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Am J Physiol Heart Circ Physiol 292: H2613-H2622, 2007. First published February 9, 2007; doi:10.1152/ajpheart.01286.2006
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Protein kinase C regulates vascular myogenic tone through activation of TRPM4

Scott Earley,1 Stephen V. Straub,2 and Joseph E. Brayden2

1Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado; and 2Department of Pharmacology, University of Vermont College of Medicine, Burlington, Vermont

Submitted 23 November 2006 ; accepted in final form 7 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myogenic vasoconstriction results from pressure-induced vascular smooth muscle cell depolarization and Ca2+ influx via voltage-dependent Ca2+ channels, a process that is significantly attenuated by inhibition of protein kinase C (PKC). It was recently reported that the melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced smooth muscle depolarization and constriction in cerebral arteries. Interestingly, PKC activity enhances the activation of cloned TRPM4 channels expressed in cultured cells by increasing sensitivity of the channel to intracellular Ca2+. Thus we postulated that PKC-dependent activation of TRPM4 might be a critical mediator of vascular myogenic tone. We report here that PKC inhibition attenuated pressure-induced constriction of cerebral vessels and that stimulation of PKC activity with phorbol 12-myristate 13-acetate (PMA) enhanced the development of myogenic tone. In freshly isolated cerebral artery myocytes, we identified a Ca2+-dependent, rapidly inactivating, outwardly rectifying, iberiotoxin-insensitive cation current with properties similar to those of expressed TRPM4 channels. Stimulation of PKC activity with PMA increased the intracellular Ca2+ sensitivity of this current in vascular smooth muscle cells. To validate TRPM4 as a target of PKC regulation, antisense technology was used to suppress TRPM4 expression in isolated cerebral arteries. Under these conditions, the magnitude of TRPM4-like currents was diminished in cells from arteries treated with antisense oligonucleotides compared with controls, identifying TRPM4 as the molecular entity responsible for the PKC-activated current. Furthermore, the extent of PKC-induced smooth muscle cell depolarization and vasoconstriction was significantly decreased in arteries treated with TRPM4 antisense oligonucleotides compared with controls. We conclude that PKC-dependent regulation of TRPM4 activity contributes to the control of cerebral artery myogenic tone.

melastatin transient receptor potential; phorbol 12-myristate 13-acetate; cerebral artery myocytes

MODULATION OF ARTERIAL TONE in response to changes in intraluminal pressure [often referred to as the vascular myogenic response (3)] is vital for autoregulation of blood flow. Myogenic constriction is mediated by pressure-induced smooth muscle cell depolarization and Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs). Inhibition of protein kinase C (PKC) activity leads to a loss of myogenic tone (13, 20, 37), suggesting an important role for this pathway in pressure-dependent vascular regulation. Further insight into the molecular mechanisms responsible for myogenic constriction was provided by a recent report from our laboratory demonstrating that TRPM4, a member of the transient receptor potential (TRP) superfamily (8), is required for pressure-induced depolarization and constriction of cerebral arteries (10). In contrast with other TRP family members, TRPM4 (23, 34) and the closely related channel TRPM5 (14, 38, 39, 51) are highly selective for monovalent cations, and activation of TRPM4 currents in arterial myocytes elicits membrane depolarization, activation of VDCCs, and vasoconstriction (10). Little is currently known about the mechanisms that regulate the activity of this channel in vascular smooth muscle cells in response to increases in intraluminal pressure.

TRPM4 activity is highly dependent on intracellular [Ca2+] (23, 34), although the level of Ca2+ required for channel activation (≥1 µM) is greater than that typically reported for average or "global" cytosolic [Ca2+] (100–300 nM) (18) in vascular smooth muscle cells. Interestingly, Nilius and co-workers (35) recently discovered that phosphorylation of TRPM4 by PKC enhances its sensitivity to intracellular Ca2+, allowing the channel to be activated by physiological, albeit elevated, levels of Ca2+. PKC activity contributes to excitability of smooth muscle and vasoconstriction (32), suggesting a potential role for the PKC pathway in the activation of depolarizing cation currents. Thus we tested the hypothesis that PKC-dependent increases in TRPM4 activity contribute to pressure-induced smooth muscle membrane depolarization and myogenic constriction. Myogenic vasoconstriction was diminished by PKC inhibition and was enhanced by elevated levels of PKC activity. Consistent with our hypothesis, PKC activation increased the [Ca2+] sensitivity of TRPM4-dependent currents in freshly isolated cerebral artery myocytes. In addition, stimulation of PKC activity in cerebral arteries induced smooth muscle cell depolarization and vasoconstriction that was attenuated by knockdown of TRPM4 expression. These findings demonstrate that PKC stimulates TRPM4-dependent currents in vascular smooth muscle through increasing the Ca2+ sensitivity of the channel and that this mechanism functions as a critical mediator of myogenic tone in cerebral arteries.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats (250–350 g; Charles River Laboratories; St. Constant, Quebec, Canada, or Harlan, Indianapolis, IN) were used for these studies. Animals were deeply anesthetized with pentobarbital sodium (50 mg ip) and euthanized by exsanguination according to a protocol approved by the Institutional Animal Care and Use Committees (IACUC) of the University of Vermont and Colorado State University. Brains were isolated in ice-cold 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered saline (in mM): 3 MOPS (pH 7.4), 145 NaCl, 5 KCl, 1 MgSO4, 2.5 CaCl2, 1 KH2PO4, 0.02 EDTA, 2 pyruvate, and 5 glucose and 1% bovine serum albumin. Cerebral and cerebellar arteries were dissected from the brain, cleaned of connective tissue, and stored in MOPS-buffered saline before further manipulation.

Cerebral artery smooth muscle cell preparation. To isolate smooth muscle cells, vessels were cut into 2-mm segments and placed in the following cell isolation solution (in mM): 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgCl2, 10 glucose, and 10 HEPES (pH 7.2). Arterial segments were initially incubated at room temperature in 1 mg/ml papain (Worthington), 1 mg/ml dithioerythritol, and 0.5 mM CaCl2 for 30 min, followed by 25 min incubation at 37°C in 2 mg/ml type II collagenase (Worthington) and 0.5 mM CaCl2. The digested segments were then washed three times in ice-cold cell isolation solution and triturated to release smooth muscle cell. Cells were stored on ice in isolation solution for use the same day.

Whole cell patch clamp studies. Whole cell currents were recorded from freshly isolated arterial myocytes using an Axopatch 200B amplifier equipped with a CV203BU headstage (Axon Instruments). Recording electrodes (resistance, 3–5 M{Omega}) were pulled from borosilicate glass (1.5 mm OD, 1.17 mm ID; Sutter Instruments, Novato, CA) and coated with wax to reduce capacitance. Currents were filtered at 1 kHz, digitized at 40 kHz, and stored for subsequent analysis. Clampex and Clampfit versions 9.0 (Axon Instruments) were used for data acquisition and analysis, respectively. Cells were initially held at a membrane potential (Em) of 0 mV, and single channel currents were recorded during voltage ramps between –150 and +100 mV. Voltage ramps were initiated as soon as whole cell conditions were established and were repeated every 2 s during the experiment. All recordings were performed at room temperature (22°C). The bathing solution contained (in mM) 156 NaCl, 5 CaCl2, 10 HEPES (pH 7.4), and 10 glucose. This solution also contained the dihydropyridine voltage-dependent Ca2+ channel (VDCC) blocker nimodipine (1 µM), and for most experiments, the selective large conductance Ca2+-activated (BKCa) channel blocker iberiotoxin (IbTx) (300 nM). Because TRPM4 channels are Ca2+ dependent, high intracellular [Ca2+] was required to activate the channel. To prevent smooth muscle cell contraction under these conditions, arterial myocytes were pretreated with the myosin light chain kinase inhibitor wortmannin (10 µM; 5 min) before patch clamp experiments. The pipette solution contained (in mM) 156 CsCl, 1 MgCl2. and 10 HEPES (pH 7.2). For experiments where intracellular [Ca2+] was 30 or 100 µM, the appropriate amount of CaCl2 was added directly to the pipette solution. When intracellular [Ca2+] was 3 or 10 µM, the Ca2+ chelator N-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (5 mM) and the appropriate amount of CaCl2 was added to the pipette solution. When intracellular [Ca2+] was 0.1 µM, the Ca2+ chelator EGTA (5 mM) and the appropriate amount of CaCl2 were added to the pipette solution. The amount of CaCl2 required to achieve the desired free [Ca2+] under buffered conditions was calculated using the program Sliders 2.1 (http://www.stanford.edu/~cpatton/maxc.html). For some experiments, cells were treated with the PKC activator phorbol 12-myristate 13-acetate (PMA; 1 µM) (6) for at least 10 min before experimentation.

Oligonucleotide sequences and reverse permeabilization of cerebral arteries. Antisense oligonucleotides (oligos) for TRPM4 were designed based on a published sequence (Gen Bank Accession AF497623) and were identical to those used for a previous study from our laboratory (10). Two antisense oligos used were TRPM4 AS-1, 5'-GTGTGCATCGCTGTCCCACA-3', and TRPM4 AS-2, 5'-CTGCGATAGCACTCGCCAAA-3'. The last three bases on the 5' and 3' ends of the oligos were phosphorothioated to limit degradation by cellular nucleases. All oligos were obtained from Operon Biotechnologies and were dissolved at a concentration of 2 mM in nuclease-free water. Oligos were introduced into intact cerebral arteries using a reversible permeabilization procedure (24). To permeabilize the arteries, segments were first incubated for 20 min at 4°C in the following solution (in mM): 120 KCl, 2 MgCl2, 10 EGTA, 5 Na2ATP, and 20 N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 6.8). Arteries were then placed in a similar solution containing oligos (2 µM) for 90 min at 4°C and then in a similar oligo containing solution with elevated MgCl2 (10 mM). Permeabilization was reversed by placing the arteries for 30 min in a MOPS-buffered physiological solution containing (in mM): 140 NaCl, 5 KCl, 10 MgCl2, 5 glucose, and 2 MOPS (pH 7.1, 22°C). [Ca2+] was gradually increased in the latter solution from nominally calcium free to 0.01, 0.1, and 1.8 mM over a 45-min period. After the reversible permeabilization procedures, arteries were organ cultured for 2 to 3 days in DMEM-F-12 culture media supplemented with L-glutamine (2 mM), penicillin (50 U/ml), and streptomycin (50 µg/ml).

Isolated vessel experiments. Arterial segments were cleaned and transferred to a vessel chamber (University of Vermont Instrumentation Facility). The proximal end of the vessel was cannulated with a glass micropipette and secured, blood was gently rinsed from the lumen, and the distal end of the vessel was cannulated and secured. Vessels were pressurized to 20 mmHg with physiological saline solution (PSS) (in mM): 119 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgSO4, 24 NaHCO3, 0.2 KH2PO4, 10.6 glucose, and 1.1 EDTA and superfused (5 ml/min) with warmed (37°C) PSS aerated with a normoxic gas mixture (21% O2-6% CO2, balance N2). After a 15-min equilibration period, intraluminal pressure was slowly increased to 100 mmHg, vessels were stretched to remove bends, and pressure was reduced to 20 mmHg for an additional 15-min equilibration period. Inner diameter was continuously monitored using video microscopy and edge-detection software (Ionoptix). Before experimentation, endothelial function was disrupted by passage of air through the vessel lumen. To assess smooth muscle cell depolarization-induced constrictor responses, arteries were exposed to isotonic PSS containing 60 mM KCl. To determine PMA-induced constrictor responses, vessels were pressurized to 60 mmHg, and increasing concentrations of PMA were added to the superfusate. To assess myogenic tone, vessels were subjected to a series of pressure steps between 20 and 100 mmHg, and spontaneous myogenic tone was allowed to develop at each step until a stable diameter was achieved, usually ~3 min. After completion of the pressure-response curve, intraluminal pressure was maintained at 20 mmHg, and vessels were superfused with Ca2+-free PSS (in mM): 119 NaCl, 4.7 KCl, 1.2 MgSO4, 24 NaHCO3, 0.2 KH2PO4, 10.6 glucose, 3 EGTA, and 0.01 diltiazem. The pressure-response curve was repeated under Ca2+-free conditions to obtain passive responses. Myogenic tone was calculated as the percent difference in diameter observed for Ca2+-containing versus Ca2+-free PSS at each pressure. A single artery was studied from each rat, thus for isolated vessel experiments, values of n refer to the number of animals used for a particular experimental group.

Smooth muscle cell membrane potential. For measurement of smooth muscle cell membrane potential, cerebral arteries were isolated and pressurized, and smooth muscle cells were impaled through the adventitia with glass intracellular microelectrodes (tip resistance 100–200 M{Omega}). A WPI Intra 767 amplifier was used for recording membrane potential (Em). Analog output from the amplifier was recorded using Axotape software (sample frequency 20 Hz). Criteria for acceptance of Em recordings were 1) an abrupt negative deflection of potential as the microelectrode was advanced into a cell; 2) stable membrane potential for at least 1 min; and 3) an abrupt change in potential to ~0 mV after the electrode was retracted from the cell. Smooth muscle cell Em was recorded for TRPM4 sense- and antisense-treated arteries under control conditions and in the presence of PMA (1 µM) at intraluminal pressures of 20 and 80 mmHg.

Calculations and statistics. All data are means ± SE. Values of n refer to number of cells for patch clamp experiments or number of animals for isolated vessel experiments. Differences in whole cell current magnitude between sense- and antisense-treated cells were compared using the Mann-Whitney rank sum test. Data from isolated vessel experiments were compared using one-way repeated measures ANOVA followed by the Student-Newman-Keuls post hoc test. Data from membrane potential experiments were compared by two-way ANOVA followed by the Student-Newman-Keuls post hoc test. A level of P ≤ 0.05 was accepted as statistically significant for all experiments.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PKC activity is required for myogenic constriction of cerebral arteries. The mechanisms responsible for PKC-induced vasoconstriction were investigated using intact pressurized cerebral arteries. Activation of PKC with PMA induced concentration-dependent vasoconstriction (Fig. 1, A and C). The vehicle for PMA (DMSO) did not alter arterial diameter (not shown), whereas vasoconstriction in response to PMA was attenuated following pretreatment with the PKC inhibitor chelerythrine (3 µM, 15 min) (Fig. 1, B and C). Vasoconstriction in response to PMA (0.1 µM) was rapidly reversed (to maximal passive diameter) when arteries were superfused with Ca2+-free PSS containing PMA (0.1 µM) and the VDCC blocker diltiazem (10 µM) (Fig. 1D). In addition, PMA (0.1 µM) did not constrict arteries superfused with Ca2+-free PSS containing diltiazem (10 µM) (n = 3, data not shown). These findings demonstrate that PKC-induced constriction of cerebral arteries requires extracellular Ca2+ influx, likely via VDCCs.


Figure 1
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  		Fig. 1. Protein kinase C (PKC) activation constricts cerebral arteries. A: effect of phorbol 12-myristate 13-acetate (PMA) administration on the luminal diameter of a cerebral artery pressurized to 60 mmHg. B: effect of PMA administration on the luminal diameter of a cerebral artery pretreated with the PKC-inhibitor chelerythrine (3 µM, 15 min). C: summary data showing change in luminal diameter (expressed as percentage of baseline diameter) as a function of PMA concentration for control vessels (n = 8) and vessels pretreated with chelerythrine (3 µM; 15 min) (n = 3). *P ≤ 0.05 vs. control. D: rapid reversal of PMA (0.1 µM)-induced constriction by superfusion with Ca2+-free physiological saline solution (PSS) containing PMA (0.1 µM) and the voltage-dependent Ca2+ channel blocker diltiazem (10 µM) (representative of 3 experiments).

 
Further experiments were performed to elucidate a potential role for PKC activity in the regulation of myogenic tone. Consistent with previous reports, PKC inhibition (chelerythrine, 3 µM) attenuated myogenic constriction in cerebral arteries (Fig. 2, A and B), suggesting that PKC activity is required for this response. Chelerythrine (3 µM) did not diminish vasoconstriction in response to elevated (60 mM) extracellular KCl, demonstrating that this treatment does not inhibit voltage-dependent Ca2+ influx or the smooth muscle contractile apparatus. [KCl-induced constriction = 55.7 ± 4.9% (control) vs. 48.2 ± 8.8% (chelerythrine), n = 5 at an intraluminal pressure of 80 mmHg and 41.8 ± 3.5% vs. 41.8 ± 6.4, n = 5 at an intraluminal pressure of 20 mmHg].


Figure 2
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  		Fig. 2. PKC activity influences myogenic tone. A: effect of intraluminal pressure on cerebral artery diameter under control conditions, following pretreatment with the PKC inhibitor chelerythrine (3 µM; 15 min) or in the presence of Ca2+-free PSS. B: summary data. n = 3 for all groups. *P ≤ 0.05 vs. control. Myogenic tone (%) = [(luminal diameter in Ca2+-free PSS – luminal diameter in Ca2+-replete PSS)/luminal diameter in Ca2+-free PSS] x 100. C: effect of intraluminal pressure on cerebral artery diameter under control conditions, following pretreatment with the PKC activator PMA (0.1 µM; 15 min), or in the presence of Ca2+-free PSS. D: summary data. n = 5 for all groups. *P ≤ 0.05 vs. control.

 
In contrast with the effects of PKC inhibition, elevated levels of PKC activity following pretreatment with PMA (0.1 µM, 20 min) resulted in enhanced development of myogenic constriction (Fig. 2, C and D). These findings demonstrate that the magnitude of myogenic vasoconstriction is directly related to PKC activity levels in cerebral arteries.

TRPM4 whole cell currents in cerebral artery myocytes. Activation of TRPM4 in cerebral arteries elicits smooth muscle cell depolarization, Ca2+ influx via VDCCs, and vasoconstriction (10). In the present study, the conventional whole cell patch clamp technique was used to examine PKC and Ca2+-dependent regulatory mechanisms of TRPM4 in these cells. The defining biophysical characteristics of whole cell currents recorded from cultured cells overexpressing TRPM4 channels include dependence on high intracellular [Ca2+] for activity, strong outward rectification, and rapid inactivation (35, 46). BKCa channels, abundant in vascular smooth muscle, have a large unitary conductance, and, like TRPM4, are outwardly rectifying and strongly activated by intracellular [Ca2+] (40). It was therefore necessary to establish conditions that would allow TRPM4-like whole cell currents to be recorded from vascular smooth muscle cells without contamination from BKCa-dependent currents. To diminish BKCa current magnitude, the pipette (intracellular) solution used for patch clamp recordings contained Cs+, which have low permeation through BKCa channels (<5% vs. K+) (4), as the only monovalent cation. For initial experiments, [Ca2+] in the pipette solution was 10 µM, near the EC50 for Ca2+ activation of expressed TRPM4 channels in whole cell experiments (15 µM) (35). Outwardly rectifying whole cell currents were recorded from freshly isolated cerebral artery myocytes under these conditions [peak outward current (+100 mV) = 186.4 ± 80.9 pA/pF, n = 3]. Peak outward current magnitude was significantly decreased when [IbTX] in the bathing solution was 100 nM (35.1 ± 8.7 pA/pF, n = 4) and was further diminished when [IbTx] was increased to 300 nM (15.6 ± 8.7 pA/pF, n = 4) but not further inhibited when [IbTx] was increased to 1,000 nM (13.2 ± 2.3 pA/pF, n = 3), or when the nonselective K+ channel blocker tetraethylammonium (TEA) (10 mM) was present in the bathing solution (14.8 ± 5.4 pA/pF, n = 4). Thus all patch clamp experiments were performed in the presence of IbTx (300 nM) to block BKCa activity.

TRPM4-dependent currents exhibit rapid inactivation following the establishment of whole cell conditions when Ca2+ is present in the patch pipette solution (35). In patch clamp experiments using cerebral artery myocytes, strong outwardly rectifying currents were observed within a few seconds after whole cell conditions were established when the [Ca2+] of the pipette (intracellular) solution was 10 µM (Fig. 3A). Voltage ramps (–150 mV to +100 mV) were performed every 2 s after whole cell conditions were established to examine the time course of the IbTX-insensitive current. Outward currents became maximal approximately 4 s after whole cell conditions were established, followed by a rapid decay to steady-state levels within about 20 s (Fig. 3B). This current was not observed when the [Ca2+] of the pipette solution was 0.1 µM, suggesting that activation is dependent on elevated intracellular [Ca2+] (Fig. 3C).


Figure 3
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  		Fig. 3. Whole cell transient receptor potential (TRP)M4 currents in cerebral artery myocytes. A: whole cell current voltage relationships for a cell with intracellular [Ca2+] ([Ca2+]i) clamped at 10 µM. Currents are shown during peak current activation and 60 s after whole cell conditions were established. B: time course of peak current activation and deactivation for the same cell as shown in Fig. 1A (representative of 8 cells). C: current-voltage relationship for a cell with [Ca2+]i clamped at 0.1 µM at the peak of current activation and 60 s after whole cell conditions were established (representative of 4 cells). D and E: representative current-voltage traces for cerebral myocytes isolated from TRPM4 sense- (D) and antisense-treated (E) arteries. Currents are shown at peak activation and 60 s after whole cell conditions were established. [Ca2+]i = 10 µM. F: mean peak outward currents (pA/pF) for cerebral artery myocytes from sense and antisense-treated arteries. n = 9 (sense) or n = 11 (antisense) *P ≤ 0.05 vs. sense treated. Cm, membrane capacitance.

 
Selective pharmacological inhibitors of TRPM4 are not currently available. Therefore, we utilized antisense technology to suppress TRPM4 channel expression in isolated cerebral arteries and cerebral myocytes. The oligonucleotide sequences used for these experiments have previously been shown to selectively diminish TRPM4 expression in cerebral arteries and inhibit pressure-induced smooth muscle depolarization and myogenic vasoconstriction (10). In whole cell patch clamp experiments, currents consistent with TRPM4 were observed in cerebral artery myocytes isolated from arteries treated with sense (control) oligonucleotides (Fig. 3D). In contrast, the magnitude of the peak outward current in myocytes from vessels treated with antisense oligonucleotides (6.7 ± 3.5 pA/pF, n = 11) was significantly (P ≤ 0.05) less than that of cells from sense-treated arteries (14.0 ± 3.7 pA/pF, n = 9) (Fig. 3, E and F), demonstrating that TRPM4-dependent currents were decreased by antisense treatment.

These experiments show that biophysical properties of this IbTX-insensitive current, namely the Ca2+ dependence, rapid inactivation, and outward rectification, are consistent with the reported properties of whole cell currents recorded from cultured cells overexpressing cloned TRPM4 channels (35). In addition, knockdown of TRPM4 expression with antisense oligonucleotides decreases the magnitude of this current. These findings strongly suggest that the molecular identity of the ion channel conducting this Ca2+-activated current is TRPM4.

PKC activation increases the Ca2+ sensitivity of TRPM4 in arterial smooth muscle cells. PKC activity increases the Ca2+ sensitivity of cloned TRPM4 channels expressed in HEK cells, shifting the intracellular [Ca2+] required for half-maximal activation from ~15 µM under control conditions to ~4 µM following treatment with the PKC activator PMA (35). Whole cell patch clamp experiments were performed to examine the effect of PKC activity on Ca2+-dependent regulation of TRPM4 in cerebral artery myocytes. Under control conditions, only small currents were observed when intracellular [Ca2+] was <3 µM (Fig. 4A). Activation was half-maximal when intracellular [Ca2+] was ~10 µM and reached a maximum at intracellular [Ca2+] of 100 µM (Fig. 4C). Current activation occurred at lower levels of intracellular [Ca2+] following PKC stimulation. In cells pretreated with the PKC activator PMA (1 µM, 10 min), whole cell currents were observed when intracellular [Ca2+] was ≥3 µM (Fig. 4B), and currents became near maximal when intracellular [Ca2+] was 10 µM. Peak outward current magnitude (normalized to maximum current) was plotted as a function of intracellular [Ca2+], and the data were fitted to a Boltzmann function (Fig. 4C). From these data, the [Ca2+] required for 50% of maximal current activation (EC50) under control conditions was calculated to be 10.1 ± 0.3 µM, and following PMA treatment, it was 5.0 ± 1.1 µM. Although the difference in EC50 is not statistically significant, owing to the variability in TRPM4 currents at high intracellular [Ca2+], in the presence of PMA, the trend in the shift of Ca2+ sensitivity for TRPM4 activation following PMA treatment is apparent. These values are consistent with those previously reported by Nilius et al. (35) for cloned TRPM4 channels expressed by HEK cells (EC50 = 15 µM under control conditions vs. 4 µM following treatment with PMA). Enhanced sensitivity to intracellular [Ca2+] in cerebral myocytes versus HEK cells overexpressing TRPM4 under control conditions [EC50 = 15 µM for HEK cells overexpressing TRPM4 (35) vs. 10.1 µM for cerebral myocytes] may reflect greater basal PKC activity and/or expression of different kinase isoforms in freshly isolated smooth muscle cells compared with HEK cells.


Figure 4
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  		Fig. 4. PKC activation increases the Ca2+ sensitivity of TRPM4 in arterial smooth muscle cells. A and B: current-voltage relationships recorded from a cell under control conditions (A) and from a cell pretreated with PMA (1 µM; 10 min) (B). [Ca2+]i = 3 µM. Currents are shown at peak activation and 60 s after whole cell conditions were established. C: peak outward current as a function of [Ca2+]i recorded from control cells or from cells pretreated with PMA. Data shown were normalized to the mean current magnitude from cells with [Ca2+]i = 100 µM. n = 3–8 cells per group.

 
PKC-induced smooth muscle cell depolarization and vasoconstriction is mediated by TRPM4. To investigate a role for PKC-dependent activation of TRPM4 in the regulation of smooth muscle excitability, intracellular microelectrodes were used to measure membrane potential in isolated cerebral arteries pressurized to 20 mmHg. In sense-treated (control) arteries, PMA (1 µM) depolarized smooth muscle cells by ~15 mV (–49 ± 2.2 mV vs. –34 ± 0.6 mV, n = 4), whereas PMA did not significantly alter the membrane potential of smooth muscle cells in antisense-treated vessels (–50 ± 1.9 mV vs. –48 ± 4.1 mV, n = 4) (Fig. 5A). Experiments were also performed on vessels pressurized to 60 mmHg. Under these conditions, diminished PMA-induced depolarization was also observed in antisense compared with sense-treated arteries [sense –38.0 ± 1.2 mV (control) vs. –31.8 ± 0.6 mV (PMA); antisense –45.0 ± 1.5 mV (control) vs. –41.5 ± 3.0 mV (PMA); n = 4 for all groups]. Under control conditions, resting membrane potential for smooth muscle cells in antisense-treated vessels was more hyperpolarized (–45.0 ± 1.5 mV) compared with sense-treated arteries (–38.0 ± 1.2 mV) in this experiment because, as previously reported, pressure-induced membrane depolarization is attenuated by TRPM4 antisense (10). These data demonstrate that PKC activation causes smooth muscle depolarization by a mechanism that requires TRPM4 channel expression.


Figure 5
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  		Fig. 5. Suppression of TRPM4 in isolated cerebral arteries attenuates PMA-induced smooth muscle depolarization and vasoconstriction. A: smooth muscle resting membrane potential recorded from isolated cerebral arteries pressurized at 20 mmHg before and after PMA (1 µM) administration. n = 4 for all groups. *P ≤ 0.05 vs. sense, vehicle-treated; #P ≤ 0.05 vs. sense-PMA treated. B and C: PMA-induced vasoconstriction in TRPM4 sense- (B) or antisense (C)-treated cerebral arteries. D: cerebral artery constriction as a function of PMA concentration for TRPM4 sense- and antisense-treated arteries. Vasoconstriction of antisense arteries undergoing vasomotion was calculated from the mean luminal diameter beginning ~3 min after PMA administration through the end of the experiment. n = 6 for each group. *P ≤ 0.05 vs. sense treated.

 
The effects of TRPM4 knockdown on PKC-induced vasoconstriction were examined in isolated cerebral arteries. PMA administration induced concentration-dependent vasoconstriction of sense-treated arteries (Fig. 5, B and D) similar in magnitude to that of freshly isolated vessels. In contrast, several (3 of 6) arteries treated with TRPM4 antisense failed to achieve a stable luminal diameter, but rather exhibited oscillatory diameter changes after exposure to PMA (Fig. 5C), and vasoconstriction resulting from PMA administration was significantly attenuated following knockdown of TRPM4 expression (Fig. 5, C and D). These findings demonstrate that PKC activation constricts cerebral arteries through a mechanism involving TRPM4.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The vascular myogenic response is an important intrinsic mechanism regulating blood flow during changes in perfusion pressure (3). Myogenic constriction results from smooth muscle depolarization (12) and Ca2+-influx via VDCCs following increases in intraluminal pressure (19). In addition, previous studies (13, 20, 37) and the current findings (Fig. 2, A and B) show that inhibition of PKC activity blocks pressure-induced vasoconstriction in isolated blood vessels. Furthermore, stimulation of PKC activity enhances the development of myogenic tone (Fig. 2, C and D), providing additional support for a link between PKC activity and pressure-induced vasoconstriction. These findings pose mechanistic questions about how PKC influences myogenic vasoconstriction. A prior study demonstrated that intraluminal pressure can induce PKC translocation to the smooth muscle plasma membrane in coronary arteries, suggesting that PKC activity is elevated by increasing perfusion pressure (9). Furthermore, PKC inhibition reverses pressure-induced smooth muscle cell depolarization in cerebral arteries, demonstrating that PKC activity contributes to increased arterial myocyte excitability in response to pressure (43). Activity of expressed TRPM4 channels can be regulated by PKC (35), and an earlier report from our group demonstrated that TRPM4 expression is required for smooth muscle cell depolarization and vasoconstriction in response to increased intraluminal pressure (10). Thus the goal of the current study was to investigate the functional implications of PKC-dependent regulation of TRPM4 in the vasculature. The major findings reported here are 1) the extent of myogenic vasoconstriction is related to the level of PKC activity; 2) PKC activation increases the sensitivity of TRPM4 to intracellular [Ca2+] in arterial smooth muscle cells; and 3) suppression of TRPM4 expression attenuates PKC-induced smooth muscle depolarization and vasoconstriction. Thus the current findings are consistent with the hypothesis that pressure-induced increases in PKC activity contribute to smooth muscle depolarization and myogenic constriction in cerebral arteries through a mechanism involving Ca2+ sensitization of TRPM4.

At least 12 different PKC isoforms have been reported in mammalian tissue, and no fewer than six are present in vascular smooth muscle cells (41). The influence of PKC on smooth muscle excitability and contractility has been studied extensively (32), and it has been demonstrated that PKC can alter vascular tone through a number of signaling pathways. For example, several studies report that PKC can constrict isolated arteries without altering intracellular [Ca2+], presumably by increasing the Ca2+ sensitivity of myosin light chain kinase or by modifying other proteins involved in smooth muscle contraction (28, 49). In contrast, other studies suggest that PKC vasoconstriction requires extracellular Ca2+ influx (7, 16, 25, 29). PKC can directly increase VDCC activity independent of changes in resting membrane potential (1). Additionally, in pressurized cerebral arteries, the phorbol compound PMA induces smooth muscle cell depolarization that is blocked by PKC inhibition (43). These findings suggest that PKC activation can induce Ca2+ influx and vasoconstriction via VDCCs. The current study provides further evidence that Ca2+ influx-dependent mechanisms are primarily responsible for vasoconstriction of cerebral arteries during PKC activation. Superfusion with Ca2+-free PSS containing the VDCC blocker diltiazem rapidly reversed vasoconstriction in response to PMA (0.1 µM) (Fig. 1D), suggesting that, under these conditions, Ca2+ influx is required to maintain arterial tone. Stimulation of PKC activity resulted in dramatic membrane depolarization that was essentially eliminated by knockdown of TRPM4 expression (Fig. 5A). In agreement with this finding, vasoconstriction resulting from PMA administration was also significantly attenuated by TRPM4 knockdown (Fig. 5, B–D). We conclude that PKC-induced vasoconstriction results from TRPM4-dependent membrane depolarization, leading to Ca2+ influx via VDCCs.

TRPM4 is present in, and physiologically important for, normal function of a number of excitable cells, including T-lymphocytes (22), cardiac myocytes (11), and vascular smooth muscle cells (10). TRPM4 and the closely related channel TRPM5 (14, 27, 38, 39, 51) are the only members of the TRP superfamily that are activated by intracellular [Ca2+]. Widely ranging values for the intracellular [Ca2+] required for TRPM4 activation have been reported. The initial study describing the biophysical properties of cloned TRPM4 channels expressed in cultured cells reported that the EC50 for Ca2+-dependent activation of the channel was ~0.4 µM in whole cell experiments (23), whereas studies by Nilius and co-workers reported an EC50 value of 370 µM in excised membrane patches (36) and 15–20 µM for TRPM4-dependent whole cell currents (35, 46). In cerebral artery smooth muscle cells, the EC50 for TRPM4 Ca2+ activation in inside-out membrane patches was reported as ~200 µM (10), and the current study shows that the whole cell EC50 for Ca2+ in these cells is ~10 µM (Fig. 4B). Thus, in both cultured and native cells, the Ca2+ sensitivity of TRPM4 appears to be consistently lower in experiments using the inside-out patch clamp technique versus conventional whole cell patch clamp experiments. This difference in Ca2+ sensitivity could be due to dilution or loss of important Ca2+-dependent regulatory factors when membrane patches are excised, whereas under whole cell conditions, these putative factors may be better maintained. In any case, most prior reports and the current study (Fig. 4, B and C) agree that intracellular [Ca2+] >1 µM is required to activate TRPM4. This is much greater than the average, or global, intracellular [Ca2+] typically reported for vascular smooth muscle cells (100–300 nM) (18), leading to questions regarding the source of Ca2+ for TRPM4 activation. Dynamic Ca2+ events due to Ca2+ release from intracellular stores through ryanodine receptors ("Ca2+ sparks") (17) or inositol 1,4,5-trisphosphate (IP3)-coupled receptors ("Ca2+ waves") (5) located on the sarcoplasmic reticulum are a possible source of Ca2+ for activation of TRPM4. These events are common in vascular smooth muscle cells (17), and Ca2+ sparks are responsible for activation of BKCa channels in arterial myocytes under physiological conditions (33), supporting the concept that dynamic Ca2+ events can regulate the activity of Ca2+-dependent channels in vascular smooth muscle. It is also possible that TRPM4 could be closely coupled to a Ca2+-permeable ion channel and that extracellular Ca2+ influx activates TRPM4. TRPC6, a Ca2+-permeable member of the canonical TRP subfamily, is also required for myogenic constriction (48). Both TRPC6 (15) and PKC are activated by diacylglycerol (DAG), and synthesis of DAG by phospholipase C (PLC) has been proposed as a possible mechanism for activation of TRPC6 by intraluminal pressure (48). Consistent with this idea, Slish et al. (43) report that activation of cation currents by a DAG analog is blocked by PKC inhibition in cerebral artery myocytes. Thus increased PLC (and DAG) activity in response to pressure elevation could also be responsible for enhanced PKC activity. Interestingly, a recent report suggests that TRPC6 may also be inherently mechanosensitive (44). If TRPC6 is located in intracellular microdomains proximal to TRPM4, stretch-activated Ca2+ influx via TRPC6 could in turn initiate depolarizing Na+ currents via TRPM4, resulting in further Ca2+ influx through VDCCs. In this hypothetical signaling pathway, TRPM4, PKC, and VDCCs could act to amplify the initial TRPC6 Ca2+ signal. Although the source of Ca2+ that activates TRPM4 is not known, it is clear that PKC increases the sensitivity of the channel to activation by intracellular [Ca2+] in both cultured cells (35) and in freshly isolated cerebral artery myocytes (Fig. 4). In contrast, TRPC6-dependent currents appear to be inhibited by PKC activity (42). Thus it appears that TRPM4 functions as an integrator of PKC activity and intracellular [Ca2+] to effect a physiological response in excitable cells, and that this mechanism contributes to the regulation of myogenic vasoconstriction.

PKC-induced smooth muscle hyperexcitability has been implicated in pathological conditions associated with deranged vascular function. For example, PKC appears to be involved in hypoxia-induced pulmonary vasoconstriction (HPV) of small arteries that is associated with pulmonary hypertension (2, 45, 47). Weissmann and colleagues (47) found that the PKC inhibitor bisindolylmaleimide I selectively blocked hypoxia-induced, but not agonist-induced, increases in pulmonary vascular resistance without influencing basal tone. These findings were extended by a report demonstrating that HPV was diminished in PKC-{epsilon} null mice (26). It was recently reported that TRPM4 is present in the pulmonary artery smooth muscle (50), consistent with the possibility that PKC-dependent activation of TRPM4 could contribute to HPV. A number of studies report that PKC inhibitors block or attenuate cerebral vasospasm in models of subarachnoid hemorrhage (30, 31), suggesting that PKC activity has a prominent role in the development of this pathology (21). Our findings that PKC activates TRPM4 in cerebral artery vascular smooth muscle, which serves to depolarize these cells and induce vasoconstriction, are consistent with a potential role for the channel in PKC-related vascular pathologies and suggest that TRPM4 might be a novel, putative pharmacological target for the treatment of such conditions.

In summary, the current study demonstrates an important functional role for PKC-dependent activation of TRPM4 in cerebral artery myocytes. PKC activity promotes TRPM4-dependent currents in these cells by increasing the Ca2+ sensitivity of the channel. These findings strongly support the possibility that regulation of TRPM4 activity by PKC could play an important role in the control of myogenic tone under normal conditions and could contribute to disrupted arterial function during certain pathophysiological situations.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants F32 HL-075995 and RO1 HL-58231 (J. E. Brayden), by American Heart Association Grant 0535226N (to S. Earley), and by the Totman Medical Trust.


   ACKNOWLEDGMENTS
 
The authors thank Katherine Lutz, Rachael C. Crnich, and Stewart A. Berry for technical assistance and Dr. Mike Tamkun for critical comments on the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Earley, Dept. of Biomedical Sciences, Colorado State Univ., Fort Collins, CO USA 80523-1680 (e-mail: Scott.Earley{at}colostate.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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