This document discusses a study examining the regulation of transient receptor potential melastatin 6 and 7 (TRPM6 and TRPM7) cation channels in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) compared to Wistar Kyoto (WKY) rats. The study found that while TRPM6 expression was similar between SHR and WKY VSMCs, TRPM7 expression and activity were lower in SHR cells. This was associated with reduced intracellular magnesium levels in SHR VSMCs. Angiotensin II increased TRPM6 expression similarly in both cell types but increased TRPM7 expression only in WKY cells, not SHR cells. The findings suggest down

1. CALL FOR PAPERS Cardiovascular-Kidney Interactions in Health and Disease
Differential regulation of transient receptor potential melastatin 6 and 7 cation
channels by ANG II in vascular smooth muscle cells from spontaneously
hypertensive rats
Rhian M. Touyz,1
Ying He,1
Augusto C. I. Montezano,1,2
Guoying Yao,1
Vladimir Chubanov,3
Thomas Gudermann,3
and Glaucia E. Callera1
1
Kidney Research Centre, University of Ottawa, Ontario, Canada; 2
Department of Pharmacology, University of
Saˆo Paulo, Saˆo Paulo, Brazil, 3
Institut Pharmakologie Toxikologie, Philipps Universita¨t, Marburg, Germany
Submitted 19 July 2005; accepted in final form 16 August 2005
Touyz, Rhian M., Ying He, Augusto C. I. Montezano, Guoying
Yao, Vladimir Chubanov, Thomas Gudermann, and Glaucia E.
Callera. Differential regulation of transient receptor potential
melastatin 6 and 7 cation channels by ANG II in vascular smooth
muscle cells from spontaneously hypertensive rats. Am J Physiol
Regul Integr Comp Physiol 290: R73–R78, 2006. First published
August 18, 2005; doi:10.1152/ajpregu.00515.2005.—Intracellular
Mg2ϩ
depletion has been implicated in vascular dysfunction in hy-
pertension. We demonstrated that transient receptor potential melasta-
tin 7 (TRPM7) cation channels mediate Mg2ϩ
influx in VSMCs.
Whether this plays a role in [Mg2ϩ
]i deficiency in hypertension is
unclear. Here, we tested the hypothesis that downregulation of
TRPM7 and its homologue TRPM6 is associated with reduced
[Mg2ϩ
]i and that ANG II negatively regulates TRPM6/7 in vascular
smooth muscle cells (VSMCs) from spontaneously hypertensive rats
(SHR). Cultured VSMCs from Wistar Kyoto (WKY) and SHR were
studied. mRNA and protein expression of TRPM6 and TRPM7 were
assessed by RT-PCR and immunoblotting, respectively. Translocation
of annexin-1, specific TRPM7 substrate, was measured as an index of
TRPM7 activation. [Mg2ϩ
]i was determined using mag fura-2.
VSMCs from WKY and SHR express TRPM6 and TRPM7. Basal
TRPM6 expression was similar in WKY and SHR, but basal TRPM7
content was lower in VSMCs from SHR vs. WKY. This was associ-
ated with significantly reduced [Mg2ϩ
]i in SHR cells (P Ͻ 0.01).
ANG II time-dependently increased TRPM6 expression, with similar
responses in WKY and SHR. ANG II significantly increased TRPM7
expression in WKY (P Ͻ 0.05), but not in SHR. Annexin-1 translo-
cation was reduced 1.5–2-fold in SHR vs. WKY. Our findings
demonstrate that TRPM6 and TRPM7 are differentially regulated in
VSMCs from SHR and WKY. Whereas TRPM6 is unaltered in SHR,
expression of TRPM7 is blunted. This was associated with attenuated
annexin-1 translocation and decreased VSMC [Mg2ϩ
]i in SHR.
Downregulation of TRPM7, but not TRPM6, may play a role in
altered Mg2ϩ
homeostasis in VSMCs from SHR.
magnesium; hypertension; mesenteric arteries; ion transport
MAGNESIUM (Mg2ϩ
), THE SECOND most abundant intracellular
cation, is critically involved in regulating vascular smooth
muscle cell (VSMC) function (34, 44). Through its Ca2ϩ
antagonistic properties Mg2ϩ
negatively modulates intracellu-
lar free Ca2ϩ
concentration ([Ca2ϩ
)]i, a major determinant of
VSMC contraction/dilation. Mg2ϩ
influences VSMC growth
and apoptosis through its modulatory actions on MAP kinases,
tyrosine kinases/phosphatases, gene expression, and cell cycle
proteins (1, 38, 45a). Changes in [Mg2ϩ
]i are associated with
endothelial dysfunction, inflammatory responses, altered vas-
cular tone, and structural remodeling, characteristic features of
hypertensive vascular damage (20, 44a). In particular low
Mg2ϩ
conditions contribute to increased vascular tone, en-
hanced responses to vasoconstrictor agents, blunted vasodila-
tion, oxidative stress, vascular remodeling, and increased blood
pressure (14, 37).
Accumulating evidence indicates reduced [Mg2ϩ
]i in vascu-
lar and circulating cells from experimental models of hyper-
tension and essential hypertensive patients (37). Intracellular
Mg2ϩ
deficiency contributes to vascular dysfunction in hyper-
tension, as normalization of [Mg2ϩ
]i improves vascular tone,
attenuates oxidative stress and inflammation, regresses vascu-
lar remodeling, and prevents development of hypertension
(14, 39).
Despite the fact that intracellular Mg2ϩ
is the most abundant
divalent intracellular cation and that it modulates over 320
enzymes, processes regulating cellular Mg2ϩ
homeostasis re-
main elusive. Some studies suggested that transmembrane
Mg2ϩ
transport occurs through the Naϩ
/Ca2ϩ
antiporter (21,
32), while others demonstrated that Mg2ϩ
efflux is linked to
Naϩ
/Hϩ
exchange (35). Renal Mg2ϩ
transport is mediated via
paracellin-1, through a paracellular process (30, 43). We re-
ported that Mg2ϩ
efflux is regulated by a Naϩ
-dependent
Mg2ϩ
exchanger linked to the Naϩ
/Hϩ
antiporter in VSMCs
(33, 35, 36).
Until recently, specific proteins mediating transcellular
Mg2ϩ
influx were unknown. In 2001, the mitochondrial Mg2ϩ
transporter Mrs2 was suggested as a candidate transporter (46).
In 2003, Mrs2p was identified as an essential component of the
major electrophoretic Mg2ϩ
influx system in mitochondria
(11). More recently, transient receptor potential melastatin
(TRPM) 6 and 7 were found to be master regulators of cellular
Mg2ϩ
influx. Confirmation of the importance of TRPM6 and
TRPM7 in Mg2ϩ
homeostasis is evidenced by findings that
mutations in TRPM6 cause hypomagnesemia with secondary
hypocalcemia (3, 27, 41), that thiazide-induced Mg2ϩ
wasting
Address for reprint requests and other correspondence: R. M Touyz, Kidney
Research Centre, Univ. of Ottawa, Rm. 1333A, 451 Smyth Road, K1H 8M5,
Ottawa, ON (e-mail rtouyz@uottawa.ca).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Regul Integr Comp Physiol 290: R73–R78, 2006.
First published August 18, 2005; doi:10.1152/ajpregu.00515.2005.
0363-6119/06 $8.00 Copyright © 2006 the American Physiological Societyhttp://www.ajpregu.org R73

2. is linked to TRPM6 downregulation (19), and that mutant
TRPM7 in dwarf zebrafish is associated with defective skel-
etogenesis, altered mineral metabolism, and kidney stone for-
mation (5).
TRPM6 and TRPM7, cloned and characterized by two
independent groups (24, 28), are novel dual-function proteins,
comprising an ion channel related to the TRP proteins fused
with a Ser/Thr kinase (16, 29). These proteins are the only
known examples of a kinase domain incorporated in the same
molecule with an ion-channel pore (2, 26). They are Mg2ϩ
-
permeable channels that facilitate Mg2ϩ
influx (15, 40).
TRPM6 expression is restricted mainly to epithelial cells,
whereas TRPM7 expression is widespread (12). In various cell
lines, TRPM7 is negatively regulated by intracellular levels of
Mg ATP (6, 18), and activity is modulated through its endog-
enous kinase in a cAMP-, PKA- and Src-dependent manner
(10, 31) and is inactivated by PIP2 hydrolysis in cardiac
fibroblasts (25). The only known substrate for TRPM7 kinase
is annexin 1 (4). We recently demonstrated that TRPM7 is
present and functionally active in VSMCs and that it is a major
regulator of Mg2ϩ
influx (9).
Because vascular [Mg2ϩ
]i homeostasis is altered in hyper-
tension (37), we questioned whether TRPM6 and TRPM7 may
play a role in this process. Here, we sought to determine
whether TRPM6 and TRPM7 are differentially regulated in
VSMCs from Wistar Kyoto (WKY) and spontaneously hyper-
tensive rats (SHR) and whether this is associated with altered
vascular [Mg2ϩ
]i status in SHR. Findings from our study
demonstrate that whereas TRPM6 is equally expressed in
VSMCs from WKY and SHR, TRPM7 abundance and activity
are reduced, ANG II-induced expression of TRPM7 is blunted
and [Mg2ϩ
]i is attenuated in SHR. These novel data suggest
that downregulation of TRPM7-mediated Mg2ϩ
transport may
play an important role in intracellular Mg2ϩ
deficiency in
VSMCs from SHR.
MATERIALS AND METHODS
Cell culture. The study was approved by the Animal and Human
Ethics Committee of the University of Ottawa and carried out accord-
ing to the recommendations of the Canadian Council for Animal Care.
VSMCs from mesenteric arteries from 16-wk-old WKY and SHR
were isolated by enzymatic digestion and cultured as we have de-
scribed (38). Cells were maintained in DMEM containing 10% FCS.
Low passaged cells (passages 2–7) were used.
RT-PCR. Expression of the TRPM6 and TRPM7 genes was studied
by RT-PCR. Primers are detailed in Table 1. Cells were stimulated
with vehicle (water) and ANG II (10Ϫ7
mol/l) for 2–24 h. 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 ϫ first-strand buffer, 1.0 ␮l oligo-(dT)12–18 primer (0.5
␮g/␮l), 1.0 ␮l of 200 U/␮l M-MLV reverse transcriptase (GIBCO-
BRL, Carlsbad, CA), 1.0 ␮l of rRNasin (RNase inhibitor, 40 U/␮l), 2
␮l dithiothreitol (0.1 mol/l), for 1 h, 37°C. The reaction was stopped
by heating at 70°C for 15 min. Two microliters of resulting cDNA
mixture was amplified using specific primers (table). TRPM6 and
TRPM7 amplification by PCR involved: 95°C for 5 s, 35 cycles of
95°C for 30 s, 54°C for 30 s, 72°C for 30 s and extension for 5 min
at 72°C. GAPDH amplification by PCR involved 94°C for 5 min, 30
cycles of 94°C, 30 s, 57°C for 30 s, 72°C for 45 s and extension for
5 min, at 72°C. Amplification products were electrophoresed on 1%
agarose gel containing ethidium bromide (0.5 ␮g/ml). Bands corre-
sponding 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.
TRPM6 and TRPM7 protein expression. Total protein was ex-
tracted from VSMCs as we described (38). Briefly, cells were washed
with cold PBS and then harvested in HEPES buffer containing (in
mmol/l), 10 HEPES, pH 7.4, 50 NaF, 50 NaCl, 5 EDTA, 5 EGTA, 50
Na pyrophosphate, containing Triton X-100 0.5%, 1 mmol/l phenyl-
methylsulfonyl fluoride, 1 ␮g/ml, leupeptin, and 1 ␮g/ml aprotinin.
Cells were disrupted by brief sonication. Samples were then centri-
fuged (500 g, 10 min, 4°C) to remove nuclei. Proteins (20 ␮g) were
separated by electrophoresis on polyacrylamide gel (7.5%) and trans-
ferred onto a polyvinylidene difluoride membrane. Nonspecific bind-
ing sites were blocked with 5% skim milk in Tris-buffered saline
solution with Tween (TBS-T) (1 h, room temperature). Membranes
were incubated with anti-TRPM6 antibody (1:500) (kind gift from T.
Gudermann) and anti-TRPM7 antibody (1:750) (Abcam Cambridge,
MA) in TBS-T-milk at 4°C overnight with agitation. Monoclonal
antibody to alpha-actin (Sigma-Aldrich, St. Louis, MO) was used as
an internal control. Washed membranes were incubated with horse-
radish peroxidase-conjugated second antibody (1:2000) in TBS-T-
Milk (room temperature, 1 h). Membranes were washed, and immu-
noreactive proteins detected by chemiluminescence. Blots were ana-
lyzed densitometrically (Image-Quant software, Molecular Dynamics,
Sunnyvale, CA).
Assessment of annexin-1 activity. Annexin-1 activation was as-
sessed by determining translocation from the cytosol to the mem-
brane. Cells, stimulated with ANG II (10Ϫ7
mol/l) for 10 min, were
fractionated to obtain cytosol- and membrane-rich fractions. Homog-
enates were centrifuged at 50,000 g for 30 min at 4°C, thereby
isolating cytosolic fraction in the supernatant. The particulate fraction
was incubated under constant shaking for 30 min at 4°C in lysis buffer
containing 1% Triton X-100 and recentrifuged at 50,000 g for 30 min
at 4°C. Western blotting was performed as described above using
anti-annexin-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA,
1:500).
Measurement of [Mg2ϩ
]i in VSMCs. The selective fluorescent
probe, mag fura-2 AM, was used to measure [Mg2ϩ
]i. (22). Cells were
washed with modified Hank’s buffered saline solution containing (in
mmol/l): 137 NaCl, 5.4 KCl, 4.2 NaHCO3, 3 Na2HPO4, 0.4 KH2PO4,
1.3 CaCl2, 0.5 MgCl2, 0.8 MgSO4, 10 glucose, 5 N-2-hydroxyethyl-
piperazine-NЈ-2-ethanesulfonic acid (HEPES), pH 7.4, and loaded
with mag-fura-2 AM (4 ␮mol/l), which was dissolved in dimethyl
sulfoxide with 0.02% pluronic acid. Cells were incubated for 30 min
at room temperature and then washed with warmed buffer and
incubated for a further 15 min to ensure complete deesterification.
Under these loading conditions, the ratiometric (343/380 nm) fluo-
rescence cell images were homogeneous, indicating that there was no
significant intracellular compartmentalization of mag-fura-2. The cov-
erslip-containing cells was placed in a stainless steel chamber and
mounted on the stage of an inverted microscope (Axiovert 135, Zeiss,
Table 1. Primer sequence for TRPM6 and TRPM7
amplification by RT-PCR in rat VSMCs
Gene Primer Sequence
Fragment Size,
bp
TRPM6
(S) 5Ј-GAAACTGTGGCTATTGGC-3Ј 317
(AS) 5Ј-CCAATACGGCTCAAAGAC-3Ј 317
TRPM7
(S) 5Ј-GCAAATGACTCCACTCTC-3Ј 422
(AS) 5Ј-GATTCTCTCTTCACTCCCAG-3Ј 422
GAPDH
(S) 5Ј-TTGTTGCCATCAACGACCCC-3Ј 435
(AS) 5Ј-ATGAGCCCTTCCACAATGCC-3Ј 435
S, sense; AS, antisense.
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3. West Germany) as previously described (35). [Mg2ϩ
]i was measured
in basal and in ANG II-stimulated cells (10Ϫ7
mol/l, 2 h).
Statistical analysis. Data are presented as means Ϯ SE. Groups
were compared using one-way ANOVA or Student’s t-test as appro-
priate. Tukey-Kramer’s correction was used to compensate for mul-
tiple testing procedures. P Ͻ 0.05 was significant.
RESULTS
mRNA expression of TRPM6 and TRPM7. Basal mRNA
expression of TRPM6 was similar in VSMCs from WKY and
SHR (Fig. 1). Basal TRPM7 mRNA content was significantly
reduced in VSMCs from SHR compared with WKY (Fig. 2).
Exposure of cells to ANG II for 2–24 h induced a slight
increase in TRPM6 in WKY and SHR cells. However, signif-
icance was not achieved. On the other hand, ANG II time-
dependently increased TRPM7 expression in WKY cells, but
not in SHR VSMCs. In all experiments, GAPDH was used as
an internal housekeeping gene, and data are expressed as the
TRPM6/7 ratio relative to GAPDH expression.
TRPM6 and TRPM7 abundance in VSMCs. Western blot
analysis demonstrated the presence of TRPM6 and TRPM7 in
VSMCs. Basal expression of TRPM6 tended to be lower in
WKY cells compared with SHR, although significance was not
achieved (Fig. 3). Basal content of TRPM7 was significantly
reduced in SHR vs. WKY VSMCs (Fig. 4). ANG II induced a
modest, nonsignificant increase in TRPM6 expression in WKY
and SHR (Fig. 3). TRPM7 abundance was time-dependently
increased by ANG II in WKY, but not in SHR cells (Fig. 4).
Activation of annexin-1. Translocation of annexin-1, the
only known specific substrate of TRMP7, was assessed as an
index of TRPM7 activity. As shown in Fig. 5, annexin-1
translocation from cytosol to membrane was attenuated in SHR
cells compared with WKY, both in basal and ANG II-stimu-
lated conditions.
VSMC [Mg2ϩ
]i in SHR and WKY. Basal and ANG II-
stimulated [Mg2ϩ
]i, as assessed by mag fura-2 methodology,
was significantly lower in VSMCs from SHR compared with
WKY (Fig. 6). Compared with basal [Mg2ϩ
]i, ANG II-stimu-
lated [Mg2ϩ
]i was elevated in VSMCs from WKY but not in
cells from SHR (Fig. 6).
Fig. 1. Transient receptor potential melastatin (TRPM)6 mRNA expression in
very smooth muscle cells (VSMCs) from Wistar Kyoto (WKY) and sponta-
neously hypertensive rats (SHR). Cells were stimulated with ANG II for 2–24
h. GAPDH was used as a housekeeping gene. Results are presented as the ratio
between TRPM6 and GAPDH expression. Veh, vehicle. Results are expressed
as means Ϯ SE of four experiments.
Fig. 2. TRPM7 mRNA expression in VSMCs from WKY and SHR. Cells
were stimulated with ANG II for 2–24 h. GAPDH was used as a housekeeping
gene. Results are presented as the ratio between TRPM7 and GAPDH expres-
sion. Results are expressed as means Ϯ SE of four experiments. *P Ͻ 0.05 vs.
Veh counterpart. ϩP Ͻ 0.05 vs. WKY counterpart.
Fig. 3. TRPM6 protein content in VSMCs from WKY and SHR. Cells were
stimulated with ANG II for 4–24 h. Results are presented as the ratio between
TRPM6 and ␤-actin content. Results are means Ϯ SE of three experiments.
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4. DISCUSSION
Major findings from the present study demonstrate that 1)
VSMCs possess both TRPM6 and TRPM7 cation channels, 2)
TRPM7 is the predominant TRPM in VSMCs and 3) ANG II
influences TRPM6/7 regulation. In addition, we provide evi-
dence that TRPM6 is equally abundant in VSMCs from WKY
and SHR, whereas basal and ANG II-stimulated TRPM7 ex-
pression and activity are attenuated in SHR. These novel
findings suggest that TRPM6 and TRPM7 are differentially
regulated in VSMCs and that TRPM7 may be the major
Mg2ϩ
-permeant cation channel responsible for transcellular
Mg2ϩ
transport in VSMCs. Downregulation of TRPM7 may be
responsible, at least in part, for reduced VSMC [Mg2ϩ
]i in
SHR. Such processes may contribute to altered regulation of
VSMC Mg2ϩ
, which may contribute to vascular dysfunction in
hypertension.
TRPM6 shows ϳ50% homology with TRPM7 and has a
restricted expression pattern predominantly present in epithelia
(8, 42). TRPM6 is preferentially expressed in the small intes-
tine, colon and kidney, participating in gastrointestinal and
renal Mg2ϩ
absorption (13, 27, 28, 40). Here, we demonstrate
for the first time that TRPM6, both at the gene and protein
levels, is present in VSMCs and that its expression is regulated
by ANG II. Unlike TRPM7, TRPM6 was not differentially
expressed in cells from WKY and SHR. The exact functional
significance of vascular TRPM6 remains unclear, and its role
in Mg2ϩ
transport in VSMCs still awaits clarification. Because
expression patterns were equal between WKY and SHR, we
propose that vascular TRPM6 may not be critically involved in
altered VSMC Mg2ϩ
homeostasis in hypertension.
Expression of TRPM7 is widespread with transcripts in
brain, spleen, lung, kidney, heart, and liver (17, 24). It is also
expressed in lymphoid-derived cell lines, hematopoietic cells,
granulocytes, leukemia cells and microglia (10, 29), and we
recently reported that TRPM7 is present and functionally
active in human and mouse VSMCs (9). Here, we show that
TRPM7 is also present in rat VSMCs and that it is differen-
tially expressed in cells from WKY and SHR. Basal TRPM7
content, both at the mRNA and protein levels, was lower in
SHR compared with WKY. Stimulation by ANG II increased
TRPM7 abundance in WKY, but not in SHR. These findings
indicate diminished vascular TRPM7 expression and blunted
regulation of TRPM7 by ANG II in SHR.
To investigate whether altered TRPM7 status translates to
changes in TRPM7 activity, we examined annexin-1, the only
known specific downstream target of TRPM7 (4). Annexin-1 is
a Ca2ϩ
- and phospholipid-binding protein that translocates to
the cell membrane upon activation (23). It influences cell
growth and differentiation and plays a role in anti-inflamma-
tory actions of glucocorticoids (23). Within 10 min of ANG II
stimulation, annexin-1 translocated from the cytosol to the cell
Fig. 4. TRPM7 protein content in VSMCs from WKY and SHR. Cells were
stimulated with ANG II for 4–24 h. Results are presented as the ratio between
TRPM7 and ␤-actin content. Results are means Ϯ SE of three experiments.
*P Ͻ 0.05 vs. Veh counterpart. ϩP Ͻ 0.05 vs. WKY counterpart.
Fig. 5. Annexin-1 translocation in VSMCs from WKY and SHR. Cells were
stimulated with ANG II (10Ϫ7
mol/l), and annexin-1 expression was deter-
mined by immunoblotting in the membrane and cytosolic fractions. Results are
presented as the annexin-1 content in membrane fraction:cytosolic fraction.
Results are expressed as means Ϯ SE. *P Ͻ 0.05 vs. basal, **P Ͻ 0.01 vs.
WKY counterpart.
Fig. 6. Intracellular free Mg2ϩ
concentration ([Mg2ϩ
]i) in VSMCs from WKY
and SHR in basal and ANG II-stimulated conditions. Cells were exposed to
ANG II (10Ϫ7
mol/l) for 2 h. Results are expressed as means Ϯ SE of 3–6
experiments. *P Ͻ 0.05, **P Ͻ 0.01 vs. WKY counterpart, ϩP Ͻ 0.05 vs.
basal counterpart.
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5. membrane, indicating activation. This acute response was tem-
porally disassociated from changes in TRPM7 expression,
which was evident a few hours after stimulation. However,
TRPM7 is rapidly activated through an autophosphorylation
process (26). Hence, rapid annexin-1 stimulation may be linked
to acute-phase activation of TRPM7, followed by a more
prolonged activation state. The exact kinetics between an-
nexin-1 and TRPM7 await clarification. Activation of VSMC
annexin-1, measured as a cytosolic:membrane translocation,
was lower in SHR vs. WKY. Although this phenomenon may
be due to multiple factors, we propose here that it is related to
TRPM7 downregulation in SHR. This is supported by the
findings that both TRPM7 expression and annexin-1 translo-
cation are attenuated in basal conditions in SHR vs. WKY.
Furthermore, in WKY, but not in SHR, ANG II-induced
upregulation of TRPM7 was associated with an increase in
annexin-1 activity.
Considering that TRPM7 is a Mg2ϩ
-permeant channel re-
sponsible for transcellular Mg2ϩ
transport (28) and an impor-
tant mediator of cell growth (7, 39), it is possible that altered
cellular Mg2ϩ
homeostasis and abnormal VSMC function in
hypertension may be related to defective TRPM7 expression/
activity. We previously demonstrated that TRPM7 knockdown
by siRNA in VSMCs was associated with reduced [Mg2ϩ
]i and
altered cellular growth, which was reversible when Mg2ϩ
was
normalized (9). Here, we show that basal and ANG II-stimu-
lated [Mg2ϩ
]i is markedly reduced in VSMCs from SHR. This
may be due, at least in part, to downregulation of TRPM7 and
consequent decreased transmembrane Mg2ϩ
transport. It is
also possible that defective TRPM7 activity contributes to
vascular dysfunction in hypertension independently of changes
in [Mg2ϩ
]i. However, this awaits further clarification.
In conclusion, findings from the present study demonstrate
that TRPM6 and TRPM7 are present in rat VSMCs, that
TRPM6 is similarly expressed in WKY and SHR, and that
TRPM7 content and activity are reduced in SHR. Furthermore,
we show that ANG II influences TRPM6 and TRPM7 and that
ANG II-induced effects are blunted in SHR. Together with the
observation that VSMC [Mg2ϩ
]i is reduced in SHR, we sug-
gest that downregulation of TRPM7 may play a role in altered
vascular Mg2ϩ
homeostasis in SHR. These novel findings
provide new insights into putative mechanisms underlying
cellular regulation of Mg2ϩ
in hypertension.
GRANTS
This study was funded by grant T5735 from the Heart and Stroke Foun-
dation of Canada. G. Callera received a fellowship from the Canadian Insti-
tutes of Health Research and A.C.I. Montezano was supported by a studentship
from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior.
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