Na+-independent Mg2+ transport sensitive to 2-aminoethoxydiphenyl borate (2-APB) in vascular smooth muscle cells: involvement of TRPM-like channels

Abstract Magnesium is associated with several important cardiovascular diseases. There is an accumulating body of evidence verifying the important roles of Mg2+-permeable channels. In the present study, we estimated the intracellular free Mg2+ concentration ([Mg2+]i) using 31P-nuclear magnetic resonance (31P-NMR) in porcine carotid arteries. pHi and intracellular phosphorus compounds were simultaneously monitored. Removal of extracellular divalent cations (Ca2+ and Mg2+) in the absence of Na+ caused a gradual decrease in [Mg2+]i to ∼60% of the control value after 125 min. On the other hand, the simultaneous removal of extracellular Ca2+ and Na+ in the presence of Mg2+ gradually increased [Mg2+]i in an extracellular Mg2+-dependent manner. 2-aminoethoxydiphenyl borate (2-APB) attenuated both [Mg2+]i load and depletion caused under Na+- and Ca2+-free conditions. Neither [ATP]i nor pHi correlated with changes in [Mg2+]i. RT-PCR detected transcripts of both TRPM6 and TRPM7, although TRPM7 was predominant. In conclusion, the results suggest the presence of Mg2+-permeable channels of TRPM family that contribute to Mg2+ homeostasis in vascular smooth muscle cells. The low, basal [Mg2+]i level in vascular smooth muscle cells is attributable to the relatively low activity of this Mg2+ entry pathway.


Introduction
From both clinical and epidemiologic aspects, Mg 2+deficiency is related to cardiovascular diseases [1][2][3] driven by the Na + -gradient, and the Na + -independent 'passive' Mg 2+ transport via Mg 2+ -permeable channels [5][6][7][8]. Since the molecular identification of the latter Mg 2+ pathway, such as melastatin-type transient receptor potential (TRPM) homologue channels, there has been an accumulating body of evidence for the crucial role that this pathway plays in Mg 2+ homeostasis [9][10][11]. Also, TRPM homologue channels are bifunctional proteins, which contain a kinase domain in the C-terminus.
[Mg 2+ ]i is known to change slowly, and thereby act as a chronic regulator. In addition, changes in the intracellular milieu, such as the intracellular pH (pHi) and [ ]i regulation during relatively short durations has been assessed using fluorescent Mg 2+ indicators. In the present study, we thus utilized 31 P-NMR to estimate slow changes in [Mg 2+ ]i over several hours in carotid arteries, which are now frequently used as a model to evaluate arteriosclerotic changes, and assessed the contribution of TRPM-like Mg 2+ -permeable channels.

Materials and methods Preparation
Porcine carotid arteries were collected at an abattoir. The arteries were stripped of fat and connective tissue, and cut into segments of approximately 30 mm in length. The lumen was exposed by cutting the artery segments into two strips along the longitudinal direction. The endothelium was removed by scratching with a cotton-tipped stick. The resultant pig carotid artery strips (~2 g wet weight) were mounted in a sample tube of 10 mm in diameter. This study was approved by the institutional committee of animal experiments.

P-NMR
The methods employed for the 31 P-NMR measurements were essentially the same as those previously described [12]. NMR spectrometers (GSX270W: JEOL, Tokyo, Japan; UNITY-500plus: Varian, Tokyo, Japan) were operated at 109.4 and 202.3 MHz, respectively. The temperature of the sample was maintained at 32°C. Radio frequency pulses corresponding to a flip angle of 30 were applied every 0.6 sec. 31 P-NMR spectra were obtained by accumulating 2500 signals (free induction decays) over 25 min. Before Fourier transformation, a broadening factor of 20 Hz was applied to enhance the signal-to-noise ratio. Spectral peak resonances (frequencies) were measured relative to that of phosphocreatine (PCr) in p.p.m. Control spectra were acquired in the absence of Ca 2+ . Then, experiments were carried out in the absence of extracellular Na + to rule out the contribution of Na + -coupled Mg 2+ transport, that is, Na + -Mg 2+ exchange. Six major peaks were observed ( Fig. 1): phosphomonoesters (PME), inorganic phosphate (Pi), PCr and the ␥-, ␣and ␤-phosphorus atoms of ATP (␥-, ␣and ␤-ATP).
Eq (3) In Table 2, [Mg 2+ ]i and pHi values were also estimated from the chemical shifts of ␤and ␥-ATP [12,17]. For the chemical shift of ␥-ATP, an equation analogous to Eq(3) can be written: ␦ f ␥ and ␦ b ␥ are the chemical shifts of metal-free and Mg 2+ -bound forms of ␥-ATP, respectively, and these parameters are also expressed as pH-functions. [Mg 2+ ]i and pHi were estimated by solving the Eq(3) and Eq(4) simultaneously.
Similar to the effect of Mg 2+ , Ca 2+ -binding to ATP changes the chemical shifts of the ATP peaks. However, basal [Ca 2+ ]i is maintained at ~100 nM in smooth muscle cells under physiological conditions. Furthermore, experiments in the present study were carried out mainly under Ca 2+ -free conditions. Therefore, the effect of Ca 2+ -binding to ATP is considered negligible.

RT-PCR
The procedures for RT-PCR were essentially the same as previously described [18]. Total RNA was extracted from porcine carotid arteries. After treatment with RQ1 DNase (Promega, Madison, WI, USA), the total RNA was subjected to an RT (reverse-transcription) reaction. RT was performed using a random hexamer (12 pmol/reaction) and Moloney murine leukemia virus (MMLV) reverse transcriptase 5 (100 U/reaction) according to the manufacture's instructions (Gibco-BRL, Rockville MD, U.S.A.). The cycling condition was 3 min of initial denaturation at 95°C followed by 35 cycles of 95°C for 30 sec, 54°C for 30 sec and 72°C for 35 sec. The RT sample was then used as a template for the PCR reaction. The amplicons (5 l were run on a 2.5% agarose gel and stained with ethidium bromide. Because the cDNA sequences for porcine TRP (transient receptor potential) homologue cation channels have not been published, the PCR primers were designed by using the conserved sequences in humans and mice. The primers used are listed in Table 1.

Statistics
Numerical data are expressed as the mean (S.D. Differences between groups with different experimental protocols were evaluated by use of ANOVA for repeated measures. When a significant difference was identified between the groups (P<0.05), individual comparisons at the same time point were performed using an unpaired t-test.

2+
] i via Na + -independent Mg 2+ pathways 31 P-NMR was used to continuously measure phosphorus compounds in porcine carotid artery smooth muscle ( Fig. 1). In this study, we mainly estimated [Mg 2+ ]i from the chemical shift of the ␤-ATP peak, and correction was made by pHi estimated from the chemical shift of the Pi peak.
During exposure to a divalent cation-free solution (i.e. 0 Ca ]i under divalent cation-free conditions. On the other hand, pHi decreased from 7.09 ± 0.05 to 6.92 ± 0.05 (n = 7) after 125 min in the absence of Na + (Fig. 2B, ), presumably due to the inhibition of Na + -coupled pHi regulatory mechanisms, such as Na + -H + exchange and Na + -HCO3 co-transport.   (B) Exposure to a divalent cation-and Na + -free solution containing 2-APB (n = 7).
In the present experiments, Na + was frequently substituted with equimolar K + .

2+ ] i and pH i from the ␤ and ␥-ATP peaks
To confirm the changes in [Mg 2+ ]i and pHi estimated above, we used a different procedure; specifically,  ]i, were comparable between the two analyses ( Table 2). [Mg 2+ ]i estimated from ␤and ␥-ATP were slightly smaller because of the higher pHi estimated; i.e. the apparent dissociation constant for MgATP is smaller in a higher pH.

High-energy phosphates
ATP is known to affect the activity of TRPM7 and to act as an important intracellular Mg 2+ buffer.

2+
]i are not correlated regardless of the presence of extracellular Mg 2+ (Fig. 6). Also, [PCr]i did not change significantly throughout (Supplementary Table S1).

RT-PCR
To confirm the transcription of genes for Mg 2+ -permeable channels, RT-PCR was performed. Because the cDNA sequences for porcine TRP (transient receptor potential) homologue cation channels have not been published, the PCR primers were designed by using the conserved sequences from humans and mice. Of TRPM2, 4, 6 and 7, the TRPM7 was predominant. TRPM6 was also detectable under the same PCR condition ( Fig. 7; see also Supplementary  Fig. S3). ]i to approximately 60% of the control after 125 min even in the absence of Na + (Fig. 2)  this procedure maintained the negative membrane potential, unlike Na + substitution with K + , which was conditions, in which the negative resting membrane potential is preserved (probably several tens of mV). TRPM6 and TRPM7 are known Mg 2+ -permeable non-selective cation channels. It has been shown that TRPM6 is abundant in the kidney and small intestine, while TRPM7 is expressed ubiquitously in numerous tissues and organs [9,21,22]. It is thought that the former and latter are responsible for Mg 2+ regulation at the organ and cellular levels, respectively. In line with this notion, RT-PCR examinations revealed that TRPM7 was a major compo-nent of TRPM homologues in porcine carotid arteries, but TRPM6 was also detectable (Fig. 7). Previous patch clamp studies have shown that the removal of extracellular divalent cations facilitates TRPM7-like current [9,11,23], a finding that is in good agreement with our [Mg 2+ ]i measurements, that is Figure 5. 2-APB is known to block store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels as well as Mg 2+ -permeable channels [24]. However, Mg 2+ transport via CRAC channels is considered negligible, because these channels have even greater Ca 2+ selectivity than voltage-sensitive Ca 2+ channels [25].

Discussion
In the present study, Mg 2+ transport via Mg 2+ -permeable channels demonstrated as Na ]i changes, was not completely suppressed by 2-APB at <150 µM, which is three times greater than the IC50 (~50 µM) reported for TRPM7-like cation currents [11,23]. This discrepancy may be explained as follows: (1) The IC50 of 2-APB in TRPM7 may be altered by the intracellular milieu, namely, [ATP]i is maintained in our 31 P-NMR measurements, while no ATP was added in the intracellular solution of the previous patch clamp experiments in which the effect of 2-APB was examined [11,23]; (2) Native TRP channels may act as multimeric proteins of complex compositions [26]; 3) some other, as yet unknown Mg 2+ transport mechanisms operate in parallel.
Recently, it has been shown that the application of 2-APB does not block, but rather facilitates TRPM6 [27] ]i in smooth muscle cells are lower than found in the other two muscle types, that is, skeletal and cardiac muscles. Furthermore, among smooth muscles distributed in numerous tissues and organs, we estimated [Mg 2+ ]i in vascular smooth muscle cells to be the lowest after correcting for the pHi and temperature [8]: (0.8-0.85 mM in taenia caeci; 0.7-0.9 mM in the uterus; 0.8 mM in the urinary bladder and 0.65-0.75 mM in the carotid   ]i [29]. Also, Mg 2+ -permeable non-selective cation channels, such as TRPM7, have been recently shown to play a crucial role in generating spontaneous rhythmicity in pacemaker interstitial cells of intestinal smooth muscle tissues [30,31]. Taken together, these facts warrant systematic investigation into cell-and tissue-specific roles of TRPM channels in numerous smooth muscle tissues containing pacemaker-like interstitial cells [32][33][34][35].
Functional mutations of ion channels are known to cause numerous diseases. Functional mutations of TRPM7 result in a significant effect on cellular Mg 2+ homeostasis. It has been experimentally shown that mutations of the kinase domain in TRPM7 modulate the sensitivity to [Mg 2+ ]i. Furthermore, knockout of TRPM7 causes intracellular Mg 2+ -deficiency in cultured cells [36]. In clinical fields, it has long been recognized that Mg 2+ is associated with several important diseases, e.g. diabetes mellitus, hypertension, and cardiovascular and cerebrovascular diseases [37,38]. Subgroups of these diseases may involve functional mutations of Mg 2+ -permeable channels contributing to passive Mg 2+ transport via the electrochemical gradient. Similarly to the studies on TRPM6 [39,40], future genetic analyses to clarify the contribution of TRPM7-like Mg 2+ -permeable channels to cardiovascular diseases will be of great interest. Indeed, a TRPM7 variant has been recently reported for two Guamanian neurodegenerative disorders [41].
In ]i in vascular smooth muscle cells. From the inhibitory effect of 2-APB and the results of RT-PCR, TRPM7 appears to play a predominant role in the passive Mg 2+ transport.

Supplementary material
The following supplementary material is available for this article: Material S1 Details of estimation procedures.