Transient Receptor Potential Melastatin 7 Ion Channels Regulate Magnesium Homeostasis in Vascular Smooth Muscle Cells
http://www.100md.com
循环研究杂志 2005年第2期
the CIHR Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, Canada.
Abstract
Magnesium modulates vascular smooth muscle cell (VSMC) function. However, molecular mechanisms regulating VSMC Mg2+ remain unknown. Using biochemical, pharmacological, and genetic tools, the role of transient receptor 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 immunoblotting demonstrated TRPM7 presence in VSMCs (membrane and cytosol). Angiotensin II (Ang II) and aldosterone increased TRPM7 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 by assessing [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 in control, but not in siRNA-transfected VSMCs. Ang IIeCinduced DNA and protein synthesis, measured by 3[H]-thymidine and 3[H]-leucine incorporation, respectively, were increased in control and nonsilencing cells, but not in TRPM7 knockdown VSMCs. Our data indicate that VSMCs possess membrane-associated, Ang IIeC, and aldosterone-regulated TRPM7 channels, which play a role in regulating basal [Mg2+]i, transmembrane Mg2+ transport and DNA and protein synthesis. These novel findings identify TRPM7 as a functionally important regulator of Mg2+ homeostasis and growth in VSMCs.
Key Words: cations TRPM channels vessels aldosterone angiotensin II siRNA
Introduction
Magnesium plays a major role in regulating vascular smooth muscle cell (VSMC) function. Increased intracellular Mg2+ concentration ([Mg2+]i) causes vasodilation and attenuates agonist-induced vasoconstriction, whereas reduced [Mg2+]i has opposite effects, leading to hypercontractility and impaired vasorelaxation.1eC4 Mg2+ also influences growth processes associated with remodeling and fibrosis, characteristic features of vascular damage in hypertension, atherosclerosis, and diabetes.5,6 At the subcellular level, these effects occur, at least in part, through Mg2+-dependent regulation of mitogen-activated protein (MAP) kinases, tyrosine kinases, and reactive oxygen species, important signaling molecules involved in VSMC proliferation, fibrosis, and inflammation.7eC9 Microarray studies demonstrated that changes in [Mg2+]i have potent modulatory actions on expression of various growth signaling molecules.10 We recently reported that altered [Mg2+]i influences cell cycle progression and VSMC growth by modulating cyclin-dependent kinases and MAP kinases.11
Intracellular Mg2+ homeostasis is tightly regulated. In VSMCs, basal [Mg2+]i is maintained at 0.5 to 0.6 mmol/L.11,12 Although Mg2+ is the second most abundant intracellular cation and the predominant divalent cation, molecular mechanisms regulating cellular Mg2+ remain elusive.13 Some studies suggested that transmembrane Mg2+ transport occurs through the Na+/Ca2+ antiporter,14 whereas others demonstrated 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 a Na+-dependent Mg2+ exchanger linked to the Na+/H+ antiporter in VSMCs.15,18
Although studies of Mg2+ fluxes in mammalian cells have indicated the presence of functionally active plasma membrane Mg2+ transport mechanisms, proteins responsible for these fluxes have not been identified.19 Recent investigations suggested that two novel ion channels of the long, or melastatin-related, transient receptor potential (TRPM) ion channel subfamily, TRPM6 and TRPM7, are critically involved 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 spanning domains with an adjacent coiled coil region, a long, highly conserved cytoplasmic N-terminal region, and a cytoplasmic C terminus, which has enzymatic activity.22eC24 TRPM6 and TRPM7, which have an overall amino acid sequence homology of 52%, harbor serine/threonine kinase domains in their C termini.21,25,26 TRPM6 is preferentially expressed in small intestine, colon, and kidney, participating in gastrointestinal and renal Mg2+ absorption.21,27,28 Mutations in TRPM6 cause hypomagnesemia with secondary hypocalcemia.27,29,30
Expression of TRPM7 is widespread with transcripts in brain, spleen, lung, kidney, heart, and liver.22,31eC33 It is also expressed in lymphoid-derived cell lines, hematopoietic cells, granulocytes, leukemia cells, and microglia.20,25 In various cell lines, TRPM7 is regulated by intracellular levels of Mg-ATP and is strongly activated when Mg-ATP falls below 1 mmol/L.32,34 Studies in microglial and HEK293-transfected cells demonstrated that TRPM7 activity is also modulated through its endogenous kinase in a cAMP-, PKA-, and Src-dependent manner25,35 and is inactivated by PIP2 hydrolysis in cardiac fibroblasts.36
To our knowledge nothing is known about the status of TRPM7 in the vasculature. It is unclear whether this cation channel influences Mg2+ transport in vascular cells and whether vasoactive agents regulate TRPM7. To gain insights into the putative role of TRPM7 in vascular Mg2+ homeostasis, we used a combination of biochemical, pharmacological, molecular and genetic approaches to investigate the presence and functional significance of TRPM7 in VSMCs. Our data demonstrate that VSMCs possess functionally active membrane-associated TRPM7 channels that are regulated by angiotensin II (Ang II) and aldosterone. Findings from this study identify for the first time that TRPM7 is a key modulator of vascular Mg2+ homeostasis and that it plays an important role in regulating VSMC function.
Materials and Methods
Cell Culture
The study was approved by the Animal and Human Ethics Committee of the Clinical Research Institute of Montreal and performed according to the recommendations of the Canadian Council for Animal Care. VSMCs from mesenteric arteries and aorta from Wistar Kyoto rats (WKY; Taconic Farms, Germantown, NY) and C57/B6J mice (Jackson Laboratory, Bar Harbor, Me) were isolated by enzymatic digestion and cultured as we described.11 VSMCs from healthy humans were derived from small arteries obtained from gluteal biopsies as we detailed.37 Cells were maintained in DMEM containing 10% fetal calf serum (FCS). Low passaged cells (passages 2 to 7) were studied.
Reverse Transcriptase Polymerase Chain Reaction
Expression of TRPM7 gene was studied by reverse transcriptase polymerase chain reaction (RT-PCR). Primers for rat, mouse, and human TRPM7 are detailed in the Table. Cells were stimulated with vehicle (water), Ang II (10eC7 mol/L), or aldosterone (10eC7 mol/L) for 2 to 24 hours. Total RNA was extracted from cells (TRIzol Reagent). Reverse transcription was performed in 20 e蘈 containing 2 e RNA, 1.0 e蘈 of 10 mmol/L dNTP, 4 e蘈 of 5x first strand buffer, 1.0 e蘈 oligo-(dT)12 to 18 primer (0.5 e/e蘈), 1.0 e蘈 of 200 U/e蘈 M-MLV reverse transcriptase (GIBCO-BRL), 1.0 e蘈 of rRnasin (Rnase inhibitor, 40 U/e蘈), 2 e蘈 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 using specific primers (Table). TRPM7 amplification by PCR involved 95°C for 5 seconds, 35 cycles of 95°C for 30 seconds, 54°C for 30 seconds, 72°C for 30 seconds, and extension for 5 minutes at 72°C. GAPDH amplification by PCR involved 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 57°C for 30 seconds, 72°C for 45 seconds, and extension for 5 minutes, at 72°C. Amplification products were electrophoresed on 1% agarose gel containing ethidium bromide (0.5 e/mL). Bands corresponding to RT-PCR products were visualized by UV light and digitized using AlphaImager software. Band intensity was quantified using the ImageQuant (version 3.3, Molecular Dynamics) software.
TRPM7 Protein Expression
Total protein was extracted from VSMCs as we described.11,37 Briefly, cells were washed with cold PBS and then harvested in HEPES buffer containing (in mmol/L), HEPES 10, pH 7.4, NaF 50, NaCl 50, EDTA 5, EGTA 5, Na pyrophosphate 50, containing triton-X 100 0.5%, phenylmethylsulfonyl fluoride (PMSF) 1mmol/L, leupeptin 1 e/mL, and aprotinin 1 e/mL. Cells were disrupted by brief 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 cell membrane was washed once with the buffer described above and then resuspended in buffer containing 100 mmol/L Tris-HCl, 300 mmol/L NaCl, 1% Triron X-100, and 0.1% SDS containing 2 mmol/L EDTA, 2 mmol/L PMSF, and 0.8 e/mL leupeptin. Proteins (20 e) were separated by electrophoresis on polyacrylamide gel (7.5%) and transferred onto a polyvinylidene diflouride membrane. Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline solution with Tween (TBS-T) (1 hour, room temperature). Membranes were incubated with anti-TRPM7 antibody (1:750) (Abcam Inc) in TBS-T-milk at 4°C overnight with agitation. Washed membranes were incubated with horse-radish peroxidase-conjugated second antibody (1:2000) in TBS-T-Milk (room temperature, 1 hour). Membranes were washed and immunoreactive proteins detected by chemiluminescence. Blots were analyzed densitometrically (Image-Quant software, Molecular Dynamics).
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 (see online data supplement available at http://circres.ahajournals.org).15,18,38eC40 [Mg2+]i responses to increasing concentrations of extracellular Mg2+ (0 to 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 to 20 minutes before addition of extracellular Mg2+. In some experiments, Ang II effects were assessed in Na+-free Hanks’ buffer (Na+ isosmotically replaced with N-methylglucamine).39 [Ca2+]i responses to ionomycin (10eC6 mol/L) were determined in cells incubated for 15 to 20 minutes in modified Hank’s buffer (as earlier) containing Mg2+ (mmol/L, MgCl2 0.5 and MgSO4 0.8).
RNA Interference and Cell Transfection
High-performance purity grade (>90% pure) small interfering RNAs (siRNA) were generated against TRPM7. Sequences identical in human and mouse but that do not match other sequences in GenBank were used. siRNAs for knocking down TRPM7 were synthesized by QIAGEN Inc. The DNA target sequences of the annealed double stand siRNA that we used were as follows: 5'-AACCGG-AGGTCAGGTCGAAAT-3' (1884eC1904), which has 100% homology to mouse gene only, and 5'-AAGCAGAGTGACCT-GGTAGAT-3', which has 100% homology to both mouse and human gene. siRNA, with a nonsilencing oligonucleotide sequence (nonsilencing siRNA) that does not recognize any known homology to mammalian genes, was also generated as a negative control.
Cells were seeded at a density of 1.6x105 cells/well in 6-well plates and grown in DMEM containing 10% FCS and antibiotics. One day after seeding, cells were transfected with siRNA using RNAiFect Transfection Reagent (Qiagen Inc) according to the manufacturer’s instructions. Briefly, siRNA (5 e) was mixed with EC-R buffer (Qiagen Inc) (100 e蘈) to which RNAiFect transfection reagent (15 e蘈) was added. After mixing (15 minutes), the lipid-formulation was added dropwise onto the cells. Control cells were exposed to transfectant in the absence of siRNA. Forty eight hours after transfection, gene silencing was monitored at the mRNA and protein levels by RT-PCR and Western blotting, respectively.
Measurement of DNA and Protein Synthesis by Ang II in TRPM7 siRNA-Transfected VSMCs
Cells were seeded, at an initial concentration of 1x105 cells/mL, into 24-well multiwell plates and grown in DMEM containing 10% FCS and antibiotics. One day after seeding, cells were exposed to transfectant alone (control cells) or transfected with siRNA or nonsilencing siRNA as described earlier. Thirty six hours after transfection, cells were stimulated with Ang II (10eC11 to 10eC6 mol/L, 24 hours) in the absence and presence of valsartan, selective AT1 receptor blocker (10eC5 mol/L). DNA and protein synthesis were evaluated by measuring incorporation of 3[H]-thymidine (5 e藽i/mL) and 3[H]-leucine (2 e藽i/mL, respectively, as we described.12 Briefly, after incubation, radioactive medium was removed, cells washed with ice cold physiological buffered saline, incubated with trichloroacetic acid (TCA) (0.75 mol/L, 15 minutes), washed with cold TCA, and incubated with NaOH (0.2 mol/L for thymidine, 1 mol/L for leucine, 60 minutes, room temperature). Relative incorporation of 3[H]-thymidine and 3[H]-leucine was determined by liquid scintillation counting.
Statistical Analysis
Data are presented as mean±SEM. Groups were compared using one-way ANOVA or Student t test as appropriate. Tukey-Kramer correction was used to compensate for multiple testing procedures. A value of P<0.05 was significant.
Results
TRPM7 Expression in VSMCs
Presence of TRPM7 transcript was assessed by RT-PCR. GAPDH mRNA was used as an internal housekeeping gene and 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 increased TRPM7 mRNA expression (Figure 1B). Maximal agonist-induced responses were obtained within 4 to 6 hours of stimulation.
To evaluate TRPM7 protein content in VSMCs, immunoblotting was performed using anti-TRPM7 antibody. -Actin was used as an internal control. As shown in Figure 2A, SDS-PAGE analysis of total cell homogenate revealed a major band migrating at the 160- to 170-kDa position, corresponding to TRPM7. To establish whether TRPM7 is membrane-associated, we also probed for TRPM7 content in membrane-rich fractions. Figure 2B demonstrates that TRPM7 is present in VSMC membranes and that Ang II stimulation significantly increases membrane TRPM7 content (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+ Influx
The functional significance of TRPM7 in VSMC [Mg2+]i regulation was assessed in cells in which TRPM7 gene was silenced by siRNA. TRPM7 mRNA expression and protein abundance were markedly reduced in human and mouse VSMCs transfected with siRNA, but not in control cells or cells 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-transfected cells (0.51±0.02 mmol/L). Results were not significantly different between control and nonsilencing transfected cells.
Exposure of VSMCs to increasing concentrations of extracellular Mg2+ resulted in a significant [Mg2+]i rise (P<0.01) in control cells and in cells transfected with nonsilencing siRNA (Figure 4). Maximal [Mg2+]i responses were obtained at 3 mmol/L extracellular Mg2+, above which [Mg2+]i did not increase further. Exposure of siRNA-transfected cells to increasing concentrations of extracellular Mg2+ induced a modest, but nonsignificant increase, in [Mg2+]i (Figure 4).
To evaluate whether TRPM7 regulates Ca2+ homeostasis in VSMCs, [Ca2+]i effects of ionomycin were assessed in TRPM7 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±10 nmol/L) versus control (94±4 nmol/L) and nonsilencing siRNA-transfected cells (102±6 nmol/L). Although ionomycin-mediated [Ca2+]i responses were slightly reduced in TRPM7-deficient cells, responses were not significantly different from control cells (Figure 1, online data supplement).
TRPM7 Plays a Role in Chronic, but Not in Acute, Ang IIeCMediated [Mg2+]i Regulation
Short-term VSMC exposure (5 to 10 minutes) to Ang II resulted in a rapid [Mg2+]i decrease (Figure 5). This effect was abrogated in Na+-free conditions (Figure 5B). Results were similar in control, siRNA-transfected, and nonsilencing siRNA-transfected cells (Figure 5A). Long-term Ang II stimulation (24 to 30 hours) was associated with an increase in [Mg2+]i in control and nonsilencing transfected cells, but not in TRPM7 knockdown cells (Figure 5C).
Essential Role of TRPM7 in Ang IIeCStimulated VSMC Growth
To evaluate the functional significance of Ang IIeCregulated TRPM7, growth effects of Ang II were assessed in TRPM7 knockdown VSMCs. As demonstrated in Figure 6, Ang II dose-dependently increased incorporation of 3[H]-thymidine and 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 selective AT1 receptor blocker, inhibited Ang IIeCmediated cell growth (Figure 2, online data supplement).
Discussion
In this study, we provide the first evidence that VSMCs possess TRPM7 channels, that TRPM7 is functionally active, and that this ion channel plays an essential role in regulating Mg2+ influx and maintaining intracellular Mg2+ levels. We also demonstrate that Ang II and aldosterone, which modulate vascular tone and structure, influence TRPM7 abundance. Finally, we report that Ang IIeCregulated TRPM7 is important in the long-term, but not in the short-term regulation of VSMC [Mg2+]i and that it plays a fundamental role in cell growth. Our findings suggest that TRPM7 is a highly regulated transmembrane Mg2+ transporter, critically involved in VSMC Mg2+ homeostasis and growth.
Transient receptor potential channels, particularly of the TRPC (for canonical TRP) and TRPV (for vanilloid TRP) subfamilies, have been demonstrated to be functionally important in regulating Ca2+ entry in VSMCs and endothelial cells.41eC43 To our knowledge, nothing is known about TRPM7 status in vascular cells, although these ion channels have been well characterized in renal and gastric epithelial cells and in numerous mammalian immortalized cell lines.21,22,27,28,33 In the present study, we demonstrate that mouse, rat, and human VSMCs possess membrane-associated TRPM7.
Many vasoactive agents influence VSMC function, of which Ang II is particularly important. Ang II stimulates vascular contraction, growth, and inflammation, in part through Mg2+-dependent processes.39,44 Aldosterone, a mineralocorticoid hormone that influences cellular Mg2+ metabolism,45,46 is increasingly being recognized as an important modulator of vascular function.47,48 In this study, we demonstrate that Ang II and aldosterone regulate TRPM7 mRNA and protein content. Exact mechanisms whereby these agonists control TRPM expression are unknown. However, both peptides stimulate activity of many transcription factors, which could play a role in de novo TRPM production.
To evaluate the functional significance of TRPM7 in VSMCs, we generated siRNAs to selectively reduce TRPM7 expression and used two independent assays to test siRNA activity and specificity. First, we questioned whether siRNA would attenuate TRPM7 content, and second, we used fluorescence digital imaging to test whether siRNA prevents Mg2+ and Ca2+ transmembrane transport. Using a lipid-based system for siRNA transfection, almost 100% TRPM7 gene silencing was obtained as evidenced by significantly reduced mRNA and protein expression. Previous studies reported that cultured cells rendered TRPM7-deficient via cre-loxeCmediated destruction of the TRPM7 gene, undergo growth arrest and die after a few days in culture.20,49 In our investigations, cells remained viable for the duration of our studies. This may relate to the fact that TRPM7 was transiently downregulated and that experiments were performed 48 hours after transfection. Cell death was previously reported to occur 48 to 72 hours after TRPM7 gene silencing in TRPM7-deficient cell lines.20
Basal [Mg2+]i was reduced in TRPM7-deficient cells, confirming the importance of TRPM7 in VSMC Mg2+ homeostasis. Similar findings were observed in stably transfected 293-HEK cells expressing human TRPM7 mutants and in TRPM7 knockout cell lines.20 Increasing concentrations of extracellular Mg2+ resulted in a marked [Mg2+]i rise in control cells. In contrast, in TRPM7-depeleted cells (by siRNA), [Mg2+]i did not change significantly when exposed to high extracellular Mg2+ levels. These findings suggest that TRPM7 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 concentrations of extracellular Mg2+ above 3 mmol/L. Possible reasons for this may relate first to the reciprocal interaction between intracellular Mg2+ and channel activity and second to the presence of regulatory Mg2+ efflux systems. There is now compelling evidence that intracellular Mg2+ negatively influences TRPM7 channel activity.32,34 As [Mg2+]i increases, TRPM7 activity declines. This inhibitory feedback loop between excess intracellular Mg2+ and channel activity may be important in maintaining [Mg2+]i within a physiological range and in protecting cells against Mg2+ overload. It is also possible that as cellular Mg2+ levels increase, Mg2+ efflux systems, such as the Na+/Mg2+ exchanger, are activated.
Data from studies in cell lines demonstrate that TRPM7 has a unique permeation profile with a permeability sequence of Zn2+Ni2+>Ba2+>Co2+>Mg2+Mn2+Sr2+Cd2+Ca2+.31 Because Ca2+ plays a fundamental regulatory role in VSMC function, and because previous studies demonstrated that TRPM7 is a Ca2+-permeant ion channel,52,53 we questioned whether TRPM7, in addition to regulating Mg2+ influx, influences transmembrane Ca2+ transport in VSMCs. Basal [Ca2+]i was unaltered in TRPM7-deficient cells, indicating that this ion channel may not be important in ambient Ca2+ homeostasis in VSMCs. These findings are in contrast to neuronal-derived cells, where TRPM7 seems to be a major regulator of Ca2+ influx.25,52 Ionomycin increased [Ca2+]i in both control and TRPM7 knockdown cells. Although responses were slightly attenuated in TRPM7-deficient cells, these effects were not significantly different from control cells. Taken together, our data indicate that TRPM7 channel is a major regulator of VSMC Mg2+ homeostasis and that it may be less important in maintaining [Ca2+]i. Our findings confirm others demonstrating that TRPM7 channels are ion selective and that sensitivity for Mg2+ is greater than that for Ca2+.52,53
A major finding in our study relates to the temporal regulation of TRPM7 and [Mg2+]i by Ang II. Short-term exposure of cells to Ang II resulted in a rapid [Mg2+]i decrease, which was abrogated in Na+-free conditions. Responses 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-deficient cells. These findings suggest that acute Ang II stimulation mediates transmembrane Mg2+ transport through Na+-dependent, TRPM7-independent pathways, whereas chronic stimulation leads to TRMP7-dependent Mg2+ influx. Putative mechanisms underlying these events could relate to initial activation of the Na+/Mg2+ exchanger by Ang II, resulting in Mg2+ efflux, as we previously reported,18,44 followed by activation of TRPM7 leading to Mg2+ influx and increased [Mg2+]i. (Figure 7). Upregulation of Ang IIeCstimulated TRPM7 may be due, in part, to increased TRPM7 content, as we demonstrated at the gene and protein levels. It is also possible that Ang IIeCinduced [Mg2+]i reduction stimulates TRPM7, because intracellular Mg2+ negatively influences TRPM7 channel activity.32,34
At the functional level, we demonstrate that TRPM7 is important in Ang IIeCregulated growth of VSMCs. Incorporation of 3[H]-thymidine and 3[H]-leucine was significantly increased by Ang II in control but not in TRPM7 knockdown cells, indicating that Ang IIeCstimulated DNA and protein synthesis in VSMCs require functionally active TRPM7. These processes are mediated through AT1 receptors, because valsartan inhibited Ang IIeCinduced effects. Although previous studies suggested that TRPM7 is involved in cell growth,20,54 our findings here are the first to demonstrate that TRPM7/Mg2+-dependent pathways play a role in Ang IIeCregulated growth of VSMCs.
In summary, using a combination of biochemical, pharmacological, and genetic tools, we provide evidence that VSMCs possess functionally active TRPM7 ion channels that play an important role in modulating VSMC Mg2+ homeostasis and growth. Furthermore, we demonstrate that Ang II regulates VSMC [Mg2+]i in a temporal and biphasic fashion such that acute Ang II stimulation mediates Mg2+ efflux through the Na+/Mg2+ exchanger, whereas chronic stimulation induces Mg2+ influx through TRPM7-sensitive pathways. To our knowledge, these are the first data to identify a putative regulator of transmembrane Mg2+ transport in VSMCs. These findings contribute to the further understanding of molecular mechanisms involved in Mg2+ homeostasis in vascular cells and to the signaling mechanisms underlying Ang IIeCmediated growth of VSMCs.
Acknowledgments
This study was supported by grant 57786 from the Canadian Institutes for Health Research and a grant from the Heart and Stroke Foundation of Canada. R.M.T. is a scholar of the Fonds de la Recherchee en Sante du Quebec.
References
Laurant P, Touyz RM, Schiffrin EL. Effect of magnesium on vascular tone and reactivity in pressurized mesenteric resistance arteries from spontaneously hypertensive rats. Can J Physiol Pharmacol. 1997; 75: 293eC300.
Northcott CA, Watts SW. Low [Mg2+]e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension. Hypertension. 2004; 43: 125eC129.
Yang ZW, Wang J, Zheng T, Altura BT, Altura BM. Low [Mg2+]o induces contraction and [Ca2+]i rises in cerebral arteries: roles of Ca2+, PKC, and PI3. Am J Physiol Heart Circ Physiol. 2000; 279: H2898eCH2907.
Touyz RM, Laurant P, Schiffrin EL. Effect of magnesium on calcium responses to vasopressin in vascular smooth muscle cells of spontaneously hypertensive rats. J Pharmacol Exp Ther. 1998; 284: 998eC1005.
Altura BM, Kostellow AB, Zhang A, Li W, Morrill GA, Gupta RK, Altura BT. Expression of the nuclear factor-kappaB and proto-oncogenes c-fos and c-jun are induced by low extracellular Mg2+ in aortic and cerebral vascular smooth muscle cells: possible links to hypertension, atherogenesis, and stroke. Am J Hypertens. 2003; 16: 701eC707.
Yue H, Lee JD, Shimizu H, Uzui H, Mitsuke Y, Ueda T. Effects of magnesium on the production of extracellular matrix metalloproteinases in cultured rat vascular smooth muscle cells. Atherosclerosis. 2003; 166: 271eC277.
Waas WF, Rainey MA, Szafranska AE, Cox K, Dalby KN. A kinetic approach towards understanding substrate interactions and the catalytic mechanism of the serine/threonine protein kinase ERK2: identifying a potential regulatory role for divalent magnesium. Biochim Biophys Acta. 2004; 1697: 81eC87.
Touyz RM, Yao G. Up-regulation of vascular and renal mitogen-activated protein kinases in hypertensive rats is normalized by inhibitors of the Na+/Mg2+ exchanger. Clin Sci (Lond). 2003; 105: 235eC242.
Waas WF, Dalby KN. Physiological concentrations of divalent magnesium ion activate the serine/threonine specific protein kinase ERK2. Biochemistry. 2003; 42: 2960eC2970.
Petrault I, Zimowska W, Mathieu J, Bayle D, Rock E, Favier A, Rayssiguier Y, Mazur A. Changes in gene expression in rat thymocytes identified by cDNA array support the occurrence of oxidative stress in early magnesium deficiency. Biochim Biophys Acta. 2002; 1586: 92eC98.
Touyz RM, Yao G. Modulation of vascular smooth muscle cell growth by magnesium-role of mitogen-activated protein kinases. J Cell Physiol. 2003; 197: 326eC335.
Quamme GA, Dai LJ, Rabkin SW. Dynamics of intracellular free Mg2+ changes in a vascular smooth muscle cell line. Am J Physiol. 1993; 265: H281eCH288.
Wolf FI. TRPM7: Channeling the Future of Cellular Magnesium Homeostasis Sci STKE. 2004: PE23.
Tashiro M, Konishi M, Iwamoto T, Shigekawa M, Kurihara S. Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts. Pflugers Arch. 2000; 440: 819eC827.
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: 24353eC14358.
Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science. 1999; 285: 103eC106.
Wong V, Goodenough DA. Paracellular channels! Science. 1999; 285: 62eC64.
Touyz RM, Schiffrin EL. Activation of the Na+-H+ exchanger modulates angiotensin IIeCstimulated Na+-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension. Hypertension. 1999; 34: 442eC449.
Konrad M, Schlingmann KP, Gudermann T. Insights into the molecular nature of magnesium homeostasis. Am J Physiol Renal Physiol. 2004; 286: F599eCF605.
Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg AM. Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell. 2003; 114: 191eC200.
Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ, Hoenderop JG. TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem. 2004; 279: 19eC25.
Runnels LW, Yue L, Clapham DE. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science. 2001; 291: 1043eC1047.
Cahalan MD. Cell biology. Channels as enzymes. Nature. 2001; 411: 542eC543.
Levitan IB, Cibulsky SM. Biochemistry. TRP ion channelseCtwo proteins in one. Science. 2001; 293: 1270eC1271.
Jiang X, Newell EW, Schlichter LC. Regulation of a TRPM7-like current in rat brain microglia. J Biol Chem. 2003; 278: 42867eC42876.
Ryazanova LV, Dorovkov MV, Ansari A, Ryazanov AG. Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel. J Biol Chem. 2004; 279: 3708eC3716.
Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet. 2002; 31: 166eC170.
Schmitz C, Perraud A-L, Fleig A, Scharenberg AM. Dual-function ion channel/protein kinases: novel components of vertebrate magnesium regulatory mechanisms. Ped Res. 2004; 55: 734eC737.
Chubanov V, Waldegger S, Mederos y Schnitzler M, Vitzthum H, Sassen MC, Seyberth HW, Konrad M, Gudermann T. Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. Proc Natl Acad Sci U S A. 2004; 101: 2894eC2899.
Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Gen. 2002; 31: 171eC174.
Monteilh-Zoller MK, Hermosura MC, Nadler MJ, Scharenberg AM, Penner R, Fleig A. TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions. J Gen Physiol. 2003; 121: 49eC60.
Monteilh-Zoller MK, Scharenberg AM, Penner R, Fleig A. Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNum. J Physiol. 2002; 539: 445eC458.
Gwanyanya A, Amuzescu B, Zakharov SI, Macianskiene R, Sipido KR, Bolotina VM, Vereecke J, Mubagwa K. Magnesium-inhibited, TRPM6/7-like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation. J Physiol. 2004; 559: 761eC766.
Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature. 2001; 411: 590eC595.
Takezawa R, Schmitz C, Demeuse P, Scharenberg AM, Penner R, Fleig A. Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain. Proc Natl Acad Sci. 2004; 10: 6009eC6014.
Runnels LW, Yue L, Clapham DE. The TRPM7 channel is inactivated by PIP hydrolysis. Nat Cell Biol. 2002; 4: 329eC336.
Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res. 2002; 90: 1205eC1213.
Raju B, Murphy E, Levy LA, Hall RD, London RE. A fluorescent indicator for measuring cytosolic free magnesium. Am J Physiol. 1989; 256: C540eCC548.
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: 13657eC13663.
Grynkiewicz G, Poene M, Tsien TY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985; 260: 3440eC3450.
Landsberg JW, Yuan JX. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci. 2004; 19: 44eC50.
Wang J, Shimoda LA, Sylvester JT. Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L848eC858.
Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, Yuan JX. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol. 2003; 284: C316eC330.
Touyz RM, Yao G. Inhibitors of Na+/Mg2+ exchange activity attenuate the development of hypertension in angiotensin IIeCinduced hypertensive rats. J Hypertens. 2003; 21: 337eC344.
Ahokas RA, Warrington KJ, Gerling IC, Sun Y, Wodi LA, Herring PA, Lu L, Bhattacharya SK, Postlethwaite AE, Weber KT. Aldosteronism and peripheral blood mononuclear cell activation: a neuroendocrine-immune interface. Circ Res. 2003 93: 124eC135.
Delva P, Pastori C, Degan M, Montesi G, Brazzarola P, Lechi A. Intralymphocyte free magnesium in patients with primary aldosteronism: aldosterone and lymphocyte magnesium homeostasis. Hypertension. 2000; 35: 113eC117.
Mazak I, Fiebeler A, Muller DN, Park JK, Shagdarsuren E, Lindschau C, Dechend R, Viedt C, Pilz B, Haller H, Luft FC. Aldosterone potentiates angiotensin IIeCinduced signaling in vascular smooth muscle cells. Circulation. 2004; 109: 2792eC2800.
Liu SL, Schmuck S, Chorazcyzewski JZ, Gros R, Feldman RD. Aldosterone regulates vascular reactivity: short-term effects mediated by phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation. Circulation. 2003; 108: 2400eC2406.
Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, Tymianski M. A key role for TRPM7 channels in anoxic neuronal death. Cell. 2003; 115: 863eC877.
Montell C. Mg2+ homeostasis: the Mg2+nificent TRPM chanzymes. Curr Biol. 2003; 13: R799eC801.
Kerschbaum HH, Kozak JA, Cahalan MD. Polyvalent cations as permeant probes of MIC and TRPM7 pores. Biophys J. 2003; 84: 2293eC2305.
Nicotera P, Bano D. The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell. 2003; 115: 768eC770.
Nijenhuis T, Hoenderop JG, Bindels RJ. Downregulation of Ca2+ and Mg2+ transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia. J Am Soc Nephrol. 2004; 15: 549eC557.
Hanano T, Hara Y, Shi J, Morita H, Umebayashi C, Mori E, Sumimoto H, Ito Y, Mori Y, Inoue R. Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells. J Pharmacol Sci. 2004; 95: 403eC419.(Ying He, Guoying Yao, Car)
Abstract
Magnesium modulates vascular smooth muscle cell (VSMC) function. However, molecular mechanisms regulating VSMC Mg2+ remain unknown. Using biochemical, pharmacological, and genetic tools, the role of transient receptor 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 immunoblotting demonstrated TRPM7 presence in VSMCs (membrane and cytosol). Angiotensin II (Ang II) and aldosterone increased TRPM7 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 by assessing [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 in control, but not in siRNA-transfected VSMCs. Ang IIeCinduced DNA and protein synthesis, measured by 3[H]-thymidine and 3[H]-leucine incorporation, respectively, were increased in control and nonsilencing cells, but not in TRPM7 knockdown VSMCs. Our data indicate that VSMCs possess membrane-associated, Ang IIeC, and aldosterone-regulated TRPM7 channels, which play a role in regulating basal [Mg2+]i, transmembrane Mg2+ transport and DNA and protein synthesis. These novel findings identify TRPM7 as a functionally important regulator of Mg2+ homeostasis and growth in VSMCs.
Key Words: cations TRPM channels vessels aldosterone angiotensin II siRNA
Introduction
Magnesium plays a major role in regulating vascular smooth muscle cell (VSMC) function. Increased intracellular Mg2+ concentration ([Mg2+]i) causes vasodilation and attenuates agonist-induced vasoconstriction, whereas reduced [Mg2+]i has opposite effects, leading to hypercontractility and impaired vasorelaxation.1eC4 Mg2+ also influences growth processes associated with remodeling and fibrosis, characteristic features of vascular damage in hypertension, atherosclerosis, and diabetes.5,6 At the subcellular level, these effects occur, at least in part, through Mg2+-dependent regulation of mitogen-activated protein (MAP) kinases, tyrosine kinases, and reactive oxygen species, important signaling molecules involved in VSMC proliferation, fibrosis, and inflammation.7eC9 Microarray studies demonstrated that changes in [Mg2+]i have potent modulatory actions on expression of various growth signaling molecules.10 We recently reported that altered [Mg2+]i influences cell cycle progression and VSMC growth by modulating cyclin-dependent kinases and MAP kinases.11
Intracellular Mg2+ homeostasis is tightly regulated. In VSMCs, basal [Mg2+]i is maintained at 0.5 to 0.6 mmol/L.11,12 Although Mg2+ is the second most abundant intracellular cation and the predominant divalent cation, molecular mechanisms regulating cellular Mg2+ remain elusive.13 Some studies suggested that transmembrane Mg2+ transport occurs through the Na+/Ca2+ antiporter,14 whereas others demonstrated 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 a Na+-dependent Mg2+ exchanger linked to the Na+/H+ antiporter in VSMCs.15,18
Although studies of Mg2+ fluxes in mammalian cells have indicated the presence of functionally active plasma membrane Mg2+ transport mechanisms, proteins responsible for these fluxes have not been identified.19 Recent investigations suggested that two novel ion channels of the long, or melastatin-related, transient receptor potential (TRPM) ion channel subfamily, TRPM6 and TRPM7, are critically involved 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 spanning domains with an adjacent coiled coil region, a long, highly conserved cytoplasmic N-terminal region, and a cytoplasmic C terminus, which has enzymatic activity.22eC24 TRPM6 and TRPM7, which have an overall amino acid sequence homology of 52%, harbor serine/threonine kinase domains in their C termini.21,25,26 TRPM6 is preferentially expressed in small intestine, colon, and kidney, participating in gastrointestinal and renal Mg2+ absorption.21,27,28 Mutations in TRPM6 cause hypomagnesemia with secondary hypocalcemia.27,29,30
Expression of TRPM7 is widespread with transcripts in brain, spleen, lung, kidney, heart, and liver.22,31eC33 It is also expressed in lymphoid-derived cell lines, hematopoietic cells, granulocytes, leukemia cells, and microglia.20,25 In various cell lines, TRPM7 is regulated by intracellular levels of Mg-ATP and is strongly activated when Mg-ATP falls below 1 mmol/L.32,34 Studies in microglial and HEK293-transfected cells demonstrated that TRPM7 activity is also modulated through its endogenous kinase in a cAMP-, PKA-, and Src-dependent manner25,35 and is inactivated by PIP2 hydrolysis in cardiac fibroblasts.36
To our knowledge nothing is known about the status of TRPM7 in the vasculature. It is unclear whether this cation channel influences Mg2+ transport in vascular cells and whether vasoactive agents regulate TRPM7. To gain insights into the putative role of TRPM7 in vascular Mg2+ homeostasis, we used a combination of biochemical, pharmacological, molecular and genetic approaches to investigate the presence and functional significance of TRPM7 in VSMCs. Our data demonstrate that VSMCs possess functionally active membrane-associated TRPM7 channels that are regulated by angiotensin II (Ang II) and aldosterone. Findings from this study identify for the first time that TRPM7 is a key modulator of vascular Mg2+ homeostasis and that it plays an important role in regulating VSMC function.
Materials and Methods
Cell Culture
The study was approved by the Animal and Human Ethics Committee of the Clinical Research Institute of Montreal and performed according to the recommendations of the Canadian Council for Animal Care. VSMCs from mesenteric arteries and aorta from Wistar Kyoto rats (WKY; Taconic Farms, Germantown, NY) and C57/B6J mice (Jackson Laboratory, Bar Harbor, Me) were isolated by enzymatic digestion and cultured as we described.11 VSMCs from healthy humans were derived from small arteries obtained from gluteal biopsies as we detailed.37 Cells were maintained in DMEM containing 10% fetal calf serum (FCS). Low passaged cells (passages 2 to 7) were studied.
Reverse Transcriptase Polymerase Chain Reaction
Expression of TRPM7 gene was studied by reverse transcriptase polymerase chain reaction (RT-PCR). Primers for rat, mouse, and human TRPM7 are detailed in the Table. Cells were stimulated with vehicle (water), Ang II (10eC7 mol/L), or aldosterone (10eC7 mol/L) for 2 to 24 hours. Total RNA was extracted from cells (TRIzol Reagent). Reverse transcription was performed in 20 e蘈 containing 2 e RNA, 1.0 e蘈 of 10 mmol/L dNTP, 4 e蘈 of 5x first strand buffer, 1.0 e蘈 oligo-(dT)12 to 18 primer (0.5 e/e蘈), 1.0 e蘈 of 200 U/e蘈 M-MLV reverse transcriptase (GIBCO-BRL), 1.0 e蘈 of rRnasin (Rnase inhibitor, 40 U/e蘈), 2 e蘈 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 using specific primers (Table). TRPM7 amplification by PCR involved 95°C for 5 seconds, 35 cycles of 95°C for 30 seconds, 54°C for 30 seconds, 72°C for 30 seconds, and extension for 5 minutes at 72°C. GAPDH amplification by PCR involved 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 57°C for 30 seconds, 72°C for 45 seconds, and extension for 5 minutes, at 72°C. Amplification products were electrophoresed on 1% agarose gel containing ethidium bromide (0.5 e/mL). Bands corresponding to RT-PCR products were visualized by UV light and digitized using AlphaImager software. Band intensity was quantified using the ImageQuant (version 3.3, Molecular Dynamics) software.
TRPM7 Protein Expression
Total protein was extracted from VSMCs as we described.11,37 Briefly, cells were washed with cold PBS and then harvested in HEPES buffer containing (in mmol/L), HEPES 10, pH 7.4, NaF 50, NaCl 50, EDTA 5, EGTA 5, Na pyrophosphate 50, containing triton-X 100 0.5%, phenylmethylsulfonyl fluoride (PMSF) 1mmol/L, leupeptin 1 e/mL, and aprotinin 1 e/mL. Cells were disrupted by brief 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 cell membrane was washed once with the buffer described above and then resuspended in buffer containing 100 mmol/L Tris-HCl, 300 mmol/L NaCl, 1% Triron X-100, and 0.1% SDS containing 2 mmol/L EDTA, 2 mmol/L PMSF, and 0.8 e/mL leupeptin. Proteins (20 e) were separated by electrophoresis on polyacrylamide gel (7.5%) and transferred onto a polyvinylidene diflouride membrane. Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline solution with Tween (TBS-T) (1 hour, room temperature). Membranes were incubated with anti-TRPM7 antibody (1:750) (Abcam Inc) in TBS-T-milk at 4°C overnight with agitation. Washed membranes were incubated with horse-radish peroxidase-conjugated second antibody (1:2000) in TBS-T-Milk (room temperature, 1 hour). Membranes were washed and immunoreactive proteins detected by chemiluminescence. Blots were analyzed densitometrically (Image-Quant software, Molecular Dynamics).
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 (see online data supplement available at http://circres.ahajournals.org).15,18,38eC40 [Mg2+]i responses to increasing concentrations of extracellular Mg2+ (0 to 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 to 20 minutes before addition of extracellular Mg2+. In some experiments, Ang II effects were assessed in Na+-free Hanks’ buffer (Na+ isosmotically replaced with N-methylglucamine).39 [Ca2+]i responses to ionomycin (10eC6 mol/L) were determined in cells incubated for 15 to 20 minutes in modified Hank’s buffer (as earlier) containing Mg2+ (mmol/L, MgCl2 0.5 and MgSO4 0.8).
RNA Interference and Cell Transfection
High-performance purity grade (>90% pure) small interfering RNAs (siRNA) were generated against TRPM7. Sequences identical in human and mouse but that do not match other sequences in GenBank were used. siRNAs for knocking down TRPM7 were synthesized by QIAGEN Inc. The DNA target sequences of the annealed double stand siRNA that we used were as follows: 5'-AACCGG-AGGTCAGGTCGAAAT-3' (1884eC1904), which has 100% homology to mouse gene only, and 5'-AAGCAGAGTGACCT-GGTAGAT-3', which has 100% homology to both mouse and human gene. siRNA, with a nonsilencing oligonucleotide sequence (nonsilencing siRNA) that does not recognize any known homology to mammalian genes, was also generated as a negative control.
Cells were seeded at a density of 1.6x105 cells/well in 6-well plates and grown in DMEM containing 10% FCS and antibiotics. One day after seeding, cells were transfected with siRNA using RNAiFect Transfection Reagent (Qiagen Inc) according to the manufacturer’s instructions. Briefly, siRNA (5 e) was mixed with EC-R buffer (Qiagen Inc) (100 e蘈) to which RNAiFect transfection reagent (15 e蘈) was added. After mixing (15 minutes), the lipid-formulation was added dropwise onto the cells. Control cells were exposed to transfectant in the absence of siRNA. Forty eight hours after transfection, gene silencing was monitored at the mRNA and protein levels by RT-PCR and Western blotting, respectively.
Measurement of DNA and Protein Synthesis by Ang II in TRPM7 siRNA-Transfected VSMCs
Cells were seeded, at an initial concentration of 1x105 cells/mL, into 24-well multiwell plates and grown in DMEM containing 10% FCS and antibiotics. One day after seeding, cells were exposed to transfectant alone (control cells) or transfected with siRNA or nonsilencing siRNA as described earlier. Thirty six hours after transfection, cells were stimulated with Ang II (10eC11 to 10eC6 mol/L, 24 hours) in the absence and presence of valsartan, selective AT1 receptor blocker (10eC5 mol/L). DNA and protein synthesis were evaluated by measuring incorporation of 3[H]-thymidine (5 e藽i/mL) and 3[H]-leucine (2 e藽i/mL, respectively, as we described.12 Briefly, after incubation, radioactive medium was removed, cells washed with ice cold physiological buffered saline, incubated with trichloroacetic acid (TCA) (0.75 mol/L, 15 minutes), washed with cold TCA, and incubated with NaOH (0.2 mol/L for thymidine, 1 mol/L for leucine, 60 minutes, room temperature). Relative incorporation of 3[H]-thymidine and 3[H]-leucine was determined by liquid scintillation counting.
Statistical Analysis
Data are presented as mean±SEM. Groups were compared using one-way ANOVA or Student t test as appropriate. Tukey-Kramer correction was used to compensate for multiple testing procedures. A value of P<0.05 was significant.
Results
TRPM7 Expression in VSMCs
Presence of TRPM7 transcript was assessed by RT-PCR. GAPDH mRNA was used as an internal housekeeping gene and 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 increased TRPM7 mRNA expression (Figure 1B). Maximal agonist-induced responses were obtained within 4 to 6 hours of stimulation.
To evaluate TRPM7 protein content in VSMCs, immunoblotting was performed using anti-TRPM7 antibody. -Actin was used as an internal control. As shown in Figure 2A, SDS-PAGE analysis of total cell homogenate revealed a major band migrating at the 160- to 170-kDa position, corresponding to TRPM7. To establish whether TRPM7 is membrane-associated, we also probed for TRPM7 content in membrane-rich fractions. Figure 2B demonstrates that TRPM7 is present in VSMC membranes and that Ang II stimulation significantly increases membrane TRPM7 content (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+ Influx
The functional significance of TRPM7 in VSMC [Mg2+]i regulation was assessed in cells in which TRPM7 gene was silenced by siRNA. TRPM7 mRNA expression and protein abundance were markedly reduced in human and mouse VSMCs transfected with siRNA, but not in control cells or cells 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-transfected cells (0.51±0.02 mmol/L). Results were not significantly different between control and nonsilencing transfected cells.
Exposure of VSMCs to increasing concentrations of extracellular Mg2+ resulted in a significant [Mg2+]i rise (P<0.01) in control cells and in cells transfected with nonsilencing siRNA (Figure 4). Maximal [Mg2+]i responses were obtained at 3 mmol/L extracellular Mg2+, above which [Mg2+]i did not increase further. Exposure of siRNA-transfected cells to increasing concentrations of extracellular Mg2+ induced a modest, but nonsignificant increase, in [Mg2+]i (Figure 4).
To evaluate whether TRPM7 regulates Ca2+ homeostasis in VSMCs, [Ca2+]i effects of ionomycin were assessed in TRPM7 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±10 nmol/L) versus control (94±4 nmol/L) and nonsilencing siRNA-transfected cells (102±6 nmol/L). Although ionomycin-mediated [Ca2+]i responses were slightly reduced in TRPM7-deficient cells, responses were not significantly different from control cells (Figure 1, online data supplement).
TRPM7 Plays a Role in Chronic, but Not in Acute, Ang IIeCMediated [Mg2+]i Regulation
Short-term VSMC exposure (5 to 10 minutes) to Ang II resulted in a rapid [Mg2+]i decrease (Figure 5). This effect was abrogated in Na+-free conditions (Figure 5B). Results were similar in control, siRNA-transfected, and nonsilencing siRNA-transfected cells (Figure 5A). Long-term Ang II stimulation (24 to 30 hours) was associated with an increase in [Mg2+]i in control and nonsilencing transfected cells, but not in TRPM7 knockdown cells (Figure 5C).
Essential Role of TRPM7 in Ang IIeCStimulated VSMC Growth
To evaluate the functional significance of Ang IIeCregulated TRPM7, growth effects of Ang II were assessed in TRPM7 knockdown VSMCs. As demonstrated in Figure 6, Ang II dose-dependently increased incorporation of 3[H]-thymidine and 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 selective AT1 receptor blocker, inhibited Ang IIeCmediated cell growth (Figure 2, online data supplement).
Discussion
In this study, we provide the first evidence that VSMCs possess TRPM7 channels, that TRPM7 is functionally active, and that this ion channel plays an essential role in regulating Mg2+ influx and maintaining intracellular Mg2+ levels. We also demonstrate that Ang II and aldosterone, which modulate vascular tone and structure, influence TRPM7 abundance. Finally, we report that Ang IIeCregulated TRPM7 is important in the long-term, but not in the short-term regulation of VSMC [Mg2+]i and that it plays a fundamental role in cell growth. Our findings suggest that TRPM7 is a highly regulated transmembrane Mg2+ transporter, critically involved in VSMC Mg2+ homeostasis and growth.
Transient receptor potential channels, particularly of the TRPC (for canonical TRP) and TRPV (for vanilloid TRP) subfamilies, have been demonstrated to be functionally important in regulating Ca2+ entry in VSMCs and endothelial cells.41eC43 To our knowledge, nothing is known about TRPM7 status in vascular cells, although these ion channels have been well characterized in renal and gastric epithelial cells and in numerous mammalian immortalized cell lines.21,22,27,28,33 In the present study, we demonstrate that mouse, rat, and human VSMCs possess membrane-associated TRPM7.
Many vasoactive agents influence VSMC function, of which Ang II is particularly important. Ang II stimulates vascular contraction, growth, and inflammation, in part through Mg2+-dependent processes.39,44 Aldosterone, a mineralocorticoid hormone that influences cellular Mg2+ metabolism,45,46 is increasingly being recognized as an important modulator of vascular function.47,48 In this study, we demonstrate that Ang II and aldosterone regulate TRPM7 mRNA and protein content. Exact mechanisms whereby these agonists control TRPM expression are unknown. However, both peptides stimulate activity of many transcription factors, which could play a role in de novo TRPM production.
To evaluate the functional significance of TRPM7 in VSMCs, we generated siRNAs to selectively reduce TRPM7 expression and used two independent assays to test siRNA activity and specificity. First, we questioned whether siRNA would attenuate TRPM7 content, and second, we used fluorescence digital imaging to test whether siRNA prevents Mg2+ and Ca2+ transmembrane transport. Using a lipid-based system for siRNA transfection, almost 100% TRPM7 gene silencing was obtained as evidenced by significantly reduced mRNA and protein expression. Previous studies reported that cultured cells rendered TRPM7-deficient via cre-loxeCmediated destruction of the TRPM7 gene, undergo growth arrest and die after a few days in culture.20,49 In our investigations, cells remained viable for the duration of our studies. This may relate to the fact that TRPM7 was transiently downregulated and that experiments were performed 48 hours after transfection. Cell death was previously reported to occur 48 to 72 hours after TRPM7 gene silencing in TRPM7-deficient cell lines.20
Basal [Mg2+]i was reduced in TRPM7-deficient cells, confirming the importance of TRPM7 in VSMC Mg2+ homeostasis. Similar findings were observed in stably transfected 293-HEK cells expressing human TRPM7 mutants and in TRPM7 knockout cell lines.20 Increasing concentrations of extracellular Mg2+ resulted in a marked [Mg2+]i rise in control cells. In contrast, in TRPM7-depeleted cells (by siRNA), [Mg2+]i did not change significantly when exposed to high extracellular Mg2+ levels. These findings suggest that TRPM7 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 concentrations of extracellular Mg2+ above 3 mmol/L. Possible reasons for this may relate first to the reciprocal interaction between intracellular Mg2+ and channel activity and second to the presence of regulatory Mg2+ efflux systems. There is now compelling evidence that intracellular Mg2+ negatively influences TRPM7 channel activity.32,34 As [Mg2+]i increases, TRPM7 activity declines. This inhibitory feedback loop between excess intracellular Mg2+ and channel activity may be important in maintaining [Mg2+]i within a physiological range and in protecting cells against Mg2+ overload. It is also possible that as cellular Mg2+ levels increase, Mg2+ efflux systems, such as the Na+/Mg2+ exchanger, are activated.
Data from studies in cell lines demonstrate that TRPM7 has a unique permeation profile with a permeability sequence of Zn2+Ni2+>Ba2+>Co2+>Mg2+Mn2+Sr2+Cd2+Ca2+.31 Because Ca2+ plays a fundamental regulatory role in VSMC function, and because previous studies demonstrated that TRPM7 is a Ca2+-permeant ion channel,52,53 we questioned whether TRPM7, in addition to regulating Mg2+ influx, influences transmembrane Ca2+ transport in VSMCs. Basal [Ca2+]i was unaltered in TRPM7-deficient cells, indicating that this ion channel may not be important in ambient Ca2+ homeostasis in VSMCs. These findings are in contrast to neuronal-derived cells, where TRPM7 seems to be a major regulator of Ca2+ influx.25,52 Ionomycin increased [Ca2+]i in both control and TRPM7 knockdown cells. Although responses were slightly attenuated in TRPM7-deficient cells, these effects were not significantly different from control cells. Taken together, our data indicate that TRPM7 channel is a major regulator of VSMC Mg2+ homeostasis and that it may be less important in maintaining [Ca2+]i. Our findings confirm others demonstrating that TRPM7 channels are ion selective and that sensitivity for Mg2+ is greater than that for Ca2+.52,53
A major finding in our study relates to the temporal regulation of TRPM7 and [Mg2+]i by Ang II. Short-term exposure of cells to Ang II resulted in a rapid [Mg2+]i decrease, which was abrogated in Na+-free conditions. Responses 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-deficient cells. These findings suggest that acute Ang II stimulation mediates transmembrane Mg2+ transport through Na+-dependent, TRPM7-independent pathways, whereas chronic stimulation leads to TRMP7-dependent Mg2+ influx. Putative mechanisms underlying these events could relate to initial activation of the Na+/Mg2+ exchanger by Ang II, resulting in Mg2+ efflux, as we previously reported,18,44 followed by activation of TRPM7 leading to Mg2+ influx and increased [Mg2+]i. (Figure 7). Upregulation of Ang IIeCstimulated TRPM7 may be due, in part, to increased TRPM7 content, as we demonstrated at the gene and protein levels. It is also possible that Ang IIeCinduced [Mg2+]i reduction stimulates TRPM7, because intracellular Mg2+ negatively influences TRPM7 channel activity.32,34
At the functional level, we demonstrate that TRPM7 is important in Ang IIeCregulated growth of VSMCs. Incorporation of 3[H]-thymidine and 3[H]-leucine was significantly increased by Ang II in control but not in TRPM7 knockdown cells, indicating that Ang IIeCstimulated DNA and protein synthesis in VSMCs require functionally active TRPM7. These processes are mediated through AT1 receptors, because valsartan inhibited Ang IIeCinduced effects. Although previous studies suggested that TRPM7 is involved in cell growth,20,54 our findings here are the first to demonstrate that TRPM7/Mg2+-dependent pathways play a role in Ang IIeCregulated growth of VSMCs.
In summary, using a combination of biochemical, pharmacological, and genetic tools, we provide evidence that VSMCs possess functionally active TRPM7 ion channels that play an important role in modulating VSMC Mg2+ homeostasis and growth. Furthermore, we demonstrate that Ang II regulates VSMC [Mg2+]i in a temporal and biphasic fashion such that acute Ang II stimulation mediates Mg2+ efflux through the Na+/Mg2+ exchanger, whereas chronic stimulation induces Mg2+ influx through TRPM7-sensitive pathways. To our knowledge, these are the first data to identify a putative regulator of transmembrane Mg2+ transport in VSMCs. These findings contribute to the further understanding of molecular mechanisms involved in Mg2+ homeostasis in vascular cells and to the signaling mechanisms underlying Ang IIeCmediated growth of VSMCs.
Acknowledgments
This study was supported by grant 57786 from the Canadian Institutes for Health Research and a grant from the Heart and Stroke Foundation of Canada. R.M.T. is a scholar of the Fonds de la Recherchee en Sante du Quebec.
References
Laurant P, Touyz RM, Schiffrin EL. Effect of magnesium on vascular tone and reactivity in pressurized mesenteric resistance arteries from spontaneously hypertensive rats. Can J Physiol Pharmacol. 1997; 75: 293eC300.
Northcott CA, Watts SW. Low [Mg2+]e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension. Hypertension. 2004; 43: 125eC129.
Yang ZW, Wang J, Zheng T, Altura BT, Altura BM. Low [Mg2+]o induces contraction and [Ca2+]i rises in cerebral arteries: roles of Ca2+, PKC, and PI3. Am J Physiol Heart Circ Physiol. 2000; 279: H2898eCH2907.
Touyz RM, Laurant P, Schiffrin EL. Effect of magnesium on calcium responses to vasopressin in vascular smooth muscle cells of spontaneously hypertensive rats. J Pharmacol Exp Ther. 1998; 284: 998eC1005.
Altura BM, Kostellow AB, Zhang A, Li W, Morrill GA, Gupta RK, Altura BT. Expression of the nuclear factor-kappaB and proto-oncogenes c-fos and c-jun are induced by low extracellular Mg2+ in aortic and cerebral vascular smooth muscle cells: possible links to hypertension, atherogenesis, and stroke. Am J Hypertens. 2003; 16: 701eC707.
Yue H, Lee JD, Shimizu H, Uzui H, Mitsuke Y, Ueda T. Effects of magnesium on the production of extracellular matrix metalloproteinases in cultured rat vascular smooth muscle cells. Atherosclerosis. 2003; 166: 271eC277.
Waas WF, Rainey MA, Szafranska AE, Cox K, Dalby KN. A kinetic approach towards understanding substrate interactions and the catalytic mechanism of the serine/threonine protein kinase ERK2: identifying a potential regulatory role for divalent magnesium. Biochim Biophys Acta. 2004; 1697: 81eC87.
Touyz RM, Yao G. Up-regulation of vascular and renal mitogen-activated protein kinases in hypertensive rats is normalized by inhibitors of the Na+/Mg2+ exchanger. Clin Sci (Lond). 2003; 105: 235eC242.
Waas WF, Dalby KN. Physiological concentrations of divalent magnesium ion activate the serine/threonine specific protein kinase ERK2. Biochemistry. 2003; 42: 2960eC2970.
Petrault I, Zimowska W, Mathieu J, Bayle D, Rock E, Favier A, Rayssiguier Y, Mazur A. Changes in gene expression in rat thymocytes identified by cDNA array support the occurrence of oxidative stress in early magnesium deficiency. Biochim Biophys Acta. 2002; 1586: 92eC98.
Touyz RM, Yao G. Modulation of vascular smooth muscle cell growth by magnesium-role of mitogen-activated protein kinases. J Cell Physiol. 2003; 197: 326eC335.
Quamme GA, Dai LJ, Rabkin SW. Dynamics of intracellular free Mg2+ changes in a vascular smooth muscle cell line. Am J Physiol. 1993; 265: H281eCH288.
Wolf FI. TRPM7: Channeling the Future of Cellular Magnesium Homeostasis Sci STKE. 2004: PE23.
Tashiro M, Konishi M, Iwamoto T, Shigekawa M, Kurihara S. Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts. Pflugers Arch. 2000; 440: 819eC827.
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: 24353eC14358.
Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science. 1999; 285: 103eC106.
Wong V, Goodenough DA. Paracellular channels! Science. 1999; 285: 62eC64.
Touyz RM, Schiffrin EL. Activation of the Na+-H+ exchanger modulates angiotensin IIeCstimulated Na+-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension. Hypertension. 1999; 34: 442eC449.
Konrad M, Schlingmann KP, Gudermann T. Insights into the molecular nature of magnesium homeostasis. Am J Physiol Renal Physiol. 2004; 286: F599eCF605.
Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg AM. Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell. 2003; 114: 191eC200.
Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ, Hoenderop JG. TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem. 2004; 279: 19eC25.
Runnels LW, Yue L, Clapham DE. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science. 2001; 291: 1043eC1047.
Cahalan MD. Cell biology. Channels as enzymes. Nature. 2001; 411: 542eC543.
Levitan IB, Cibulsky SM. Biochemistry. TRP ion channelseCtwo proteins in one. Science. 2001; 293: 1270eC1271.
Jiang X, Newell EW, Schlichter LC. Regulation of a TRPM7-like current in rat brain microglia. J Biol Chem. 2003; 278: 42867eC42876.
Ryazanova LV, Dorovkov MV, Ansari A, Ryazanov AG. Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel. J Biol Chem. 2004; 279: 3708eC3716.
Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet. 2002; 31: 166eC170.
Schmitz C, Perraud A-L, Fleig A, Scharenberg AM. Dual-function ion channel/protein kinases: novel components of vertebrate magnesium regulatory mechanisms. Ped Res. 2004; 55: 734eC737.
Chubanov V, Waldegger S, Mederos y Schnitzler M, Vitzthum H, Sassen MC, Seyberth HW, Konrad M, Gudermann T. Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. Proc Natl Acad Sci U S A. 2004; 101: 2894eC2899.
Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Gen. 2002; 31: 171eC174.
Monteilh-Zoller MK, Hermosura MC, Nadler MJ, Scharenberg AM, Penner R, Fleig A. TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions. J Gen Physiol. 2003; 121: 49eC60.
Monteilh-Zoller MK, Scharenberg AM, Penner R, Fleig A. Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNum. J Physiol. 2002; 539: 445eC458.
Gwanyanya A, Amuzescu B, Zakharov SI, Macianskiene R, Sipido KR, Bolotina VM, Vereecke J, Mubagwa K. Magnesium-inhibited, TRPM6/7-like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation. J Physiol. 2004; 559: 761eC766.
Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature. 2001; 411: 590eC595.
Takezawa R, Schmitz C, Demeuse P, Scharenberg AM, Penner R, Fleig A. Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain. Proc Natl Acad Sci. 2004; 10: 6009eC6014.
Runnels LW, Yue L, Clapham DE. The TRPM7 channel is inactivated by PIP hydrolysis. Nat Cell Biol. 2002; 4: 329eC336.
Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res. 2002; 90: 1205eC1213.
Raju B, Murphy E, Levy LA, Hall RD, London RE. A fluorescent indicator for measuring cytosolic free magnesium. Am J Physiol. 1989; 256: C540eCC548.
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: 13657eC13663.
Grynkiewicz G, Poene M, Tsien TY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985; 260: 3440eC3450.
Landsberg JW, Yuan JX. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci. 2004; 19: 44eC50.
Wang J, Shimoda LA, Sylvester JT. Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L848eC858.
Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, Yuan JX. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol. 2003; 284: C316eC330.
Touyz RM, Yao G. Inhibitors of Na+/Mg2+ exchange activity attenuate the development of hypertension in angiotensin IIeCinduced hypertensive rats. J Hypertens. 2003; 21: 337eC344.
Ahokas RA, Warrington KJ, Gerling IC, Sun Y, Wodi LA, Herring PA, Lu L, Bhattacharya SK, Postlethwaite AE, Weber KT. Aldosteronism and peripheral blood mononuclear cell activation: a neuroendocrine-immune interface. Circ Res. 2003 93: 124eC135.
Delva P, Pastori C, Degan M, Montesi G, Brazzarola P, Lechi A. Intralymphocyte free magnesium in patients with primary aldosteronism: aldosterone and lymphocyte magnesium homeostasis. Hypertension. 2000; 35: 113eC117.
Mazak I, Fiebeler A, Muller DN, Park JK, Shagdarsuren E, Lindschau C, Dechend R, Viedt C, Pilz B, Haller H, Luft FC. Aldosterone potentiates angiotensin IIeCinduced signaling in vascular smooth muscle cells. Circulation. 2004; 109: 2792eC2800.
Liu SL, Schmuck S, Chorazcyzewski JZ, Gros R, Feldman RD. Aldosterone regulates vascular reactivity: short-term effects mediated by phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation. Circulation. 2003; 108: 2400eC2406.
Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, Tymianski M. A key role for TRPM7 channels in anoxic neuronal death. Cell. 2003; 115: 863eC877.
Montell C. Mg2+ homeostasis: the Mg2+nificent TRPM chanzymes. Curr Biol. 2003; 13: R799eC801.
Kerschbaum HH, Kozak JA, Cahalan MD. Polyvalent cations as permeant probes of MIC and TRPM7 pores. Biophys J. 2003; 84: 2293eC2305.
Nicotera P, Bano D. The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death. Cell. 2003; 115: 768eC770.
Nijenhuis T, Hoenderop JG, Bindels RJ. Downregulation of Ca2+ and Mg2+ transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia. J Am Soc Nephrol. 2004; 15: 549eC557.
Hanano T, Hara Y, Shi J, Morita H, Umebayashi C, Mori E, Sumimoto H, Ito Y, Mori Y, Inoue R. Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells. J Pharmacol Sci. 2004; 95: 403eC419.(Ying He, Guoying Yao, Car)