Shear Stress-mediated Extracellular Signal-regulated Kinase Activation Is Regulated by Sodium in Endothelial Cells

Abstract

Fluid shear stress is an important regulator of endothelial cell (EC) function. To determine whether mechanosensitive ion channels participate in the EC response to shear stress, we characterized the role of ion transport in shear stress-mediated extracellular signal-regulated kinase (ERK1/2) stimulation. Replacement of all extracellular Na+ with eitherN-methyl-d-glucamine or choline chloride increased the ERK1/2 stimulation in response to shear stress by 1.89 ± 0.1-fold. The Na+ effect was concentration-dependent (maximal effect, ≤12.5 mm) and was specific for shear stress-mediated ERK1/2 activation as epidermal growth factor-stimulated ERK1/2 activation was unaffected by removal of extracellular Na+. Shear stress-mediated ERK1/2 activation was potentiated by the voltage-gated sodium channel antagonist, tetrodotoxin (100 nm), to a magnitude similar to that achieved with extracellular Na+withdrawal. Transfection of Chinese hamster ovary cells with a rat brain type IIa voltage-gated sodium channel completely inhibited shear stress-mediated ERK1/2 activation in these cells. Inhibition was reversed by performing the experiment in sodium-free buffer or by including tetrodotoxin in the buffer. Western blotting of bovine and human EC lysates with SP19 antibody detected a 250-kDa protein consistent with the voltage-gated sodium channel. Degenerate polymerase chain reaction of cDNA from primary human EC yielded transcripts whose sequences were identical to the sodium channel SCN4a and SCN8a α subunit genes. These results indicate that shear stress-mediated ERK1/2 activation is regulated by extracellular sodium and demonstrate that ion transport via Na+ channels modulates EC responses to shear stress. Fluid shear stress is an important regulator of endothelial cell (EC) function. To determine whether mechanosensitive ion channels participate in the EC response to shear stress, we characterized the role of ion transport in shear stress-mediated extracellular signal-regulated kinase (ERK1/2) stimulation. Replacement of all extracellular Na+ with eitherN-methyl-d-glucamine or choline chloride increased the ERK1/2 stimulation in response to shear stress by 1.89 ± 0.1-fold. The Na+ effect was concentration-dependent (maximal effect, ≤12.5 mm) and was specific for shear stress-mediated ERK1/2 activation as epidermal growth factor-stimulated ERK1/2 activation was unaffected by removal of extracellular Na+. Shear stress-mediated ERK1/2 activation was potentiated by the voltage-gated sodium channel antagonist, tetrodotoxin (100 nm), to a magnitude similar to that achieved with extracellular Na+withdrawal. Transfection of Chinese hamster ovary cells with a rat brain type IIa voltage-gated sodium channel completely inhibited shear stress-mediated ERK1/2 activation in these cells. Inhibition was reversed by performing the experiment in sodium-free buffer or by including tetrodotoxin in the buffer. Western blotting of bovine and human EC lysates with SP19 antibody detected a 250-kDa protein consistent with the voltage-gated sodium channel. Degenerate polymerase chain reaction of cDNA from primary human EC yielded transcripts whose sequences were identical to the sodium channel SCN4a and SCN8a α subunit genes. These results indicate that shear stress-mediated ERK1/2 activation is regulated by extracellular sodium and demonstrate that ion transport via Na+ channels modulates EC responses to shear stress. Mechanical stimuli are important modulators of cellular function in tissues, particularly in the cardiovascular system. A key physical force experienced by EC 1The abbreviations ECendothelial cellERKextracellular signal-regulated kinaseBAECbovine aortic ECHUVEChuman umbilical vein ECCHOChinese hamster ovaryPCRpolymerase chain reactionEGFepidermal growth factorMEKmitogen-activated protein kinase/extracellular signal-regulated kinase by virtue of their unique location in the vascular wall is fluid shear stress created by the frictional force of blood flow (1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2337) Google Scholar). Changes in fluid shear stress have been shown to regulate EC function, including permeability of plasma lipoproteins, adhesion of leukocytes, and release of pro- and antithrombotic factors, growth factors, and vasoactive substances (reviewed in Refs. 1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2337) Google Scholar and 2Traub O. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 677-685Crossref PubMed Scopus (937) Google Scholar). These hemodynamically regulated events may contribute to the pathogenesis of vascular disease as atherosclerotic plaques are preferentially localized to areas of the vascular system that experience turbulent flow and low time-averaged shear stress (3Ku D.N. Giddens D.P. Zarins C.K. Glagov S. Arteriosclerosis. 1985; 5: 293-302Crossref PubMed Google Scholar, 4Asakura T. Karino T. Circ. Res. 1990; 66: 1045-1066Crossref PubMed Scopus (732) Google Scholar). endothelial cell extracellular signal-regulated kinase bovine aortic EC human umbilical vein EC Chinese hamster ovary polymerase chain reaction epidermal growth factor mitogen-activated protein kinase/extracellular signal-regulated kinase Our laboratory has previously reported that ERK1/2 are activated by shear stress in EC (5Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (269) Google Scholar). Whereas the mechanisms responsible for growth factor-mediated stimulation of ERK1/2 have been well characterized (6Pelech S.L. Sanghera J.S. Science. 1992; 257: 1355-1356Crossref PubMed Scopus (307) Google Scholar), the upstream signaling pathway that leads to activation of ERK1/2 by shear stress remains undefined. Of particular interest are the primary plasma membrane mechanisms by which the physical force of shear stress can be transduced into biochemical signals. Several candidate mechanotransducers have been proposed including G proteins, caveolae, integrins, and mechanosensitive ion channels (2Traub O. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 677-685Crossref PubMed Scopus (937) Google Scholar). A common mechanism that has evolved to sense changes in mechanical stimuli is the mechanosensitive ion channels (1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2337) Google Scholar, 7Morris C.E. J. Membr. Biol. 1990; 113: 93-107Crossref PubMed Scopus (475) Google Scholar). These channels are widely distributed in tissues and participate in processes such as hearing, balance, touch, and vasoregulation. EC exhibit ion channel responses to mechanical forces that are likely to participate in the signaling response to shear stress. Several different mechanosensitive ion channels are present in EC (8Adams D.J. Trends in Cardiovasc. Med. 1994; 4: 18-26Crossref PubMed Scopus (55) Google Scholar, 9Nilius B. Viana F. Droogmans G. Annu. Rev. Physiol. 1997; 59: 145-170Crossref PubMed Scopus (259) Google Scholar). Shear stress-responsive channels include a cation-selective channel (high calcium conductance) (10Lansman J.B. Hallam T.J. Rink T.J. Nature. 1987; 325: 811-813Crossref PubMed Scopus (522) Google Scholar), a potassium channel (11Olesen S.P. Clapham D.E. Davies P.F. Nature. 1988; 331: 168-170Crossref PubMed Scopus (742) Google Scholar, 12Cooke J.P. Rossitch E. Andon N.A. Loscalzo J. Dzau V.J. J. Clin. Invest. 1991; 88: 1663-1671Crossref PubMed Scopus (442) Google Scholar, 13Jacobs E.R. Cheliakine C. Gebremedhin D. Birks E.K. Davies P.F. Harder D.R. Pflugers Arch. 1995; 431: 129-131Crossref PubMed Scopus (75) Google Scholar), and a stretch-activated calcium channel (1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2337) Google Scholar,14Schwarz G. Droogmans G. Nilius B. Pflugers Arch. 1992; 421: 394-396Crossref PubMed Scopus (70) Google Scholar). Studies have shown that blockage of mechanosensitive K+ channels with barium chloride or tetraethylammonium inhibited shear stress-mediated increases in NO production (15Ohno M. Gibbons G.H. Dzau V.J. Cooke J.P. Circulation. 1993; 88: 193-197Crossref PubMed Scopus (235) Google Scholar) and transforming growth factor-β release (16Ohno M. Cooke J.P. Dzau V.J. Gibbons G.H. J. Clin. Invest. 1995; 95: 1363-1369Crossref PubMed Scopus (306) Google Scholar), suggesting that transmembrane ion flux and intracellular ion homeostasis are important mediators of the EC response to shear stress. To determine the contribution of mechanosensitive ion channels to the shear stress-mediated regulation of ERK1/2 activity in EC we characterized the effects of varying extracellular cation concentrations and the effects of specific ion transport agonists and antagonists. In the present study we report that Na+ entry through voltage-gated sodium channels inhibits shear stress-mediated ERK1/2 activation and demonstrate expression of two voltage-gated sodium channels (identified as SCN4a and SCN8a) in human umbilical vein EC. Bovine aortic EC (BAEC) were isolated from fetal calf aortas and maintained in M199 (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Cells used in experiments were at passages <6, as ERK1/2 kinase activation decreased in later passages. Human umbilical vein EC (HUVEC) were obtained as previously described (17Gimbrone Jr., M.A. Prog. Hemostasis. Thromb. 1976; 3: 1-28PubMed Google Scholar). Cells at passages between 1 and 3 were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 20% fetal bovine serum (HyClone Laboratories, Inc.), heparin (Sigma), and EC growth factor. CHO-K1 and CNaIIA-1 cells were obtained from the laboratory of Dr. W. Catterall (University of Washington, Seattle, WA) and maintained in RPMI 1640 supplemented with 5% fetal bovine serum. G418 (200 μg/ml) was added to media used for CNaIIA-1 cells. CHO cells were placed in medium supplemented with 0.1% serum for 24 h prior to experiments to reduce base-line ERK1/2 phosphorylation. All cells were grown on tissue culture dishes coated with 2.5% gelatin (Sigma). Cells were grown in 74 × 36-cm slides of tissue culture plastic cut from the bottom of tissue culture dishes. Two days after reaching confluence, cells were rinsed free of culture media with HBSS (130 mm NaCl, 5 mm KCl, 1.5 mm CaCl2, 1.0 mm MgCl2, 20 mm HEPES, pH 7.4) with 10 mm glucose added and either maintained in a static condition or exposed to flow (shear stress = 12 dynes/cm2) in a parallel plate chamber (5Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (269) Google Scholar) or cone and plate viscometer (18Bussolari S.R. Dewey Jr., C.F. Gimbrone Jr., M.A. Rev. Sci. Instrum. 1982; 53: 1851-1854Crossref PubMed Scopus (141) Google Scholar) at 37 °C. After varying times of exposure to fluid shear stress, cells were washed gently with ice-cold phosphate-buffered saline (137 mm NaCl, 2.7 mmKCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.3), and ERK1/2 activation was determined. Cells were washed with phosphate-buffered saline and harvested in lysis buffer (50 mm sodium pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 100 μmNa3VO4, 10 mm HEPES, 0.1% mm 10 pH lysates were by and in a protein concentrations were by protein Western cell lysates or were to and were to To of proteins, the were with S. The membrane was for h at with a buffer (Life Technologies, The were at with the primary antibody ERK1/2 antibody was obtained from and were from voltage-gated sodium channel antibody SP19 was by W. Catterall and B. at the of Catterall J. Biol. 1998; PubMed Scopus (70) Google Scholar, D. D. Catterall 1988; PubMed Scopus Google Scholar), and sodium channel antibody was by Dr. D. at the of by for h with antibody were by the of the as described previously (5Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (269) Google Scholar). Cells or tissue were with lysis buffer 10 mm EDTA, 5 mm EGTA, 5 mm 10 mm 1 50 and pH 7.4) on After for 5 cells were with a (50 and was × for 1 The was as the The was washed with lysis in of lysis buffer that and for 1 h at Western as described To Na+ cell lysates of protein were with SP19 antibody at and with 20 of protein (Life Technologies, Inc.) for h on a system at °C. The were washed two times with 1 of lysis two times with 1 of buffer 100 pH 0.1% and 1 and two times with 1 of buffer HEPES, pH EGTA, 10 MgCl2, 1 and 0.1% Na+ channels were by at 37 in 50 mm pH 0.1% 10 mm MgCl2, 1 mm EGTA, or 100 in the of subunit of protein kinase A for 1 The kinase reaction was by at for 3 in mm pH 10% 10 mm and of were sequences present in the and voltage-gated sodium channels or sequences present in the sodium and and and Degenerate were for particularly on the and were at in was isolated from primary from the to the cDNA was a cDNA (Life Technologies, chain reaction was a system and the in the concentrations in the were as (200 cDNA polymerase and reaction buffer as in the A of was by for 5 at by at and °C. were by and by were into the of 50 1 of and buffer in for h at and into and grown on and were by and into media and grown were isolated by and were at the of (University of Washington, Seattle, sequences were in the the and are as ± All experiments were at were by was To of different experiments the in the shear stress was by the in the static and the was a of The for experiment was and was on the Mechanical stimuli intracellular calcium by stretch-activated channels Circ. Res. 1998; PubMed Scopus Google Scholar). Several have previously reported that shear stress increases intracellular calcium Berk B.C. J. Physiol. 1992; PubMed Google Scholar, J. Dewey Gimbrone Jr., M.A. J. Physiol. 1992; PubMed Google Scholar, J. T. In Biol. 1988; PubMed Scopus Google Scholar). calcium channels have been reported to be in EC and cells and to transport calcium in response to increased M. J. Physiol. 1993; PubMed Google Scholar). To determine whether shear stress-mediated ERK1/2 activation is on stretch-activated calcium experiments were in the of which inhibits calcium entry via stretch-activated calcium ERK1/2 was activated by shear stress for 10 with cells static concentrations of chloride effect on shear stress-mediated ERK1/2 activation in suggesting that stretch-activated channels are for (100 for 10 activated and activation was unaffected by have that shear K+ channels the release of vasoactive mediators from EC (15Ohno M. Gibbons G.H. Dzau V.J. Cooke J.P. Circulation. 1993; 88: 193-197Crossref PubMed Scopus (235) Google Scholar, M. Cooke J.P. Dzau V.J. Gibbons G.H. J. Clin. Invest. 1995; 95: 1363-1369Crossref PubMed Scopus (306) Google Scholar). of a cation with cation of of with chloride effect on shear stress-mediated ERK1/2 stimulation of the potassium channel tetraethylammonium effect on shear stress-mediated ERK1/2 activation = These results indicate that extracellular K+ shear stress-mediated ERK1/2 indicate that a sodium channel in J. B. Science. PubMed Scopus Google Scholar). of mm chloride for mm increased ERK1/2 stimulation by shear stress ± in results were obtained choline chloride was used as the or were = of the sodium that at Na+ concentrations of ≤12.5 a in shear stress-mediated ERK1/2 activation to the mm stress condition was of ERK1/2 activation ERK1/2 activation was by changes in sodium suggesting that the of shear stress-mediated ERK1/2 activation was of a extracellular sodium increased ERK1/2 activation and sodium a into the the results that sodium transport the plasma membrane is for of ERK1/2 A sodium is the cell membrane by of the which sodium of the in a the a Na+ a and an into the cell sodium are of present in including are sodium and the To determine which sodium are responsible for the effects on shear stress-mediated we characterized the effect of of sodium transport mechanisms on shear stress-mediated ERK1/2 activation in the with which intracellular of decreased shear stress-mediated ERK1/2 activation to ± of = These results that a in intracellular sodium by increased sodium or by decreased sodium inhibits shear stress-mediated ERK1/2 effect on or static of The that effect on ERK1/2 in static or cells that cell by a in intracellular sodium is to the effects on shear stress-mediated ERK1/2 these results that a in intracellular sodium is to ERK1/2 the with or the with effect on the shear stress-mediated ERK1/2 suggesting that sodium entry through these ion of To determine whether voltage-gated sodium Western was an antibody the of the of voltage-gated sodium channel α Annu. Rev. 1995; PubMed Scopus Google Scholar). Western blotting of cell lysates from and with the SP19 antibody detected an protein with a similar to that of voltage-gated sodium channel α Annu. Rev. 1995; PubMed Scopus Google Scholar) results were with lysates from rat brain and The protein was in membrane from and as with the SP19 that protein is localized to of lysates from different cell two between and suggesting that type of voltage-gated sodium channels is that in EC. To that the sodium from and lysates were with SP19 and to by protein kinase reported by Catterall J. Biol. 1998; PubMed Scopus (70) Google Scholar), protein kinase A in SP19 of voltage-gated sodium channels sodium channels were detected specific for Western of and These results that the and used for the present experiments sodium channels as reported by J. J. Physiol. 1998; Google Scholar, M. S.L. J. Biol. 1998; PubMed Scopus Google Scholar, H. Pflugers Arch. 1994; PubMed Scopus Google Scholar). To determine the effect of voltage-gated sodium channel function on shear stress-mediated ERK1/2 activation in we characterized the effects of specific sodium channel agonists and antagonists. a voltage-gated sodium channel antagonist, increased shear stress-mediated ERK1/2 activation in a concentration-dependent to a magnitude similar to extracellular Na+ ± a voltage-gated sodium channel inhibited shear stress-mediated ERK1/2 activation in a ± tetrodotoxin effect on static or that the effects of these were an sodium channel antagonist, effect on shear stress-mediated ERK1/2 activation These are consistent with the that sodium entry inhibits shear stress-mediated ERK1/2 activation via Na+ To that sodium transport via voltage-gated sodium channels inhibits shear stress-mediated ERK1/2 CHO cells were with cDNA the rat brain type Na+ channel α subunit to as to CNaIIA-1 cells M. Catterall T. Sci. S. 1994; PubMed Scopus Google Scholar). of CHO cells to flow (shear stress of 12 for 10 increased ERK1/2 activity that CHO-K1 cells were shear stress-responsive In CNaIIA-1 cells to ERK1/2 stimulation in response to shear stress. These results indicate that expression of voltage-gated sodium channels inhibits shear stress-mediated ERK1/2 activation ERK1/2 expression was similar in the two cell The sodium channels were to sodium as shown by the that removal of extracellular Na+ or of 100 shear stress-mediated ERK1/2 in CNaIIA-1 cells These that Na+ through voltage-gated sodium channels is responsible for shear stress-mediated effects on To the voltage-gated sodium in was of were on sequences in the voltage-gated sodium channel and These were used for cDNA from primary as a After two of a of the for the voltage-gated sodium were of these yielded two at for voltage-gated sodium After and 20 were at for the of two different The was to rat and SCN8a voltage-gated sodium channel of with the human SCN8a at the laboratory of Dr. M. and J. B. 1995; PubMed Scopus Google Scholar). The was completely to human SCN4a voltage-gated sodium channel Degenerate with on the sodium channel yielded with The of study are that shear stress-mediated ERK1/2 activation is by extracellular Na+ by extracellular voltage-gated sodium and sodium entry through voltage-gated sodium channels inhibits shear stress-mediated ERK1/2 activation has effect on or ERK1/2 phosphorylation. is the study to demonstrate expression of voltage-gated sodium channels in EC and to a role for these channels in The present are by that used to demonstrate an ion channel that to be a voltage-gated sodium channel in H. Pflugers Arch. 1994; PubMed Scopus Google Scholar), EC J. J. Physiol. 1998; Google Scholar), and human vein EC M. S.L. J. Biol. 1998; PubMed Scopus Google Scholar). on we a for the of ERK1/2 activation by the voltage-gated sodium channel that Na+ entry and of a upstream of pathway is of signaling activated by experiments be to the mechanisms by which Na+ inhibits is likely that shear stress sodium channel activity and Na+ entry the sodium channel tetrodotoxin potentiated ERK1/2 and the sodium channel inhibited In an shear stress two for ERK1/2 a and and a To we have a be to of ERK1/2 of such include the effects of on ERK1/2 activity M. E. Peterson T.E. J. M. J. Berk B.C. Circ. Res. 1998; PubMed Scopus Google Scholar) and the of mitogen-activated protein kinase by agonists that ERK1/2 Berk B.C. J. Biol. 1995; PubMed Scopus Google Scholar). that shear stress sodium via the sodium a In sodium regulate upstream mediators of ERK1/2 such as and which is EC are exposed to shear stress. To experiment has been as the membrane is from the with the of shear stress. ion channels have been characterized in EC. These include and (8Adams D.J. Trends in Cardiovasc. Med. 1994; 4: 18-26Crossref PubMed Scopus (55) Google Scholar, 9Nilius B. Viana F. Droogmans G. Annu. Rev. Physiol. 1997; 59: 145-170Crossref PubMed Scopus (259) Google Scholar). flow and mechanical have been shown to ion transport via channels in EC. cell an chloride channel P.F. J. Physiol. 1995; Google Scholar). Mechanical cation channels (10Lansman J.B. Hallam T.J. Rink T.J. Nature. 1987; 325: 811-813Crossref PubMed Scopus (522) Google Scholar, M. J. Physiol. 1993; PubMed Google J. J. H. J. Physiol. 1992; Scopus Google Scholar), as well as a voltage-gated J. Circ. Res. PubMed Scopus Google Scholar). Shear stress different including an (11Olesen S.P. Clapham D.E. Davies P.F. Nature. 1988; 331: 168-170Crossref PubMed Scopus (742) Google Scholar, 13Jacobs E.R. Cheliakine C. Gebremedhin D. Birks E.K. Davies P.F. Harder D.R. Pflugers Arch. 1995; 431: 129-131Crossref PubMed Scopus (75) Google Scholar) and K+ channels J. 1997; PubMed Scopus Google Scholar). is that are channels present in EC that may have described voltage-gated sodium channels in EC J. J. Physiol. 1998; Google Scholar, M. S.L. J. Biol. 1998; PubMed Scopus Google Scholar, H. Pflugers Arch. 1994; PubMed Scopus Google Scholar). The report a = 1 sodium channel in and EC from rat of the H. Pflugers Arch. 1994; PubMed Scopus Google Scholar). and M. S.L. J. Biol. 1998; PubMed Scopus Google Scholar) a = channel in vein EC. The channel was as to as the sodium on and with an antibody to the between and of sodium channel α In and J. J. Physiol. 1998; Google Scholar) characterized a channel from EC that was = 5 and different from The tetrodotoxin of the channel described 100 from of the previously described is with channels such as SCN4a and which we from on the present and that EC sodium channels whose may be regulated in the vascular sodium channels are to contribute to in EC as by J. J. Physiol. 1998; Google M. S.L. J. Biol. 1998; PubMed Scopus Google Scholar). regulate intracellular calcium via the Of particular for shear stress-mediated these sodium channels participate in between EC or between EC and cells J. Pflugers Arch. 1997; PubMed Scopus Google Scholar). may participate in that is well described in the J. Physiol. Google Scholar). have a role for Na+ transport and sodium channels in The of sodium channels in C. is to channels present in tissue M. Annu. Rev. Physiol. 1997; 59: PubMed Scopus Google Scholar). Several of the and have been characterized as Na+ channels that in C. via with the J. B. Science. PubMed Scopus Google Scholar). The is a in the system that sodium for In the cardiovascular laboratory 1993; PubMed Scopus Google Scholar, D. J. Vasc. Res. 1995; PubMed Google Scholar, D. J. Vasc. Res. 1995; PubMed Scopus Google Scholar) in vascular by changes in extracellular sodium and proposed that the primary were to extracellular The study that by shear stress to voltage-gated sodium channel activation was by J. Res. 1997; PubMed Scopus Google Scholar). These membrane of cells exposed to shear stress that was by sodium was inhibited by and previously that shear stress-mediated ERK1/2 activation in M. Berk B.C. J. Clin. Invest. PubMed Scopus Google Scholar) and that were important T. Peterson T.E. Berk B.C. Circ. Res. PubMed Scopus Google Scholar). these that changes in that regulate sodium channel activity may be a common mechanism for shear regulation of the voltage-gated sodium channel may extracellular that with the J. B. Science. PubMed Scopus Google Scholar). may by changes in channel by physical forces through the to the Several mechanisms by which sodium channels changes in intracellular sodium participate in shear may be signaling a mechanism the of signaling that are to changes in Na+ Of was reported that a activation of kinase that extracellular sodium J. Biol. 1997; PubMed Scopus Google Scholar). In the proposed a pathway of In have shown that protein kinase activity and protein kinase are inhibited by increases in intracellular Na+ E. T. F. H. 1993; PubMed Scopus Google Scholar, H. 1995; PubMed Scopus Google Scholar). protein kinase can regulate Na+ by of voltage-gated sodium channels M. T. Catterall Science. 1993; PubMed Scopus Google Scholar) and we have shown that protein kinase is activated by shear stress O. Berk B.C. J. Biol. 1997; PubMed Scopus Google Scholar), protein kinase may participate in events regulated by shear stress and Na+. changes in and cell shear stress ERK1/2 in a T. Peterson T.E. Berk B.C. Circ. Res. PubMed Scopus Google Scholar) and of M. M. J. Biol. 1997; Google Scholar), which is in Changes in sodium may these may be between the sodium channel and J. B. Science. PubMed Scopus Google Scholar). regulation of cell is regulated through Na+ sodium and cell the effect of cell is to ERK1/2 the effect of extracellular which shear stress-mediated ERK1/2 was effect of extracellular sodium or on cells maintained in static culture suggesting that changes in cell are responsible for the increased intracellular sodium may the activity of the Changes in intracellular to be important for regulation of ERK1/2 activation a in intracellular is for shear stress-mediated ERK1/2 activation (5Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (269) Google Scholar, O. Berk B.C. J. Biol. 1997; PubMed Scopus Google Scholar), and the in intracellular in EC exposed to flow extracellular Berk B.C. J. Physiol. 1992; PubMed Google Scholar). In the present that sodium entry through voltage-gated sodium channels in inhibits shear stress-mediated ERK1/2 These results may have for such as in mechanosensitive ion channel expression have been reported J. 1997; PubMed Scopus Google Scholar). experiments be to the voltage-gated sodium channels in to the regulation of the mechanosensitive sodium channel protein kinase and and to the mechanisms by which sodium entry