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(Circulation Research. 2004;95:922.)
© 2004 American Heart Association, Inc.

Cellular Biology

Critical Role for Transient Receptor Potential Channel TRPM4 in Myogenic Constriction of Cerebral Arteries

Scott Earley, Brian J. Waldron, Joseph E. Brayden

From the Department of Pharmacology, University of Vermont College of Medicine, Burlington.

Correspondence to Scott Earley, PhD, Dept of Pharmacology, University of Vermont College of Medicine, 89 Beaumont Ave, Burlington, VT. E-mail Scott.Earley{at}uvm.edu

	Abstract

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Local control of cerebral blood flow is regulated in part through myogenic constriction of resistance arteries. Although this response requires Ca²⁺ influx via voltage-dependent Ca²⁺ channels secondary to smooth muscle cell depolarization, the mechanisms responsible for alteration of vascular smooth muscle (VSM) cell membrane potential are not fully understood. A previous study from our laboratory demonstrated a critical role for a member of the transient receptor potential (TRP) superfamily of ion channels, TRPC6, in this response. Several other of the approximately 22 identified TRP proteins are also present in cerebral arteries, but their functions have not been elucidated. Two of these channels, TRPM4 and TRPM5, exhibit biophysical properties that are consistent with a role for control of membrane potential of excitable cells. We hypothesized that TRPM4/TRPM5-dependent currents contribute to myogenic vasoconstriction of cerebral arteries. Cation channels with unitary conductance, ion selectivity and Ca²⁺-dependence similar to those of cloned TRPM4 and TRPM5 were present in freshly isolated VSM cells. We found that TRPM4 mRNA was detected in both whole cerebral arteries and in isolated VSM cells whereas TRPM5 message was absent from cerebral artery myocytes. We also found that pressure-induced smooth muscle cell depolarization was attenuated in isolated cerebral arteries treated with TRPM4 antisense oligodeoxynucleotides to downregulate channel subunit expression. In agreement with these data, myogenic vasoconstriction of intact cerebral arteries administered TRPM4 antisense was attenuated compared with controls, whereas KCl-induced constriction did not differ between groups. We concluded that activation of TRPM4-dependent currents contributed to myogenic vasoconstriction of cerebral arteries.

Key Words: TRP channels • cerebral circulation • cation channels • vasoconstriction

	Introduction

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Small arteries and arterioles supplying blood to the brain constrict in response to increasing intraluminal pressure and dilate in response to decreasing pressure. This vital regulatory mechanism, known as the vascular myogenic response, ensures that blood flow remains nearly constant during moment-to-moment changes in arterial pressure. Although initially described in vivo,¹ myogenic constriction also occurs in blood vessels studied in isolation,^2,3 demonstrating that mechanisms inherent to the vascular wall are sufficient to induce this response. Disruption of the endothelium does not impair pressure-induced constriction,⁴ suggesting that sensor and effector mechanisms responsible for myogenic reactivity both reside at the level of smooth muscle cells. Increased intravascular pressure causes depolarization of the arterial myocyte cell membrane,⁵ thereby activating voltage-dependent Ca²⁺ channels, resulting in Ca²⁺ influx and subsequent vasoconstriction.⁶ Despite the essential nature of this response, signaling pathways responsible for pressure-induced smooth muscle depolarization are poorly understood. Improved comprehension of this mechanism could have significant therapeutic potential because impaired myogenic responsiveness and cerebral blood flow autoregulation are associated with a number of pathological conditions, including systemic hypertension,⁷ diabetes mellitus,⁸ stroke,⁹ and head trauma.¹⁰

Under resting conditions, vascular smooth muscle (VSM) membrane potential is a consequence of ionic homeostasis. Disruption of this equilibrium via activation of mechanosensitive ion channels could account for altered membrane potential during pressure elevation. Consistent with this hypothesis, a previous study from our laboratory¹¹ demonstrated a critical role for the transient receptor potential (TRP) channel TRPC6 in pressure-induced smooth muscle depolarization and vasoconstriction of cerebral arteries. TRPC6 is a member of the TRP superfamily of cation channels, comprising at least 22 separate genes.¹² Although message for other TRPs is present in blood vessels,¹³ potential functions of the majority of these proteins in vascular tissues have not been reported. The current study focused on TRPM4 and TRPM5, members of the melastatin TRP subfamily, which exhibit distinguishing biophysical characteristics suggesting a potential role for regulation of Ca²⁺ homeostasis of excitable cells.^14,15 When expressed in HEK cells, these channels are selective for monovalent cations, Ca²⁺-impermeant, and are activated by intracellular Ca²⁺.^14,15 Interestingly, the unitary conductance of these two TRPs (25 pico Siemens [pS]) is similar to that reported for mechanosensitive cation channels expressed by smooth muscle cells.^16–19 We, therefore, hypothesized that TRPM4 and/or TRPM5 contribute to VSM cell depolarization and vasoconstriction associated with increases in intraluminal pressure. Here we report the presence of a channel with biophysical properties similar to TRPM4 and TRPM5 in cerebral artery smooth muscle cells. Message for TRPM4 but not TRPM5 was detected in these cells. Consistent with our hypothesis, we found that downregulation of TRPM4 expression in isolated vessels impairs pressure-induced depolarization and vasoconstriction, suggesting a key role for this channel in cerebral blood flow regulation.

	Materials and Methods

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Cerebral and cerebellar arteries used for these studies were isolated from male Sprague-Dawley rats (250 to 350 g; Charles River Laboratories; St. Constant, Quebec, Canada). All animal use procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of the University of Vermont. To determine whether TRPM4 or TRPM5 like-channels were present in cerebral artery myocytes, the current-voltage characteristics and Ca²⁺-dependence of cation channels expressed by these cells were investigated. Inside-out membrane patches were obtained from enzymatically dispersed smooth muscle cells. The bathing solution (intracellular face) contained (in mmol/L) 110 Na-glutamate, 5 NaCl, 10 HEPES (pH 7.2), and 60 mannitol. CaCl₂ was added to the bath solution to achieve the concentration required for specific protocols. The pipette solution contained (in mmol/L) 110 NaCl, 10 HEPES (pH 7.4), 1.5 MgCl₂, 2 CaCl₂, 60 mannitol, and 300 nmol/L iberiotoxin. These solutions result in reversal potentials for Na⁺–1 mV, for Cl^––79 mV, and for Ca²⁺76 mV (when bath [Ca²⁺]=100 µmol/L). For some experiments, cells were treated with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) (0.5 µmol/L) for at least 10 minutes before membrane patches were excised. RT-PCR was used to determine whether TRPM4 and TRPM5 mRNA were present in cerebral arteries. Total RNA was extracted from isolated vessels or dispersed smooth muscle cells and first-strand cDNA was synthesized. Forward and reverse primers specific for TRPM4 were as follows: TRPMF, 5'-GTCATCGTGAGCAAGATGATGAA-3'; and TRPM4R, 5'-GTCCACCTTCTGGGACGTGC-3'. These primers yield a 707-bp PCR product. Forward and reverse primers specific for TRPM5 were TRPM5F 5'-CAAGTGTGACATGGTGGCCATC TT-3' and TRPM5R 5'-GCTCAGGTGGCTGAGCAGGAT-3', yielding a 639-bp PCR product. PCR products were resolved on 1% agarose gels.

Antisense oligodeoxynucleotides (ODNs) were used to downregulate TRPM4 expression in isolated cerebral arteries. Sequences of TRPM4 antisense ODNs that were used were as follows: TRPM4 AS-1, 5'-GTGTGCATCGCTGTCCCACA-3'; and TRPM4 AS-2, 5'-CTGCGATAGCACTCGCCAAA-3' (Qiagen, Inc). Complementary sequences were used as sense ODNs. Fluorescein was conjugated to the 5' terminal nucleotides to assess cellular location. ODNs were introduced into intact cerebral arteries using a reversible permeabilization procedure.²⁰ After reversal of permeabilization, arteries were organ-cultured for 2.5 days in DMEM/F-12 medium without serum. Semiquantitative RT-PCR was used to evaluate the effects of antisense ODNs on TRPM4 mRNA levels. Smooth muscle cells isolated from cultured arteries were patch-clamped in the inside-out configuration to investigate the effects of antisense ODNs on the frequency of observation of a TRPM4-like channel. In additional experiments, sense- and antisense-treated arteries were mounted in an arteriograph, endothelial cells were removed, and the effects TRPM4-downregulation on pressure-induced smooth muscle cell depolarization and vasoconstriction were evaluated.

An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

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TRPM4-/TRPM5-Like Channel Is Present in Rat Cerebral Arteries
Patch-clamp studies were performed to determine whether channels with biophysical properties similar to TRPM4 and TRPM5 were present in cerebral artery smooth muscle cells. In some inside-out membrane patches obtained from freshly isolated cells (9 of 72 cells; 12.5%; n=10 rats), channels were observed that exhibited inward currents at negative holding potentials, reversed near 0 mV, and became outward at positive holding potentials (Figure 1A; bath [Ca²⁺]=100 µmol/L). The bath and pipette solutions used for these experiments included symmetrical [Na⁺], whereas Cl^– and Ca²⁺ ions were asymmetrically distributed on either side of the membrane. In this configuration, the linear current-voltage relationship reversed at near 0 mV (Figure 1B), which was consistent with a cation channel conducting Na⁺ ions. Single channel conductance () calculated from the slope of the I–V curve was 24 pS (Figure 1B). Unlike cloned TRPM4,¹⁴ this channel did not appear to be voltage dependent (NP_o at –80 mV=0.52±0.16, n=4; and at +80 mV=0.58±0.03, n=3).

View larger version (33K): [in this window] [in a new window]   Figure 1. A, Example single-channel recordings obtained at holding potentials (VH) from +80 mV to –80 mV. Bath [Ca2+]=100 µmol/L; c indicates closed state. B, Unitary current-voltage relationship for control (n=3 to 9 at each holding potential) or PMA-treated (n=3 to 11) cells with symmetrical [Na]+ in bath and pipette solutions. Data for each group were fitted to a linear function. Control, =24 pS, Erev=–4.9±1.8 mV; PMA treated, =24 pS, Erev=–6.1±2.9 mV. C, Unitary current voltage relationship for PMA-treated cells with 110 mmol/L Na+ in the pipette solution and 100 mmol/L Ca2+ in the bath solution (n=3 at each holding potential). =14 pS; Erev=14.0±3.3 mV.

Characterization of this channel was initially protracted because of the low frequency of observation. However, it was recently reported that the PKC activator PMA increases the frequency of observation of a TRPM4-like cation channel from 6.7% to 50% in cardiac myocytes.²¹ In agreement with these data, we found that pretreatment of VSM cells with PMA before membrane patches were excised increased the frequency of channel observation from 12.5% to 47% (33 of 70 cells; n=12 rats). The current-voltage relationship, single-channel conductance, and NP_o at –40 mV (bath [Ca²⁺]=100 µmol/L) of patches from PMA-treated cells did not differ from those of untreated cells,

Cloned TRPM4 and TRPM5 channels exhibit considerable selectivity for Na⁺ versus Ca²⁺ ions. To examine the ionic specificity of the 24 pS present in cerebral arteries, we substituted Ca²⁺ for Na⁺ in the bath (intracellular) solution used for inside-out patch-clamp studies. Ca²⁺ replacement shifted the reversal potential from near 0 mV (Figure 1B) to +14 mV (Figure 1C). The relative permeability of Ca²⁺ versus Na⁺ (P_Ca/P_Na) was estimated using the Fatt-Ginsborg²² equation as 0.09, indicating that this channel is selective for Na⁺ ions.

Another distinguishing characteristic of TRPM4 and TRPM5 is that both are activated by intracellular [Ca²⁺]. We, therefore, examined the effects of bath [Ca²⁺] on NP_o of the 24 pS cation channel in excised patches. Channel openings were not observed when intracellular [Ca²⁺] was 100 nmol/L (n=4). However, channel activity was observed when bath [Ca²⁺] was 1 µmol/L. Channel activity increased with increasing Ca²⁺ concentration (Figure 2A and 2B), demonstrating Ca²⁺-dependence of the 24 pS cation channel. [Ca²⁺] for half-maximal channel activity was 200 µmol/L.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of bath [Ca2+] on channel activity. A, Example single channel recordings at bath [Ca2+] of 0.1, 10, 100, and 1000 µmol/L. VH=–40 mV; c indicates closed state. B, Open probability (NPo) as a function of bath [Ca2+]. n=4 at each concentration. Data were fitted to a Boltzman function.

The Ca²⁺-dependence, unitary conductance, and ion permeability of this cerebral artery myocyte channel were consistent with those reported for cloned TRPM4^14,15 and TRPM5.^23–26 To determine whether message encoding these channels was expressed by rat cerebral arteries, we performed RT-PCR for TRPM4 and TRPM5. Message for TRPM4 was present in RNA isolated from both whole cerebral arteries as well as smooth muscle cells isolated from these vessels (Figure 3). TRPM5 message was also present in RNA from whole cerebral arteries (Figure 3). However, we found that TRPM5 was absent in RNA extracted from isolated smooth muscle cells (Figure 3). These findings indicate that the 24 pS Ca²⁺-dependent cation channel identified in cerebral artery myocytes could be TRPM4 but not TRPM5.

View larger version (27K): [in this window] [in a new window]   Figure 3. RT-PCR for TRPM4 and TRPM5 for RNA extracted from whole cerebral artery (CA) or isolated smooth muscle cells (SMC). NT indicates no template control.

Downregulation of TRPM4 Expression in Cerebral Arteries
To date, selective inhibitors of TRP channels are not available. Therefore, to investigate a potential functional role for TRPM4 in cerebral arteries we suppressed expression of the channel using antisense technology. We found that the fluorescence of arteries that were permeabilized and exposed to fluorescein-labeled TRPM4 antisense ODNs (Figure 4A) was much greater than that of arteries that were exposed to ODNs but not permeabilized (Figure 4B) or untreated arteries (Figure 4C). Fluorescently labeled ODNs appeared to be present within smooth muscle of permeabilized arteries (Figure 4A), suggesting efficient delivery to these cells.

View larger version (38K): [in this window] [in a new window]   Figure 4. A through C, Photomicrographs demonstrating entry of fluorescein-labeled ODNs into VSM cells of permeabilized arteries. A, Cerebral artery permeabilized and exposed to fluorescein-labeled ODNs (green). B, Cerebral artery exposed to labeled ODNs but not permeabilized. C, An untreated cerebral artery. Bar=50 µm. D through E, Semiquantitative RT-PCR for arteries treated with TRPM4 sense or antisense ODNs. D, PCR for TRPM4. E, PCR for TRPC6. F through G, Semiquantitative RT-PCR for arteries treated with TRPC6 sense or antisense ODNs. F, PCR for TRPM4. G, PCR for TRPC6. For all experiments, GAPDH PCR was used to demonstrate uniformity of reaction conditions between groups.

Semiquantitative RT-PCR was used to evaluate the effects of antisense ODNs on TRPM4 mRNA levels. We found that the intensity of TRPM4 bands in reactions using cDNA derived from antisense-treated arteries was less than that of sense-treated vessels (Figure 4D), suggesting that antisense ODNs suppress TRPM4 expression. The band intensity of a housekeeping gene (GAPDH) did not differ between groups (Figure 4D). We also examined the effects of TRPM4 antisense on mRNA levels of TRPC6, previously shown to contribute to cerebral artery function.¹¹ Expression of TRPC6 was unaffected by TRPM4 antisense (Figure 4E). As an additional control, we assessed the effects of TRPC6 antisense on TRPM4 mRNA levels in cerebral arteries. TRPM4 expression was not altered by TRPC6 antisense (Figure 4F), whereas TRPC6 mRNA levels were diminished in these arteries (Figure 4G).

To further examine the possibility that the 24 pS channel is TRPM4, we studied the effects of TRPM4 antisense on the frequency of observation of the channel in patch-clamp experiments. Inside-out membrane patches were obtained from smooth muscle cells isolated from TRPM4 sense and antisense treated arteries after PMA administration. Channels (24 pS) were observed in cells obtained from TRPM4-sense treated arteries at a frequency (53%) similar to that of cells from untreated arteries (47%). In contrast, these channels were less frequently (10%) observed in cells from arteries treated with TRPM4 antisense ODNs (Table). The number of cells exhibiting TRPM4-like channels was significantly less for antisense compared with sense-treated vessels (Table). These findings further support the hypothesis that the 24 pS Ca²⁺-activated monovalent-selective cation channel expressed by cerebral artery myocytes is TRPM4.

View this table: [in this window] [in a new window]   Table 1. Frequency of Observation of TRPM4-Like Channels in Cells from Sense- and Antisense-Treated Arteries

Functional Role of TRPM4 in Cerebral Arteries
The effects of TRPM4 downregulation on pressure-induced depolarization and myogenic constriction were examined using isolated, pressurized cerebral blood vessels treated with TRPM4 sense and antisense ODNs. We found that VSM cells in TRPM4-sense treated arteries were more depolarized when the vessels were pressurized at 80 Torr compared with 20 Torr (Figure 5A and 5B). Smooth muscles cells in these vessels were also depolarized when the purinergic receptor agonist UTP (30 µmol/L) was administered. The degree of pressure and agonist-induced depolarization observed in these experiments was consistent with those reported for freshly isolated arteries.^6,27 In contrast, smooth muscle membrane potential of antisense-treated arteries did not depolarize when pressure was elevated from 20 or 80 Torr (Figure 5C and 5D). Furthermore, VSM cells in antisense-treated arteries pressurized to 80 Torr were hyperpolarized compared with sense-treated vessels at this pressure (Figure 5D). Administration of UTP (30 µmol/L) induced depolarization of smooth muscle in antisense-treated vessels equal to that in sense-treated arteries (Figure 5D). These findings show that TRPM4 contributes to pressure-, but not UTP-, dependent smooth muscle cell depolarization in cerebral arteries.

View larger version (24K): [in this window] [in a new window]   Figure 5. Membrane potential of VSM cells in pressurized cerebral arteries treated with TRPM4 sense (A and B) or antisense (C and D) ODNs. Left, Example traces at intraluminal pressures of 20 and 80 Torr. Right, Mean±SEM. *P<0.05 vs sense ODN-treated arteries at 20 Torr; #P<0.05 vs sense ODN-treated arteries at 80 Torr; **P<0.05 vs antisense-treated arteries at 20 and 80 Torr. n=4 to 6.

In subsequent studies, we found that arterial constriction resulting from administration of 60 mmol/L KCl did not differ between sense and antisense-treated arteries (Figure 6A), suggesting that TRPM4 downregulation does not impair K⁺ depolarization-induced vasoconstriction of these vessels. However, myogenic constriction of TRPM4 antisense-treated vessels was much less than that of sense-treated arteries (Figure 6B through 6C). Pressure-induced constriction of TRPM4 antisense-treated vessels was significantly (P<0.05) less than that of sense-treated arteries at intraluminal pressures 60 Torr. These findings demonstrate an important functional role for TRPM4 in pressure-induced depolarization and constriction of cerebral blood vessels.

View larger version (21K): [in this window] [in a new window]   Figure 6. A, 60 mmol/L KCl-induced constriction for cerebral arteries treated with TRPM4 sense and antisense ODNs. There was no difference between groups. n=6 for both groups. B through D, Myogenic constriction in response to increasing intraluminal pressure for cerebral arteries treated with TRPM4 sense (B) or antisense (C) ODNs. D, Summary myogenic tone data (mean ± SEM). *P<0.05 vs sense ODN-treated arteries. n=6 for both groups.

	Discussion

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Although the properties of cloned TRPM4 and TRPM5 are consistent with possible roles for these channels in the regulation of membrane potential and Ca²⁺ homeostasis of excitable cells, their function in smooth muscle has not previously been reported. The objective of this study was to determine whether these channels were present within the cerebral vasculature and how they influence arterial function. Our major findings were as follows: (1) a monovalent selective, Ca²⁺-dependent cation channel with unitary conductance of 24 pS was present in cerebral artery smooth muscle cells; (2) TRPM4 message was present in cerebral artery myocytes, whereas mRNA encoding TRPM5 was not detected in these cells; (3) suppression of TRPM4 expression in isolated cerebral arteries decreased the frequency of observation of the 24 pS channel and impaired pressure-induced smooth muscle depolarization; and (4) myogenic constriction of cerebral arteries was diminished by downregulation of TRPM4. Thus, our findings show that functional TRPM4 is present in VSM and suggest a critical role for this channel in the regulation of cerebral artery tone.

The biophysical characteristics of TRPM4 and TRPM5 are distinct from those of other TRPs. For example, other members of the TRP superfamily are nonselective or Ca²⁺-selective cation channels,¹² whereas TRPM4 and TRPM5 exhibit specificity for monovalent cations.^14,15 In addition, activation of TRPM4 and TRPM5 is dependent on intracellular [Ca²⁺],^14,15,23,25 whereas other TRPs are either indifferent to or inhibited by intracellular Ca²⁺¹². Cloned TRPM4 channels expressed in HEK cells are also voltage-dependent.¹⁴ Our data show that cation channels with unitary conductance, ionic selectivity, and Ca²⁺-dependence similar to those of TRPM4 and TRPM5 are present in cerebral artery myocytes. Unlike cloned TRPM4, these channels did not exhibit voltage dependence. Although the reason for differences in voltage sensitivity between expressed TRPM4 and the arterial smooth muscle cation channel are unclear, this may reflect differential tissue or species-specific properties of the channel, alternative splicing, or heteromultimerization of TRP channel proteins. Interestingly, a recent study demonstrates that cloned TRPM4 is not voltage dependent in the presence of decavanadate.²⁸ This activity of decavanadate was found to be dependent on a cluster of positively charged amino acid residues in the C terminus of TRPM4. These findings suggest the possibility that the voltage dependence of TRPM4 may be modulated in native cells via this C-terminal domain. Despite the apparent differences in voltage dependence, our findings that message for TRPM4, but not TRPM5, was present in isolated cerebral artery myocytes and that the frequency of observation of the 24 pS cation channel was reduced when TRPM4 expression was suppressed strongly suggests that that the Ca²⁺-dependent, monovalent-selective cation channel identified in cerebral artery smooth muscle cells is TRPM4.

Further experiments examined the functional role of TRPM4 in the cerebral vasculature. Deformation of the smooth muscle cell plasma membrane resulting in activation of mechanosensitive ion channels may contribute to depolarization associated with elevation of intraluminal pressure. In support of this possibility, a number of studies demonstrate stretch-activation of cation channels in smooth muscle cells.^16–19 The unitary conductance for Na⁺ ions of these stretch-activated channels is 25 pS, and the channels are inhibited by trivalent ions such as Gd³⁺. These properties are consistent with those of cloned TRPM4 and TRPM5 expressed in HEK cells.^14,15 To test the hypothesis that the molecular identity of these previously described, stretch-activated channels is TRPM4, we successfully downregulated expression of this TRP protein in isolated cerebral arteries. We found that decreased TRPM4 expression is specifically associated with attenuated pressure-induced smooth muscle cell depolarization and myogenic constriction of these vessels. These findings demonstrate that TRPM4 is an important determinant of pressure-induced smooth muscle cell depolarization and vasoconstriction, suggesting a key role for this channel in the control of cerebral blood flow.

A previous study from our laboratory has demonstrated a critical role for TRPC6 in myogenic constriction of cerebral arteries,¹¹ whereas the current study shows that TRPM4 is also very important in this response. These findings could be the result of nonselectivity of the antisense technology used to suppress TRP expression. For example, a recent study found that suppression of TRPM7 expression also downregulates TRPM2 in primary neuron cultures, suggesting transcriptional interdependency among TRP channels in these cells.²⁹ To examine the possibility that a similar phenomenon was responsible for our observations, we evaluated the effects of TRPM4 antisense on TRPC6 expression and found that TRPC6 mRNA levels were unaltered by this treatment. We also examined the effects of TRPC6 antisense on TRPM4 expression and found TRPM4 levels to be unchanged when TRPC6 mRNA levels were decreased. These findings support the specificity of antisense procedures used for the current and previous¹¹ studies and suggest that expression of both TRPM4 and TRPC6 is necessary for myogenic constriction of cerebral arteries. Given the diversity of biophysical properties and complex molecular biology of the TRP superfamily,¹² a number of potential mechanisms for interaction between these two channels is possible. For example, activation of TRPM4 during increases in intraluminal pressure may occur as a result of TRPC6-dependent Ca²⁺ influx. Another possibility may be the formation of channels that comprised both TRPC6 and TRPM4 protein subunits. Previous studies have shown that heteromeric channels with novel characteristics can form when multiple TRPC proteins are coexpressed.^30–32 Although heteromization of channels between different TRP families has not been reported, this could account for our observations. Of course, TRPC6 and TRPM4 channels could contribute to pressure-induced depolarization of cerebral VSM, independent of one another. The relative importance of the two channels in this response has not yet been determined, but studies of this type will provide important insights about the integrated functions and diversity of physiologic role of TRP channels in smooth muscle.

We report here that suppression of TRPM4 expression attenuates pressure-induced smooth muscle depolarization. This finding suggests that a TRPM4-dependent depolarizing current is activated by increased intravascular pressure, although the mechanism responsible for activation of this channel in smooth muscle cells is still unknown. Among TRP channels, Ca²⁺-dependence is unique to TRPM4 and TRPM5, suggesting that intracellular [Ca²⁺] may be an important regulatory stimulus under physiological conditions. However, considerable disagreement regarding the [Ca²⁺] required to open TRPM4 has been reported. Initially, Launay et al reported half-maximal P_o of the cloned channel in whole cell experiments at intracellular [Ca²⁺] of 0.4 µmol/L,¹⁵ whereas a study by Nilius et al reported that this value was 370 µmol/L for inside-out membrane patches.³³ Our finding of half-maximal activation at 200 µmol/L in cerebral artery smooth muscle cells is more in agreement with the latter study. The reason for the discrepancy in Ca²⁺ sensitivity of the channel is not known, but may be attributable to differences in experimental conditions used. Our findings suggest that the level of Ca²⁺ required to activate TRPM4 is greater than the physiological range (0.1 to 0.4 µmol/L) for global intracellular [Ca²⁺] normally encountered in smooth muscle cells. However, local [Ca²⁺] can exceed 10 µmol/L during transient Ca²⁺ events, such as Ca²⁺ sparks.³⁴ Our findings show that this level of Ca²⁺ is sufficient to activate TRPM4 in smooth muscle cells. Transient Ca²⁺ events play a critical role in the regulation of K⁺ channel function in arterial smooth muscle.³⁵ It is possible that such events also modulate TRPM4 activity, should the appropriate localization of TRPM4 channels and Ca²⁺ release sites be present in VSM.

Similar to a previous report using cardiac myocytes,²¹ we found that administration of a potent PKC activator increases the frequency of observation of a TRPM4-like channel in inside-out membrane patches from VSM cells. In addition, a PROSITE (http://us.expasy.org/prosite/) search of the mouse TRPM4 amino acid sequence identified several PKC activation sites on projected intracellular domains of the channel. These findings suggest that TRPM4 activity can be enhanced by PKC-dependent phosphorylation. Interestingly, an earlier study reports pressure-dependent increases in PKC translocation to the plasma membrane,³⁶ whereas the current study demonstrates a critical role for TRPM4 in pressure-induced smooth muscle cell depolarization. These observations raise the possibility that pressure-dependent PKC activity contributes to activation of depolarizing currents mediated by TRPM4 in VSM cells. Although not directly addressed by the current study, this possibility is in agreement with a number of early reports demonstrating impaired myogenic constriction following PKC inhibition.^37–39 Whereas evidence supporting Ca²⁺-independent pathways for PKC-induced vasoconstriction has been reported,^40,41 other studies show that smooth muscle constriction induced by PKC activation is dependent on extracellular Ca²⁺ and can be blocked by antagonist of voltage-dependent Ca²⁺ channels.^42–45 Furthermore, administration of PMA depolarizes both airway⁴⁶ and cerebral artery⁴⁷ smooth muscle, and this effect is blocked by PKC inhibition. Future investigations will examine the hypothesis that pressure-induced, PKC-dependent phosphorylation of TRPM4 constitutes a novel mechanism for myogenic depolarization and constriction of cerebral arteries.

In summary, the current study demonstrates an important role for TRPM4 in myogenic constriction of cerebral vessels. Considering the findings of our previous study show a similar role for TRPC6,¹¹ we conclude that multiple TRP channels expressed by arterial smooth muscle participate in the regulation of cerebral blood flow.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grants F32HL075995 (to S.E.) and RO1HL58231 (to J.E.B.). We thank Johann Patlak for technical assistance, Drs Adrian Bonev and Thomas Heppner for advice on patch-clamp methodology, and Drs Kevin Thorneloe and Mark T. Nelson for critical comments on the manuscript.

	Footnotes

 
Original received June 25, 2004; revision received September 24, 2004; accepted September 27, 2004.

	References

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