transient receptor potential melastatin 7 ion channels ... · transient receptor potential...

Transient Receptor Potential Melastatin 7 Ion ChannelsRegulate Magnesium Homeostasis in Vascular Smooth

Muscle CellsRole of Angiotensin II

Ying He, Guoying Yao, Carmine Savoia, Rhian M. Touyz

Abstract—Magnesium modulates vascular smooth muscle cell (VSMC) function. However, molecular mechanismsregulating VSMC Mg2� remain unknown. Using biochemical, pharmacological, and genetic tools, the role of transientreceptor potential membrane melastatin 7 (TRPM7) cation channel in VSMC Mg2� homeostasis was evaluated. Rat,mouse, and human VSMCs were studied. Reverse transcriptase polymerase chain reaction and immunoblottingdemonstrated TRPM7 presence in VSMCs (membrane and cytosol). Angiotensin II (Ang II) and aldosterone increasedTRPM7 expression. Gene silencing using small interfering RNA (siRNA) against TRPM7, downregulated TRPM7(mRNA and protein). Basal [Mg2�]i, measured by mag fura-2AM, was reduced in siRNA-transfected cells(0.39�0.01 mmol/L) versus controls (0.54�0.01 mmol/L; P�0.01). Extracellular Mg2� dose-dependently increased[Mg2�]i in control cells (Emax 0.70�0.02 mmol/L) and nonsilencing siRNA-transfected cells (Emax 0.71�0.04 mmol/L),but not in siRNA-transfected cells (Emax 0.5�0.01 mmol/L). The functional significance of TRPM7 was evaluated byassessing [Mg2�]i and growth responses to Ang II in TRPM7 knockdown cells. Acute Ang II stimulation decreased[Mg2�]i in control and TRPM7-deficient cells in a Na�-dependent manner. Chronic stimulation increased [Mg2�]i incontrol, but not in siRNA-transfected VSMCs. Ang II–induced DNA and protein synthesis, measured by 3[H]-thymidineand 3[H]-leucine incorporation, respectively, were increased in control and nonsilencing cells, but not in TRPM7knockdown VSMCs. Our data indicate that VSMCs possess membrane-associated, Ang II–, and aldosterone-regulatedTRPM7 channels, which play a role in regulating basal [Mg2�]i, transmembrane Mg2� transport and DNA and proteinsynthesis. These novel findings identify TRPM7 as a functionally important regulator of Mg2� homeostasis and growthin VSMCs. (Circ Res. 2005;96:207-215.)

Key Words: cations � TRPM channels � vessels � aldosterone � angiotensin II � siRNA

Magnesium plays a major role in regulating vascularsmooth muscle cell (VSMC) function. Increased intra-

cellular Mg2� concentration ([Mg2�]i) causes vasodilation andattenuates agonist-induced vasoconstriction, whereas reduced[Mg2�]i has opposite effects, leading to hypercontractility andimpaired vasorelaxation.1–4 Mg2� also influences growthprocesses associated with remodeling and fibrosis, character-istic features of vascular damage in hypertension, atheroscle-rosis, and diabetes.5,6 At the subcellular level, these effectsoccur, at least in part, through Mg2�-dependent regulation ofmitogen-activated protein (MAP) kinases, tyrosine kinases,and reactive oxygen species, important signaling moleculesinvolved in VSMC proliferation, fibrosis, and inflamma-tion.7–9 Microarray studies demonstrated that changes in[Mg2�]i have potent modulatory actions on expression ofvarious growth signaling molecules.10 We recently reportedthat altered [Mg2�]i influences cell cycle progression and

VSMC growth by modulating cyclin-dependent kinases andMAP kinases.11

Intracellular Mg2� homeostasis is tightly regulated. InVSMCs, basal [Mg2�]i is maintained at 0.5 to 0.6 mmol/L.11,12

Although Mg2� is the second most abundant intracellularcation and the predominant divalent cation, molecular mech-anisms regulating cellular Mg2� remain elusive.13 Somestudies suggested that transmembrane Mg2� transport occursthrough the Na�/Ca2� antiporter,14 whereas others demon-strated that Mg2� efflux is linked to Na�/H� exchange.15

Renal paracellular Mg2� transport is mediated via paracellin-1.16,17 We reported that Mg2� efflux is regulated by aNa�-dependent Mg2� exchanger linked to the Na�/H� anti-porter in VSMCs.15,18

Although studies of Mg2� fluxes in mammalian cells haveindicated the presence of functionally active plasma mem-brane Mg2� transport mechanisms, proteins responsible for

Original received August 24, 2004; revision received November 15, 2004; accepted December 1, 2004.From the CIHR Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, Canada.Correspondence to Rhian M. Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, H2W 1R7, Canada. E-mail

[email protected]© 2005 American Heart Association, Inc.

Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000152967.88472.3e

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these fluxes have not been identified.19 Recent investigationssuggested that two novel ion channels of the long, ormelastatin-related, transient receptor potential (TRPM) ionchannel subfamily, TRPM6 and TRPM7, are critically in-volved in Mg2� influx in epithelial and neuronal cells.20,21

TRPM6/7 are polypeptides with dual-function ion channel/protein kinases, characterized by six transmembrane spanningdomains with an adjacent coiled coil region, a long, highlyconserved cytoplasmic N-terminal region, and a cytoplasmicC terminus, which has enzymatic activity.22–24 TRPM6 andTRPM7, which have an overall amino acid sequence homol-ogy of 52%, harbor serine/threonine kinase domains in theirC termini.21,25,26 TRPM6 is preferentially expressed in smallintestine, colon, and kidney, participating in gastrointestinaland renal Mg2� absorption.21,27,28 Mutations in TRPM6 causehypomagnesemia with secondary hypocalcemia.27,29,30

Expression of TRPM7 is widespread with transcripts inbrain, spleen, lung, kidney, heart, and liver.22,31–33 It is alsoexpressed in lymphoid-derived cell lines, hematopoietic cells,granulocytes, leukemia cells, and microglia.20,25 In variouscell lines, TRPM7 is regulated by intracellular levels ofMg-ATP and is strongly activated when Mg-ATP falls below1 mmol/L.32,34 Studies in microglial and HEK293-transfectedcells demonstrated that TRPM7 activity is also modulatedthrough its endogenous kinase in a cAMP-, PKA-, andSrc-dependent manner25,35 and is inactivated by PIP2 hydro-lysis in cardiac fibroblasts.36

To our knowledge nothing is known about the status ofTRPM7 in the vasculature. It is unclear whether this cationchannel influences Mg2� transport in vascular cells andwhether vasoactive agents regulate TRPM7. To gain insightsinto the putative role of TRPM7 in vascular Mg2� homeosta-sis, we used a combination of biochemical, pharmacological,molecular and genetic approaches to investigate the presenceand functional significance of TRPM7 in VSMCs. Our datademonstrate that VSMCs possess functionally activemembrane-associated TRPM7 channels that are regulated byangiotensin II (Ang II) and aldosterone. Findings from thisstudy identify for the first time that TRPM7 is a keymodulator of vascular Mg2� homeostasis and that it plays animportant role in regulating VSMC function.

Materials and MethodsCell CultureThe study was approved by the Animal and Human Ethics Commit-tee of the Clinical Research Institute of Montreal and performedaccording to the recommendations of the Canadian Council forAnimal Care. VSMCs from mesenteric arteries and aorta fromWistar Kyoto rats (WKY; Taconic Farms, Germantown, NY) andC57/B6J mice (Jackson Laboratory, Bar Harbor, Me) were isolatedby enzymatic digestion and cultured as we described.11 VSMCs fromhealthy humans were derived from small arteries obtained fromgluteal biopsies as we detailed.37 Cells were maintained in DMEMcontaining 10% fetal calf serum (FCS). Low passaged cells (pas-sages 2 to 7) were studied.

Reverse Transcriptase Polymerase Chain ReactionExpression of TRPM7 gene was studied by reverse transcriptasepolymerase chain reaction (RT-PCR). Primers for rat, mouse, andhuman TRPM7 are detailed in the Table. Cells were stimulated withvehicle (water), Ang II (10�7 mol/L), or aldosterone (10�7 mol/L) for

2 to 24 hours. Total RNA was extracted from cells (TRIzol Reagent).Reverse transcription was performed in 20 �L containing 2 �g RNA,1.0 �L of 10 mmol/L dNTP, 4 �L of 5� first strand buffer, 1.0 �Loligo-(dT)12 to 18 primer (0.5 �g/�L), 1.0 �L of 200 U/�L M-MLVreverse transcriptase (GIBCO-BRL), 1.0 �L of rRnasin (Rnaseinhibitor, 40 U/�L), 2 �L dithiothreitol (0.1 mol/L), for 1 hour,37°C. The reaction was stopped by heating at 70°C for 15 minutes.Two microliters of resulting cDNA mixture was amplified usingspecific primers (Table). TRPM7 amplification by PCR involved95°C for 5 seconds, 35 cycles of 95°C for 30 seconds, 54°C for 30seconds, 72°C for 30 seconds, and extension for 5 minutes at 72°C.GAPDH amplification by PCR involved 94°C for 5 minutes, 30cycles of 94°C for 30 seconds, 57°C for 30 seconds, 72°C for 45seconds, and extension for 5 minutes, at 72°C. Amplificationproducts were electrophoresed on 1% agarose gel containingethidium bromide (0.5 �g/mL). Bands corresponding to RT-PCRproducts were visualized by UV light and digitized using AlphaIm-ager software. Band intensity was quantified using the ImageQuant(version 3.3, Molecular Dynamics) software.

TRPM7 Protein ExpressionTotal protein was extracted from VSMCs as we described.11,37

Briefly, cells were washed with cold PBS and then harvested inHEPES buffer containing (in mmol/L), HEPES 10, pH 7.4, NaF 50,NaCl 50, EDTA 5, EGTA 5, Na pyrophosphate 50, containingtriton-X 100 0.5%, phenylmethylsulfonyl fluoride (PMSF) 1mmol/L,leupeptin 1 �g/mL, and aprotinin 1 �g/mL. Cells were disrupted bybrief sonication. Samples were then centrifuged (500g, 10 minutes,4°C) to remove nuclei. For membrane and cytosol separation,samples were centrifuged at 100 000g for 1 hour at 4°C. The cellmembrane was washed once with the buffer described above andthen resuspended in buffer containing 100 mmol/L Tris-HCl,300 mmol/L NaCl, 1% Triron X-100, and 0.1% SDS containing2 mmol/L EDTA, 2 mmol/L PMSF, and 0.8 �g/mL leupeptin.Proteins (20 �g) were separated by electrophoresis on polyacryl-amide gel (7.5%) and transferred onto a polyvinylidene diflouridemembrane. Nonspecific binding sites were blocked with 5% skimmilk in Tris-buffered saline solution with Tween (TBS-T) (1 hour,room temperature). Membranes were incubated with anti-TRPM7antibody (1:750) (Abcam Inc) in TBS-T-milk at 4°C overnight withagitation. Washed membranes were incubated with horse-radishperoxidase-conjugated second antibody (1:2000) in TBS-T-Milk(room temperature, 1 hour). Membranes were washed and immuno-reactive proteins detected by chemiluminescence. Blots were ana-lyzed densitometrically (Image-Quant software, MolecularDynamics).

Measurement of [Mg2�]i and [Ca2�]i in VSMCsThe selective fluorescent probes, mag fura-2AM and fura-2AM,were used to measure [Mg2�]i and [Ca2�]i, respectively, as described(see online data supplement available at http://circres.ahajournals.org).15,18,38–40 [Mg2�]i responses to increasing concentrations of

Primer Sequence for TRPM7 Amplification by RT-PCR in Rat,Mouse, and Human VSMCs

Species Primer SequencePredicted

Size

Rat (S) 5�-GCA AAT GAC TCC ACT CTC-3� 422

(AS) 5�-GATTCTCTCTTCACTCCCAG-3� 422

Mouse (S) 5�-CTCATGGGAGGAACCTACAG-3� 198

(AS) 5�-CATCTTTGGTCTGTAGGGTTG-3� 198

Human (S) 5�-CTTATGAAGAGGCAGGTCATGG-3� 213

(AS) 5�-CATCTTGTCTGAAGGACTG-3� 213

GAPDH (S) 5�-TTGTTGCCATCAACGACCCC-3� 435

(AS) 5�-ATGAGCCCTTCCACAATGCC-3� 435

S indicates sense; AS, antisense.

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extracellular Mg2� (0 to 5 mmol/L) were measured in cells incubatedin Mg2�-free, Ca2�-containing modified Hanks’ buffer (in mmol/L,NaCl 137, NaHCO3 4.2, NaHPO4 3, KCl 5.4, KH2PO4 0.4, CaCl2 1.3,glucose 10, and HEPES 5; pH 7.4). Cells were exposed to Mg2�-freebuffer for 15 to 20 minutes before addition of extracellular Mg2�. Insome experiments, Ang II effects were assessed in Na�-free Hanks’buffer (Na� isosmotically replaced with N-methylglucamine).39

[Ca2�]i responses to ionomycin (10�6 mol/L) were determined incells incubated for 15 to 20 minutes in modified Hank’s buffer (asearlier) containing Mg2� (mmol/L, MgCl2 0.5 and MgSO4 0.8).

RNA Interference and Cell TransfectionHigh-performance purity grade (�90% pure) small interfering RNAs(siRNA) were generated against TRPM7. Sequences identical inhuman and mouse but that do not match other sequences in GenBankwere used. siRNAs for knocking down TRPM7 were synthesized byQIAGEN Inc. The DNA target sequences of the annealed doublestand siRNA that we used were as follows: 5�-AACCGG-AGGTCAGGTCGAAAT-3� (1884–1904), which has 100% homol-ogy to mouse gene only, and 5�-AAGCAGAGTGACCT-GGTAGAT-3�, which has 100% homology to both mouse andhuman gene. siRNA, with a nonsilencing oligonucleotide sequence(nonsilencing siRNA) that does not recognize any known homologyto mammalian genes, was also generated as a negative control.

Cells were seeded at a density of 1.6�105 cells/well in 6-wellplates and grown in DMEM containing 10% FCS and antibiotics.One day after seeding, cells were transfected with siRNA usingRNAiFect Transfection Reagent (Qiagen Inc) according to themanufacturer’s instructions. Briefly, siRNA (5 �g) was mixed withEC-R buffer (Qiagen Inc) (100 �L) to which RNAiFect transfectionreagent (15 �L) was added. After mixing (15 minutes), the lipid-formulation was added dropwise onto the cells. Control cells wereexposed to transfectant in the absence of siRNA. Forty eight hoursafter transfection, gene silencing was monitored at the mRNA andprotein levels by RT-PCR and Western blotting, respectively.

Measurement of DNA and Protein Synthesis byAng II in TRPM7 siRNA-Transfected VSMCsCells were seeded, at an initial concentration of 1�105 cells/mL, into24-well multiwell plates and grown in DMEM containing 10% FCSand antibiotics. One day after seeding, cells were exposed totransfectant alone (control cells) or transfected with siRNA ornonsilencing siRNA as described earlier. Thirty six hours aftertransfection, cells were stimulated with Ang II (10�11 to 10�6 mol/L,24 hours) in the absence and presence of valsartan, selective AT1

receptor blocker (10�5 mol/L). DNA and protein synthesis wereevaluated by measuring incorporation of 3[H]-thymidine (5 �Ci/mL)and 3[H]-leucine (2 �Ci/mL, respectively, as we described.12 Briefly,after incubation, radioactive medium was removed, cells washedwith ice cold physiological buffered saline, incubated with trichlo-roacetic acid (TCA) (0.75 mol/L, 15 minutes), washed with coldTCA, and incubated with NaOH (0.2 mol/L for thymidine, 1 mol/Lfor leucine, 60 minutes, room temperature). Relative incorporation of3[H]-thymidine and 3[H]-leucine was determined by liquid scintilla-tion counting.

Statistical AnalysisData are presented as mean�SEM. Groups were compared usingone-way ANOVA or Student t test as appropriate. Tukey-Kramercorrection was used to compensate for multiple testing procedures. Avalue of P�0.05 was significant.

ResultsTRPM7 Expression in VSMCsPresence of TRPM7 transcript was assessed by RT-PCR.GAPDH mRNA was used as an internal housekeeping geneand results were expressed as the ratio of TRPM7:GAPDH.As demonstrated in Figure 1A, mRNA for TRPM7 is present

in VSMCs. Ang II and aldosterone significantly increasedTRPM7 mRNA expression (Figure 1B). Maximal agonist-induced responses were obtained within 4 to 6 hours ofstimulation.

To evaluate TRPM7 protein content in VSMCs, immuno-blotting was performed using anti-TRPM7 antibody. �-Actinwas used as an internal control. As shown in Figure 2A,SDS-PAGE analysis of total cell homogenate revealed amajor band migrating at the 160- to 170-kDa position,corresponding to TRPM7. To establish whether TRPM7 ismembrane-associated, we also probed for TRPM7 content inmembrane-rich fractions. Figure 2B demonstrates thatTRPM7 is present in VSMC membranes and that Ang IIstimulation significantly increases membrane TRPM7 con-tent (P�0.05 versus control). Daudi cell lysate (Abcam Inc)was used as a positive control.

Downregulation of TRPM7 Attenuates Basal[Mg2�]i and Abrogates Mg2� InfluxThe functional significance of TRPM7 in VSMC [Mg2�]i

regulation was assessed in cells in which TRPM7 gene wassilenced by siRNA. TRPM7 mRNA expression and proteinabundance were markedly reduced in human and mouseVSMCs transfected with siRNA, but not in control cells orcells transfected with nonsilencing siRNA (Figure 3). Basal[Mg2�]i was significantly decreased (P�0.01) in siRNA-transfected cells (0.39�0.01 mmol/L) compared with control(0.54�0.01 mmol/L) and nonsilencing siRNA-transfectedcells (0.51�0.02 mmol/L). Results were not significantlydifferent between control and nonsilencing transfected cells.

Exposure of VSMCs to increasing concentrations of extra-cellular Mg2� resulted in a significant [Mg2�]i rise (P�0.01)in control cells and in cells transfected with nonsilencingsiRNA (Figure 4). Maximal [Mg2�]i responses were obtainedat 3 mmol/L extracellular Mg2�, above which [Mg2�]i did notincrease further. Exposure of siRNA-transfected cells toincreasing concentrations of extracellular Mg2� induced amodest, but nonsignificant increase, in [Mg2�]i (Figure 4).

To evaluate whether TRPM7 regulates Ca2� homeostasis inVSMCs, [Ca2�]i effects of ionomycin were assessed inTRPM7 knockdown cells. Ionomycin induced a rapid [Ca2�]i

rise as evidenced by increased fura-2 fluorescence. Basal[Ca2�]i was unaltered in siTRPM7-deficient cells (106�10nmol/L) versus control (94�4 nmol/L) and nonsilencingsiRNA-transfected cells (102�6 nmol/L). Although ionomycin-mediated [Ca2�]i responses were slightly reduced in TRPM7-deficient cells, responses were not significantly different fromcontrol cells (Figure 1, online data supplement).

TRPM7 Plays a Role in Chronic, but Not inAcute, Ang II–Mediated [Mg2�]i RegulationShort-term VSMC exposure (5 to 10 minutes) to Ang IIresulted in a rapid [Mg2�]i decrease (Figure 5). This effectwas abrogated in Na�-free conditions (Figure 5B). Resultswere similar in control, siRNA-transfected, and nonsilencingsiRNA-transfected cells (Figure 5A). Long-term Ang IIstimulation (24 to 30 hours) was associated with an increasein [Mg2�]i in control and nonsilencing transfected cells, butnot in TRPM7 knockdown cells (Figure 5C).

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Essential Role of TRPM7 in Ang II–StimulatedVSMC GrowthTo evaluate the functional significance of Ang II–regulatedTRPM7, growth effects of Ang II were assessed in TRPM7knockdown VSMCs. As demonstrated in Figure 6, Ang IIdose-dependently increased incorporation of 3[H]-thymidineand 3[H]-leucine, indices of DNA and protein synthesis,respectively, in control and nonsilencing siRNA-transfected

cells, but not in siRNA-transfected cells. Valsartan, a selec-tive AT1 receptor blocker, inhibited Ang II–mediated cellgrowth (Figure 2, online data supplement).

DiscussionIn this study, we provide the first evidence that VSMCspossess TRPM7 channels, that TRPM7 is functionally active,and that this ion channel plays an essential role in regulating

Figure 1. A, TRPM7 transcript is present in vascular smooth muscle cells (VSMC). mRNA was prepared from rat VSMCs (from aorta,Ao, and mesenteric arteries, Mes) and mouse VSMCs (from mesenteric arteries). TRPM7-specific forward and reverse primers amplifieda product of the correct size but not in negative controls (NC) when cDNA was omitted. M indicates marker; PCR, polymerase chain reaction.B, Effects of Ang II and aldosterone on TRPM7 mRNA expression. mRNA was prepared from rat VSMCs exposed to Ang II or aldosterone(10�7 mol/L, Aldo) for 2 to 24 hours. Representative scans demonstrate amplified PCR products corresponding to TRPM7 and GAPDH. Bargraphs are mean�SEM of 3 to 5 experiments. Data are presented as TRPM7:GAPDH ratio. *P�0.05 vs Control (Cont) counterpart.




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Mg2� influx and maintaining intracellular Mg2� levels. Wealso demonstrate that Ang II and aldosterone, which modulatevascular tone and structure, influence TRPM7 abundance.Finally, we report that Ang II–regulated TRPM7 is importantin the long-term, but not in the short-term regulation ofVSMC [Mg2�]i and that it plays a fundamental role in cellgrowth. Our findings suggest that TRPM7 is a highly regu-

lated transmembrane Mg2� transporter, critically involved inVSMC Mg2� homeostasis and growth.

Transient receptor potential channels, particularly of theTRPC (for canonical TRP) and TRPV (for vanilloid TRP)subfamilies, have been demonstrated to be functionally im-portant in regulating Ca2� entry in VSMCs and endothelialcells.41–43 To our knowledge, nothing is known about TRPM7status in vascular cells, although these ion channels have beenwell characterized in renal and gastric epithelial cells and innumerous mammalian immortalized cell lines.21,22,27,28,33 Inthe present study, we demonstrate that mouse, rat, and humanVSMCs possess membrane-associated TRPM7.

Many vasoactive agents influence VSMC function, ofwhich Ang II is particularly important. Ang II stimulatesvascular contraction, growth, and inflammation, in partthrough Mg2�-dependent processes.39,44 Aldosterone, a min-eralocorticoid hormone that influences cellular Mg2� metab-olism,45,46 is increasingly being recognized as an importantmodulator of vascular function.47,48 In this study, we demon-strate that Ang II and aldosterone regulate TRPM7 mRNAand protein content. Exact mechanisms whereby these ago-nists control TRPM expression are unknown. However, bothpeptides stimulate activity of many transcription factors,which could play a role in de novo TRPM production.

To evaluate the functional significance of TRPM7 inVSMCs, we generated siRNAs to selectively reduce TRPM7expression and used two independent assays to test siRNA

Figure 2. TRPM7 protein expression in VSMCs. A, Total proteinlysates from human VSMCs were prepared for immunoblotting.Membranes were probed with anti-TRPM7 antibody andreprobed with anti–�-actin antibody. Daudi cell lysate was usedas a positive control (PC). B, Membrane protein lysates fromVSMCs were probed for TRPM7 by immunoblotting. Immuno-blots are representative of 3 experiments. *P�0.05 vs control;**P�0.01 vs control.

Figure 3. TRPM7 siRNA downregulates TRPM7 expression inhuman VSMCs. mRNA expression and protein abundance wereassessed by RT-PCR (A) and Western blotting (B), respectively,in human VSMCs transfected with TRPM7 siRNA or nonsilenc-ing (NS) siRNA. Control cells were exposed to transfectant with-out siRNA. C, Bar graphs corresponding to representative im-munoblots. Data are expressed as the TRPM7:GAPDH ratio.Results are presented as mean�SEM of 4 experiments.**P�0.01 vs other groups.




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activity and specificity. First, we questioned whether siRNAwould attenuate TRPM7 content, and second, we used fluo-rescence digital imaging to test whether siRNA preventsMg2� and Ca2� transmembrane transport. Using a lipid-basedsystem for siRNA transfection, almost 100% TRPM7 genesilencing was obtained as evidenced by significantly reducedmRNA and protein expression. Previous studies reported thatcultured cells rendered TRPM7-deficient via cre-lox–medi-ated destruction of the TRPM7 gene, undergo growth arrestand die after a few days in culture.20,49 In our investigations,cells remained viable for the duration of our studies. This mayrelate to the fact that TRPM7 was transiently downregulatedand that experiments were performed 48 hours after transfec-tion. Cell death was previously reported to occur 48 to 72hours after TRPM7 gene silencing in TRPM7-deficient celllines.20

Basal [Mg2�]i was reduced in TRPM7-deficient cells,confirming the importance of TRPM7 in VSMC Mg2� ho-meostasis. Similar findings were observed in stably trans-fected 293-HEK cells expressing human TRPM7 mutants andin TRPM7 knockout cell lines.20 Increasing concentrations ofextracellular Mg2� resulted in a marked [Mg2�]i rise in controlcells. In contrast, in TRPM7-depeleted cells (by siRNA),[Mg2�]i did not change significantly when exposed to highextracellular Mg2� levels. These findings suggest thatTRPM7 facilitates transmembrane Mg2� transport in VSMCs,probably by promoting Mg2� influx through an ion channel

mechanism.32,50,51 [Mg2�]i seems to be highly regulated,because [Mg2�]i plateaued despite increasing concentrationsof extracellular Mg2� above 3 mmol/L. Possible reasons forthis may relate first to the reciprocal interaction betweenintracellular Mg2� and channel activity and second to thepresence of regulatory Mg2� efflux systems. There is nowcompelling evidence that intracellular Mg2� negatively influ-ences TRPM7 channel activity.32,34 As [Mg2�]i increases,TRPM7 activity declines. This inhibitory feedback loopbetween excess intracellular Mg2� and channel activity maybe important in maintaining [Mg2�]i within a physiologicalrange and in protecting cells against Mg2� overload. It is alsopossible that as cellular Mg2� levels increase, Mg2� effluxsystems, such as the Na�/Mg2� exchanger, are activated.

Data from studies in cell lines demonstrate that TRPM7 has aunique permeation profile with a permeability sequence ofZn2��Ni2��Ba2��Co2��Mg2��Mn2��Sr2��Cd2��Ca2�.31

Because Ca2� plays a fundamental regulatory role in VSMCfunction, and because previous studies demonstrated that TRPM7 isa Ca2�-permeant ion channel,52,53 we questioned whether TRPM7,in addition to regulating Mg2� influx, influences transmembraneCa2� transport in VSMCs. Basal [Ca2�]i was unaltered in TRPM7-deficient cells, indicating that this ion channel may not be importantin ambient Ca2� homeostasis in VSMCs. These findings are incontrast to neuronal-derived cells, where TRPM7 seems to be amajor regulator of Ca2� influx.25,52 Ionomycin increased [Ca2�]i inboth control and TRPM7 knockdown cells. Although responses

Figure 4. Effects of increasing extracellular Mg2� concentration ([Mg2�]e) on [Mg2�]i in TRPM7-deficient VSMCs. Mouse and humanVSMCs were transfected with nonsilencing siRNA and TRPM7 siRNA as described in Materials and Methods. [Mg2�]i was measuredusing mag fura-2AM (4 �mol/L). Top, Representative mag fura-2AM tracings in human control cells and cells transfected with nonsi-lencing (NS) and TRPM7 silencing RNA. Cells were incubated in Mg2�-free Ca2�-containing Hank buffer for 15 to 20 minutes beforeMg2� addition. Arrow indicates addition of Mg2� (2 mmol/L). Bottom, Line graphs demonstrate effects of increasing [Mg2�]e on [Mg2�]i incontrol and TRPM7-deficient cells. Each data point is the mean�SEM of 3 or 4 experiments with each experimental field comprising 15to 30 cells. **P�0.01 vs other groups, †P�0.001 vs other groups.




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were slightly attenuated in TRPM7-deficient cells, these effectswere not significantly different from control cells. Taken together,our data indicate that TRPM7 channel is a major regulator ofVSMC Mg2� homeostasis and that it may be less important inmaintaining [Ca2�]i. Our findings confirm others demonstrating that

TRPM7 channels are ion selective and that sensitivity for Mg2� isgreater than that for Ca2�.52,53

A major finding in our study relates to the temporalregulation of TRPM7 and [Mg2�]i by Ang II. Short-termexposure of cells to Ang II resulted in a rapid [Mg2�]i

Figure 5. TRPM7 plays a role in chronic, but not in acute, Ang II–mediated [Mg2�]i regulation. Ang II [Mg2�]i effects were assessed inVSMCs transfected with nonsilencing siRNA (NS-siRNA), siRNA against TRPM7, or in the presence of transfectant alone (Control). A,Acute Ang II–induced responses. Cells were exposed to Ang II for 5 minutes. B, Acute responses to Ang II (5 minutes) in Na�-free con-ditions. C, Chronic Ang II–induced responses. Cells were exposed to Ang II for 24 hours. *P�0.05 vs basal counterpart; **P�0.01 vsbasal counterpart; †P�0.05 vs control and NS-siRNA counterpart.

Figure 6. Essential role of TRPM7 in Ang II–stimulated VSMC growth. Effects of Ang II on 3[H]thymidine and 3[H]leucine incorporation in control,nonsilencing- (NS-siRNA), and siRNA-transfected cells. *P�0.05 vs other groups; **P�0.01 vs other groups; †P�0.05 vs NS-siRNA counterpart.




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decrease, which was abrogated in Na�-free conditions. Re-sponses were similar in control and TRPM7 knockdown cells.However, long-term Ang II exposure resulted in a significant[Mg2�]i increase, which was not evident in TRPM7-deficientcells. These findings suggest that acute Ang II stimulationmediates transmembrane Mg2� transport through Na�-dependent, TRPM7-independent pathways, whereas chronicstimulation leads to TRMP7-dependent Mg2� influx. Putativemechanisms underlying these events could relate to initialactivation of the Na�/Mg2� exchanger by Ang II, resulting inMg2� efflux, as we previously reported,18,44 followed byactivation of TRPM7 leading to Mg2� influx and increased[Mg2�]i. (Figure 7). Upregulation of Ang II–stimulatedTRPM7 may be due, in part, to increased TRPM7 content, aswe demonstrated at the gene and protein levels. It is alsopossible that Ang II–induced [Mg2�]i reduction stimulatesTRPM7, because intracellular Mg2� negatively influencesTRPM7 channel activity.32,34

At the functional level, we demonstrate that TRPM7 isimportant in Ang II–regulated growth of VSMCs. Incorpora-tion of 3[H]-thymidine and 3[H]-leucine was significantlyincreased by Ang II in control but not in TRPM7 knockdowncells, indicating that Ang II–stimulated DNA and proteinsynthesis in VSMCs require functionally active TRPM7.These processes are mediated through AT1 receptors, becausevalsartan inhibited Ang II–induced effects. Although previ-ous studies suggested that TRPM7 is involved in cellgrowth,20,54 our findings here are the first to demonstrate that

TRPM7/Mg2�-dependent pathways play a role in Ang II–regulated growth of VSMCs.

In summary, using a combination of biochemical, pharma-cological, and genetic tools, we provide evidence thatVSMCs possess functionally active TRPM7 ion channels thatplay an important role in modulating VSMC Mg2� homeosta-sis and growth. Furthermore, we demonstrate that Ang IIregulates VSMC [Mg2�]i in a temporal and biphasic fashionsuch that acute Ang II stimulation mediates Mg2� effluxthrough the Na�/Mg2� exchanger, whereas chronic stimula-tion induces Mg2� influx through TRPM7-sensitive path-ways. To our knowledge, these are the first data to identify aputative regulator of transmembrane Mg2� transport inVSMCs. These findings contribute to the further understand-ing of molecular mechanisms involved in Mg2� homeostasisin vascular cells and to the signaling mechanisms underlyingAng II–mediated growth of VSMCs.

AcknowledgmentsThis study was supported by grant 57786 from the CanadianInstitutes for Health Research and a grant from the Heart and StrokeFoundation of Canada. R.M.T. is a scholar of the Fonds de laRecherche en Sante du Quebec.

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Figure 7. Hypothetical scheme demonstrating the possible roleof TRPM7 in Ang II regulation of VSMC [Mg2�]i. Short-termexposure to Ang II results in activation of the Na�/Mg2�

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Ying He, Guoying Yao, Carmine Savoia and Rhian M. Touyzin Vascular Smooth Muscle Cells: Role of Angiotensin II

Transient Receptor Potential Melastatin 7 Ion Channels Regulate Magnesium Homeostasis

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Methods

Measurement of [Mg2+]i and [Ca2+]i in VSMCs

The selective fluorescent probes, mag fura-2AM and fura-2AM were used to measure

[Mg2+]i and [Ca2+]i respectively as described (1-4). Upon complexation with Mg2+, mag-fura 2

exhibits fluorescent changes and undergoes a shift in excitation wavelength. Mag-fura 2 has a

binding constant for Mg2+ of 1.5 mmol/L (3). We fully characterized mag fura-2 specificity for

Mg2+ in previous studies (4)

Washed cells were loaded with mag fura-2AM or fura-2AM (4 µmol/L) and incubated

for 30 minutes at room temperature. [Mg2+]i and [Ca2+]i were measured in multiple cells

simultaneously by fluorescence digital imaging system (Attofluor Ratiovision, Zeiss, West

Germany) using an emission wavelength of 520 nm and alternating excitatory wavelengths of

343 nm and 380 nm (1-5). Video images of fluorescence at 520nm emission were obtained using

an intensified charge-coupled device camera system with the output digitized to a resolution of

512 x 480 pixels. Images of fluorescence ratios were obtained by dividing, pixel by pixel, the

343 nm image after background subtraction by the 380 nm image after background subtraction.

[Mg2+]i responses to increasing concentrations of extracellular Mg2+ (0-5 mmol/L) were

measured in cells incubated in Mg2+-free, Ca2+-containing modified Hanks buffer (in mmol/L,

NaCl 137, NaHCO3 4.2, NaHPO4 3, KCl 5.4, KH2PO4 0.4, CaCl2 1.3, glucose 10 and HEPES 5,

pH 7.4). Cells were exposed to Mg2+-free buffer for 15-20 minutes before addition of

extracellular Mg2+. [Ca2+]i responses to ionomycin (10-6 mol/L) were determined in cells

incubated for 15-20 minutes in modified Hank’s buffer containing Mg2+ (mmol/L, MgCl2 0.5,

MgSO4 0.8).

References

1. Touyz RM, Schiffrin EL. Angiotensin II and vasopressin modulate intracellular free

magnesium in vascular smooth muscle cells through Na+-dependent protein kinase C

pathways. J Biol Chem. 1996;271(40):24353-14358.

2. Touyz RM, Schiffrin EL. Activation of the Na+-H+ exchanger modulates angiotensin II-

stimulated Na+-dependent Mg2+ transport in vascular smooth muscle cells in genetic

hypertension. Hypertension. 1999;34(3):442-449.

3. Raju B, Murphy E, Levy LA, Hall RD, London RE. A fluorescent indicator for

measuring cytosolic free magnesium. Am J Physiol, 1989;256:C540-C548.

4. Touyz RM, Mercure C, Reudelhuber TL. Angiotensin II type I receptor modulates

intracellular free Mg2+ in renally derived cells via Na+-dependent Ca2+-independent

mechanisms. J Biol Chem. 2001;276(17):13657-13663.

5. Grynkiewicz G, Poene M, Tsien TY. A new generation of Ca2+ indicators with greatly

improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.

Figure Legends

Figure 1. [Ca2+]i responses in TRPM7-deficient VSMCs. Human VSMCs were transfected with

non-silencing siRNA and TRPM7 siRNA as described in Methods. [Ca2+]i was measured using

fura-2AM (4 umol/L). Upper panel, representative fura-2AM tracings in control cells and cells

transfected with non-silencing (NS) and TRPM7 silencing RNA. Cells were incubated for 15-20

minutes in Mg2+-containing, Ca2+-containing Hank’s buffer. Arrow indicates addition of

ionomycin (10-6 mol/L). Lower panel, Bar graphs demonstrate maximal [Ca2+]i effect of

ionomycin in control and TRPM7-deficient cells. Results are means±SEM of 3 experiments with

each experimental field comprising 18-26 cells. **p<0.01 vs basal counterpart.

Figure 2. Effects of valsartan, AT1 receptor blocker, on Ang II-stimulated growth of VSMCs.

Incorporation of 3[H]-thymidine and 3[H]-leucine was assessed in control, non-silencing siRNA-

and siRNA-transfected VSMCs treated with Ang II (10-7 mol/L, 24 hours) with or without

vasartan (val) (10-5 mol/L, 24 hours). **p<0.01 vs corresponding control, valsartan and

valsartan+Ang II groups.

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Figure 2

transient receptor potential melastatin 7 ion channels ... · transient receptor potential...

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