective cation channels, which are involved in the endothelial response to a variety of stimuli including blood flow-dependent sheer stress, neurotransmitters and oxidative stress [1]. Redox signals are known to determine cellular functions and serve as a switch between apoptotic cell death, differentiation or proliferation [2,3], and excess generation of reactive oxygen species has been recognized as a key event in the pathogenesis of atherosclerosis [4,5]. We tested the hypothesis that canonical transient receptor potential (TRPC) species, which are able to form nonselective cation conductances [6] contribute to the redox sensitivity of the Abbreviations: Cai, intracellular Ca2+ concentration; TRP, transient receptor potential; HEK293, human embryonic kidney cells; U73122, 1-[6-((17h-3-methoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl]-1H-pyrrole2,5-dione. * Corresponding author. Department of Pharmacology and Toxicology, University of Graz, Universitaetsplatz 2, A-8010, Graz, Austria. Tel.: +43-316-380-5570; fax: +43-316-380-9890. E-mail address: [email protected] (K. Groschner). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2003.12.034
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endothelium. Our initial work suggested TRPC proteins are indeed expressed in vascular endothelium and provided the first evidence that these proteins may serve as primary redox sensors in this tissue [7]. Our recent results suggest that TRPC3 and TRPC4 proteins contribute to the nonselective cation conductance, which depolarizes vascular endothelial cells in the presence of oxidants. 2. Experimental procedures 2.1. Cell culture and cell transfection Human embryonic kidney cells (HEK293) stably expressing TRPC3 (T3 – 9) and TRPC4 (T4– 60) were kindly provided by M.X. Zhu. Vascular endothelial cells were cultured from pig aorta as described [8]. 2.2. Western blot SDS-PAGE and Western blotting was performed as described [11]. 2.3. Electrophysiology Patch-clamp experiments were performed as described [7,9]. 2.4. Materials 1-[6-((17h-3-Methoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl]-1H-pyrrole-2,5-dione (U73122) was purchased from Calbiochem, Germany. The TRPC3 and TRPC4 antibodies were purchased from Alomone Labs, Israel. 3. Results and discussion 3.1. TRPC3 and TRPC4 are coexpressed in pig aortic endothelium Pig aortic endothelial cells (ECAP) display a prominent redox-sensitive membrane conductance [10], while HEK293 cells are resistant to even strong oxidative stress (unpublished data). As illustrated in Fig. 1, expression of the two candidate cation channel proteins TRPC3 and TRPC4 is evident in pig aortic endothelium, but not in HEK293 cells.
Fig. 1. Expression of TRPC3 and TRPC4 in ECAP cells. A total of 200 Ag proteins from HEK and ECAP cell lysates was analyzed by Western blot with the TRPC antibodies shown.
A. Graziani et al. / International Congress Series 1262 (2004) 137–140
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These results prompted us to test whether overexpression of TRPC3 or TRPC4 is able to generate a redox-sensitive cation conductance in HEK293 cells, which resembles that of endothelial cells. 3.2. Overexpression of TRPC species generates a redox-sensitive cation conductance in HEK293 cells A redox-sensitive cation conductance resembling that of pig aortic endothelium was generated by either overexpression of TRPC3 (T3 –9; N = 6) or TRPC4 (T4– 60; N = 6), while vector-transfected controls (N = 6) were insensitive to the oxidant (data not shown). Thus, TRPC3 and TRPC4 confer a specific cellular redox-sensitivity, which results in membrane depolarization. Membrane depolarization reduces the driving force for Ca2 + entry and may suppress Ca2 +-dependent formation of vasoactive mediators. Nonetheless, Na+ loading, another consequence of activation of TRPC channels is likely to generate local Ca2 + gradients due to reversed mode Na+/Ca2 + exchange, which has been demonstrated to promote the formation nitric oxide [11]. 3.3. Mechanism of TRPC3 activation by peroxides The phospholipase C inhibitor U73122 (40 AM) prevented activation of TRPC3dependent redox-sensitive membrane conductances (data not shown). Phospholipase C, in particular redox-sensitive, TRPC-associated phospholipase Cg, may therefore be involved in oxidant-induced activation of TRPC channels. Since TRPC3 lacks tight functional coupling to mitochondria [12], this TRPC species may mediate oxidant-induce Ca2 + signalling events without promoting mitochondrial dysfunction and apoptosis. TRPC proteins appear to be targeted to signalplexes in caveolae [13]. Thus, it is tempting to speculate that disintegration of caveolae [14] might be involved in redox activation of TRPC species. Indeed, we observed that t-BHP (400 AM; 1 h) distorted cellular localization of caveolin-1 but not that of TRPC3. Recent reports suggest modulation of
Fig. 2. Reactive oxygen species may activate TRPC channels due to stimulation of phospholipase C activity and alterations in the structure of cholesterol and caveolin-1 (Cav-1)-rich membrane microdomains.
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phospholipase Cg signalling in response to disruption of lipid raft structures [15]. Oxidative disruption of the integrity of cholesterol-rich microdomains is likely to result in alterated phopsholipase C-dependent TRPC signalling (Fig. 2). 3.4. TRPCs represent a novel target for drug therapy We propose oxidative stress-induced stimulation of phospholipase C activity and oxidant-induced disintegration of cholesterol-rich membrane microdomains as a key signal for activation of endothelial TRPC channels. Endothelial TRPC channels appear as a key signalling component in endothelial pathophysiology and therefore as a potential target for novel cardiovascular drug therapies. Acknowledgements This study is supported by the Austrian Research Foundation (FWF), SFBBiomembranes F715 and FWF-14950. References [1] B. Nilius, G. Droogmans, Ion channels and their functional role in vascular endothelium, Physiol. Rev. 81 (2001) 1415. [2] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing, Nature 408 (2000) 239. [3] J.L. Martindale, N.J. Holbrook, Cellular response to oxidative stress: signaling for suicide and survival, J. Cell. Physiol. 192 (2002) 1. [4] A. Harris, S. Devaraj, I. Jialal, Oxidative stress, alpha-tocopherol therapy, and atherosclerosis, Curr. Atheroscler. Rep. 4 (2002) 373. [5] R.W. Alexander, The Jeremiah Metzger Lecture. Pathogenesis of atherosclerosis: redox as a unifying mechanism, Trans. Am. Clin. Climatol. Assoc. 114 (2003) 273. [6] R. Vennekens, T. Voets, R.J. Bindels, G. Droogmans, B. Nilius, Current understanding of mammalian TRP homologues, Cell Calcium 31 (2002) 253. [7] M. Balzer, B. Lintschinger, K. Groschner, Evidence for a role of TRP proteins in the oxidative stressinduced membrane conductances of porcine aortic endothelial cells, Cardiovasc. Res. 42 (1999) 543. [8] B.C. Berk, Redox signals that regulate the vascular response to injury, Thromb. Haemost. 82 (1999) 810. [9] K. Groschner, S. Hingel, B. Lintschinger, M. Balzer, C. Romanin, X. Zhu, W. Schreibmayer, TRP proteins form store-operated cation channels in human vascular endothelial cells, FEBS Lett. 437 (1998) 101. [10] S.K. Koliwad, D.L. Kunze, S.J. Elliott, Oxidant stress activates a non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells, J. Physiol. 491 (Pt. 1) (1996) 1. [11] M. Teubl, K. Groschner, S.D. Kohlwein, B. Mayer, K. Schmidt, Na(+)/Ca(2+) exchange facilitates Ca(2+)dependent activation of endothelial nitric-oxide synthase, J. Biol. Chem. 274 (1999) 29529. [12] B. Thyagarajan, M. Poteser, C. Romanin, H. Kahr, M.X. Zhu, K. Groschner, Expression of TRP3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of TRP3 as a subunit of capacitative Ca2+ entry channels, J. Biol. Chem. 276 (2001) 48149. [13] T. Lockwich, B.B. Singh, X. Liu, I.S. Ambudkar, Stabilization of cortical actin induces internalization of transient receptor potential 3 (TRP3)-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of TRP3-inositol trisphosphate receptor association, J. Biol. Chem. 276 (2001) 42401. [14] E.J. Smart, Y.S. Ying, P.A. Conrad, R.G. Anderson, Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation, J. Cell Biol. 127 (1994) 1185. [15] I.H. Jang, J.H. Kim, B.D. Lee, S.S. Bae, M.H. Park, P.G. Suh, S.H. Ryu, Localization of phospholipase Cgamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation, FEBS Lett. 491 (2001) 4.
www.ics-elsevier.com
Role of TRP channels in endothelial pathophysiology—evidence for vascular TRPs as a potential target for drug therapy Annarita Graziani a, Michael Poteser b, Christian Rosker a, Klaus Groschner a,* a
Department of Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria b Department of Hygiene and Preventive Medicine, Yamagata University, Japan
Abstract. Proteins related to the Drosophila transient receptor potential (TRP) gene product are key players in Ca2 + homeostasis of mammalian cells. TRP proteins form cation channels, which function as sensors for a variety of stimuli including oxidative stress. The canonical TRP proteins TRPC3 and TRPC4 are likely to play a role in endothelial physiology and pathophysiology. Here, we report that overexpression of TRPC3 or TRPC4 generates a redox-sensitive cation conductance in human embryonic kidney cells (HEK293). Redox activation of these TRPC species appears to be due to oxidant-induced promotion of phospholipase C activity and/or sensitisation of TRPC proteins to regulation by phospholipase C. Our results suggest TRPC species as attractive, novel targets for cardiovascular therapy. D 2003 Elsevier B.V. All rights reserved. Keywords: Endothelial cells; Oxidative stress; Nonselective cation channels; Transient receptor potential
1. Introduction The function of vascular endothelium is governed by the activity of nonselective cation channels, which are involved in the endothelial response to a variety of stimuli including blood flow-dependent sheer stress, neurotransmitters and oxidative stress [1]. Redox signals are known to determine cellular functions and serve as a switch between apoptotic cell death, differentiation or proliferation [2,3], and excess generation of reactive oxygen species has been recognized as a key event in the pathogenesis of atherosclerosis [4,5]. We tested the hypothesis that canonical transient receptor potential (TRPC) species, which are able to form nonselective cation conductances [6] contribute to the redox sensitivity of the Abbreviations: Cai, intracellular Ca2+ concentration; TRP, transient receptor potential; HEK293, human embryonic kidney cells; U73122, 1-[6-((17h-3-methoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl]-1H-pyrrole2,5-dione. * Corresponding author. Department of Pharmacology and Toxicology, University of Graz, Universitaetsplatz 2, A-8010, Graz, Austria. Tel.: +43-316-380-5570; fax: +43-316-380-9890. E-mail address: [email protected] (K. Groschner). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2003.12.034
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endothelium. Our initial work suggested TRPC proteins are indeed expressed in vascular endothelium and provided the first evidence that these proteins may serve as primary redox sensors in this tissue [7]. Our recent results suggest that TRPC3 and TRPC4 proteins contribute to the nonselective cation conductance, which depolarizes vascular endothelial cells in the presence of oxidants. 2. Experimental procedures 2.1. Cell culture and cell transfection Human embryonic kidney cells (HEK293) stably expressing TRPC3 (T3 – 9) and TRPC4 (T4– 60) were kindly provided by M.X. Zhu. Vascular endothelial cells were cultured from pig aorta as described [8]. 2.2. Western blot SDS-PAGE and Western blotting was performed as described [11]. 2.3. Electrophysiology Patch-clamp experiments were performed as described [7,9]. 2.4. Materials 1-[6-((17h-3-Methoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl]-1H-pyrrole-2,5-dione (U73122) was purchased from Calbiochem, Germany. The TRPC3 and TRPC4 antibodies were purchased from Alomone Labs, Israel. 3. Results and discussion 3.1. TRPC3 and TRPC4 are coexpressed in pig aortic endothelium Pig aortic endothelial cells (ECAP) display a prominent redox-sensitive membrane conductance [10], while HEK293 cells are resistant to even strong oxidative stress (unpublished data). As illustrated in Fig. 1, expression of the two candidate cation channel proteins TRPC3 and TRPC4 is evident in pig aortic endothelium, but not in HEK293 cells.
Fig. 1. Expression of TRPC3 and TRPC4 in ECAP cells. A total of 200 Ag proteins from HEK and ECAP cell lysates was analyzed by Western blot with the TRPC antibodies shown.
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These results prompted us to test whether overexpression of TRPC3 or TRPC4 is able to generate a redox-sensitive cation conductance in HEK293 cells, which resembles that of endothelial cells. 3.2. Overexpression of TRPC species generates a redox-sensitive cation conductance in HEK293 cells A redox-sensitive cation conductance resembling that of pig aortic endothelium was generated by either overexpression of TRPC3 (T3 –9; N = 6) or TRPC4 (T4– 60; N = 6), while vector-transfected controls (N = 6) were insensitive to the oxidant (data not shown). Thus, TRPC3 and TRPC4 confer a specific cellular redox-sensitivity, which results in membrane depolarization. Membrane depolarization reduces the driving force for Ca2 + entry and may suppress Ca2 +-dependent formation of vasoactive mediators. Nonetheless, Na+ loading, another consequence of activation of TRPC channels is likely to generate local Ca2 + gradients due to reversed mode Na+/Ca2 + exchange, which has been demonstrated to promote the formation nitric oxide [11]. 3.3. Mechanism of TRPC3 activation by peroxides The phospholipase C inhibitor U73122 (40 AM) prevented activation of TRPC3dependent redox-sensitive membrane conductances (data not shown). Phospholipase C, in particular redox-sensitive, TRPC-associated phospholipase Cg, may therefore be involved in oxidant-induced activation of TRPC channels. Since TRPC3 lacks tight functional coupling to mitochondria [12], this TRPC species may mediate oxidant-induce Ca2 + signalling events without promoting mitochondrial dysfunction and apoptosis. TRPC proteins appear to be targeted to signalplexes in caveolae [13]. Thus, it is tempting to speculate that disintegration of caveolae [14] might be involved in redox activation of TRPC species. Indeed, we observed that t-BHP (400 AM; 1 h) distorted cellular localization of caveolin-1 but not that of TRPC3. Recent reports suggest modulation of
Fig. 2. Reactive oxygen species may activate TRPC channels due to stimulation of phospholipase C activity and alterations in the structure of cholesterol and caveolin-1 (Cav-1)-rich membrane microdomains.
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phospholipase Cg signalling in response to disruption of lipid raft structures [15]. Oxidative disruption of the integrity of cholesterol-rich microdomains is likely to result in alterated phopsholipase C-dependent TRPC signalling (Fig. 2). 3.4. TRPCs represent a novel target for drug therapy We propose oxidative stress-induced stimulation of phospholipase C activity and oxidant-induced disintegration of cholesterol-rich membrane microdomains as a key signal for activation of endothelial TRPC channels. Endothelial TRPC channels appear as a key signalling component in endothelial pathophysiology and therefore as a potential target for novel cardiovascular drug therapies. Acknowledgements This study is supported by the Austrian Research Foundation (FWF), SFBBiomembranes F715 and FWF-14950. References [1] B. Nilius, G. Droogmans, Ion channels and their functional role in vascular endothelium, Physiol. Rev. 81 (2001) 1415. [2] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing, Nature 408 (2000) 239. [3] J.L. Martindale, N.J. Holbrook, Cellular response to oxidative stress: signaling for suicide and survival, J. Cell. Physiol. 192 (2002) 1. [4] A. Harris, S. Devaraj, I. Jialal, Oxidative stress, alpha-tocopherol therapy, and atherosclerosis, Curr. Atheroscler. Rep. 4 (2002) 373. [5] R.W. Alexander, The Jeremiah Metzger Lecture. Pathogenesis of atherosclerosis: redox as a unifying mechanism, Trans. Am. Clin. Climatol. Assoc. 114 (2003) 273. [6] R. Vennekens, T. Voets, R.J. Bindels, G. Droogmans, B. Nilius, Current understanding of mammalian TRP homologues, Cell Calcium 31 (2002) 253. [7] M. Balzer, B. Lintschinger, K. Groschner, Evidence for a role of TRP proteins in the oxidative stressinduced membrane conductances of porcine aortic endothelial cells, Cardiovasc. Res. 42 (1999) 543. [8] B.C. Berk, Redox signals that regulate the vascular response to injury, Thromb. Haemost. 82 (1999) 810. [9] K. Groschner, S. Hingel, B. Lintschinger, M. Balzer, C. Romanin, X. Zhu, W. Schreibmayer, TRP proteins form store-operated cation channels in human vascular endothelial cells, FEBS Lett. 437 (1998) 101. [10] S.K. Koliwad, D.L. Kunze, S.J. Elliott, Oxidant stress activates a non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells, J. Physiol. 491 (Pt. 1) (1996) 1. [11] M. Teubl, K. Groschner, S.D. Kohlwein, B. Mayer, K. Schmidt, Na(+)/Ca(2+) exchange facilitates Ca(2+)dependent activation of endothelial nitric-oxide synthase, J. Biol. Chem. 274 (1999) 29529. [12] B. Thyagarajan, M. Poteser, C. Romanin, H. Kahr, M.X. Zhu, K. Groschner, Expression of TRP3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of TRP3 as a subunit of capacitative Ca2+ entry channels, J. Biol. Chem. 276 (2001) 48149. [13] T. Lockwich, B.B. Singh, X. Liu, I.S. Ambudkar, Stabilization of cortical actin induces internalization of transient receptor potential 3 (TRP3)-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of TRP3-inositol trisphosphate receptor association, J. Biol. Chem. 276 (2001) 42401. [14] E.J. Smart, Y.S. Ying, P.A. Conrad, R.G. Anderson, Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation, J. Cell Biol. 127 (1994) 1185. [15] I.H. Jang, J.H. Kim, B.D. Lee, S.S. Bae, M.H. Park, P.G. Suh, S.H. Ryu, Localization of phospholipase Cgamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation, FEBS Lett. 491 (2001) 4.