TRPV4 Forms a Novel Ca2+ Signaling Complex With Ryanodine Receptors and BKCa Channels
http://www.100md.com
循环研究杂志 2005年第12期
the Department of Pharmacology, University of Vermont College of Medicine, Burlington.
Abstract
Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4-phorbol 12,13-didecanoate (4-PDD) (5 eol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EETeCinduced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 eol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EETeCinduced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.
Key Words: Ca2+ sparks Ca2+ transients eicosanoids ion channels ryanodine receptor vascular smooth muscle vasodilation
Introduction
Diffusible factors produced by the vascular endothelium are vital for the regulation of smooth muscle membrane potential, arterial tone, blood pressure, and blood flow. Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase products of arachidonic acid,1 cause vasodilation and account for endothelium-derived hyperpolarizing factor (EDHF) activity in some vascular beds.2,3 Critical components of signaling pathways activated by these compounds are not fully understood.1 Although evidence supportive of a receptor-dependent smooth muscle hyperpolarization mechanism has been presented,4 the molecular identity of the "EET receptor" has not been reported. Vanilloid transient receptor potential (TRPV) channels are important sensors of biochemical and physiological stimuli.5 Therefore, we tested the hypothesis that TRPV channels are involved in transducing endothelial signals to underlying vascular smooth muscle. EETs promote Ca2+ influx in HEK cells overexpressing TRPV46 and increase intracellular [Ca2+] in cultured smooth muscle cells.7 In addition, activation of TRPV4 with the phorbol compound 4-phorbol 12,13-didecanoate8 (4-PDD) increases intracellular [Ca2+] in airway smooth muscle.9 An elevation in intracellular [Ca2+] is usually associated with vasoconstriction. Thus, these reports pose a paradox: How does EET-induced Ca2+ influx cause vasodilation A number of studies suggest involvement of large-conductance Ca2+-activated K+ (BKCa) channels in EET-induced smooth muscle hyperpolarization and vasodilation.1,2 Under physiological conditions, BKCa channels in smooth muscle cells are activated by elementary Ca2+-release events (Ca2+ sparks) via ryanodine receptors (RyRs) located on the sarcoplasmic reticulum (SR). Ca2+ sparks activate nearby BKCa channels to cause transient membrane hyperpolarization and vasodilation.10 Thus, EET-induced dilation could be explained by Ca2+ sparks activating BKCa channels. We further hypothesized that Ca2+ influx through TRPV4 preferentially stimulates RyRs in the SR, generating Ca2+ sparks that signal adjacent BKCa channels to open and cause membrane hyperpolarization and vasodilation.10 Consistent with this possibility, the open probability (NPo) of SR RyRs is increased by both elevated cytoplasmic [Ca2+]11 and enhanced SR Ca2+ loading,12 which augments Ca2+ spark activity in arterial smooth muscle. Thus, Ca2+ influx via TRPV4 could stimulate SR RyRs, either directly or through increased SR Ca2+ loading, or both, to generate Ca2+ sparks and enhance BKCa channel activity, thereby causing smooth muscle hyperpolarization and relaxation. We propose that TRPV4 forms a novel Ca2+ signaling complex with RyRs and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release13,14 (CICR).
Materials and Methods
Cerebral and cerebellar arteries used for these studies were isolated from male SpragueeCDawley 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. Total RNA was extracted from whole brain, isolated cerebral arteries, and freshly dispersed cerebral artery smooth muscle cells, and RT-PCR was used to determine whether TRPV4 mRNA was present in these tissues. Forward and reverse primers specific for TRPV4 were TRPV4F (5'-GGGCAGCTCCCCAAAGTAGAA-3') and TRPV4R (5'-GTGCCTGGGCCCAAGAAA-3'). These primers yield a 578-bp PCR product.
Whole-cell patch-clamp experiments were used to determine whether ion channels with biophysical properties similar to TRPV4 were present in freshly isolated cerebral artery myocytes. Currents were recorded during voltage ramps between eC120 and +80 mV before and after administration of 4-PDD or 11,12 EET and were normalized to membrane capacitance (pA/pF). Isolated smooth muscle cells were loaded with the rapid Ca2+ indicator dye fluo-4 (Molecular Probes), and the effects of 11,12 EET on Ca2+ spark frequency were evaluated using high-speed (30 to 60 frames/s) laser-scanning confocal microscopy.15,16 Cerebral myocytes were also patch clamped in the perforated-patch configuration to examine the effects of 11,12 EET on the frequency of transient macroscopic BKCa currents ("spontaneous transient outward currents" [STOCs]).
Downregulation of TRPV4 expression in isolated cerebral arteries was accomplished with antisense oligonucleotides. The sequence of the TRPV4 antisense oligonucleotide used for these studies was 5'-CATCACCAGGATCTGCCATACTG-3'17 (Operon Biotechnologies Inc). The complementary sequence was used as the sense (control) oligonucleotide. Sense and antisense oligonucleotides were introduced into intact cerebral arteries using a reversible permeabilization procedure.18 Following reversal of permeabilization, arteries were organ cultured for 2 to 3 days in DMEM/F-12 medium without serum to allow for TRPV4 downregulation. Semiquantitative RT-PCR was used to evaluate the effects of antisense treatment on TRPV4 mRNA levels. Smooth muscle cells isolated from cultured arteries were patch clamped in whole-cell or perforated-patch configuration to investigate the effects of TRPV4 antisense on 11,12 EETeCinduced cation currents and transient BKCa channel activity. In additional experiments, sense- and antisense-treated arteries were mounted in an arteriograph, endothelial cells were removed, and the effects of TRPV4 downregulation on 11,12 EETeCinduced increases in Ca2+ spark activity, smooth muscle cell hyperpolarization, and vasodilation were evaluated.
An expanded Materials and Methods section can be found in the online supplement available at http://circres.ahajournals.org.
Results
TRPV4-Like Channels Are Present in Cerebral Artery Smooth Muscle
Message encoding TRPV4 was found in rat brain, cerebral arteries, and isolated cerebral artery smooth muscle cells (Figure 1a, inset). PCR products were not detected when template cDNA was omitted from the PCR (Figure 1a) or when reverse-transcription reactions were performed in the absence of reverse transcriptase (not shown). Because a number of transient receptor potential channels, including TRPC1,19 TRPC3,19eC21 TRPC4,19 TRPC6,19,21 and TRPM4,22 are also present in cerebral artery myocytes, conventional whole-cell patch-clamp experiments were performed to determine whether ion channels with biophysical properties similar to those of cloned TRPV4 channels were present in these cells. The TRPV4 agonist 4-PDD (5 eol/L) activated an outwardly rectifying current in these cells (Figure 1a and 1b). 11,12 EET (300 nmol/L) also rapidly (1 minute) evoked a current with similar currenteCvoltage characteristics (Figure 1c). Activation of whole-cell currents by 11,12 EET (300 nmol/L) was blocked by prior administration of the TRPV channel inhibitor ruthenium red (RR) (1 eol/L) (Figure 1d and 1e). The rapid activation and inactivation of 11,12 EETeCevoked currents in cerebral myocytes (Figure 1f) was similar to the reported time course of EETeCinduced Ca2+ influx and whole-cell currents in cultured cells overexpressing TRPV4.6 Activation of inward, but not outward, currents by 11,12 EET (300 nmol/L) was absent when N-methyl-D-glucamine (NMDG) was substituted for cations in the extracellular solution (Figure 1g and 1h). Thus, properties of this current in cerebral myocytes, such as currenteCvoltage relationships, activation by 4-PDD and 11,12 EET, and block by RR, are consistent with previously described characteristics of cloned TRPV4 channels6,8,23 and, combined with expression of TRPV4 mRNA in these cells, strongly suggests that functional TRPV4 channels are present in cerebral artery smooth muscle.
EETs Increase Ca2+ Spark and Transient BKCa Activity in Cerebral Myocytes
EETs, which activate TRPV4 channels,6 have also been shown to induce membrane potential hyperpolarization and relaxation of arteries, which is prevented by blocking BKCa channels.2 We, therefore, examined the effects of EETs on intracellular Ca2+ dynamics in cerebral artery smooth muscle cells loaded with the rapid Ca2+ indicator dye fluo-4. 11,12 EET (300 nmol/L) increased Ca2+ spark frequency nearly 2-fold in freshly isolated cerebral myocytes (Figure 2a and 2b). Previous reports demonstrate that Ca2+ sparks stimulate nearby sarcolemmal BKCa channels to cause transient K+ currents (STOCs).10 11,12 EET (100 nmol/L) increased the frequency of STOCs in voltage-clamped (0 mV) arterial smooth muscle cells (Figure 2c and 2d). 11,12 EET (300 nmol/L) also significantly (P<0.05) increased STOC frequency (0.31±0.01 versus 0.73±0.14 Hz; n=3) for cells voltage clamped at eC40 mV, a membrane potential in the physiological range for smooth muscle cells in pressurized cerebral arteries. STOC activity was not altered by the vehicle for 11,12 EET (DMSO, 0.1%) (Figure 2d) or by the metabolic precursor of 11,12 EET, arachidonic acid (1 eol/L; 101.4±27% of control; n=5). STOC activity in the presence of 11,12 EET was greatly diminished by ryanodine (5 eol/L; 0.55±0.08 versus 0.12±0.07 Hz; n=3) and the specific BKCa channel blocker iberiotoxin (100 nmol/L; 0.33±0.11 versus 0.05±0.02 Hz; n=3). In addition, 11,12 EET (300 nmol/L) did not increase the open probability (NPo) of BKCa channels in membrane patches obtained from cerebral artery myocytes (online Figure I). These findings strongly support the concept that EETs signal RyRs to increase Ca2+ sparks, which in turn activate BKCa channels.
EETeCInduced Increases in Ca2+ Sparks and Transient BKCa Activity Requires Ca2+ Influx
TRPV4 channels are Ca2+ permeable,23 consistent with the possibility that Ca2+ entry through this channel could trigger Ca2+ sparks via CICR. We, therefore, examined the effects of reduced extracellular Ca2+ on EET-induced increases in Ca2+ spark activity in arterial myocytes. In the presence of an L-type voltage-dependent Ca2+ channel (VDCC) blocker (30 eol/L diltiazem), 11,12 EET (300 nmol/L) significantly (P<0.05) elevated Ca2+ spark frequency when external [Ca2+] was maintained at physiological levels (2 mmol/L) (Figure 3a). In contrast, when extracellular [Ca2+] was reduced to 10 eol/L in the presence of diltiazem, Ca2+ spark frequency did not differ between control and 11,12 EETeCtreated cells (Figure 3a). 11,12 EET (300 nmol/L) also increased STOC frequency when external [Ca2+] was 2 mmol/L and VDCCs were blocked (1 eol/L nisoldipine), whereas when bath [Ca2+] was reduced to 10 eol/L while VDCC inhibition was maintained, STOC activity was not altered by 11,12 EET administration (Figure 3b and 3c). These findings demonstrate that EET-induced increases in Ca2+ spark and STOC frequency are dependent on Ca2+ entry via a non-VDCC channel.
EETeCInduced Increases in Transient BKCa Activity Are TRPV4 Dependent
TRPV4 channels are activated by EETs,6 consistent with the possibility that these channels could mediate EET-induced hyperpolarization in cerebral artery smooth muscle cells. The TRPV antagonist RR (1 eol/L), applied externally, blocked 11,12 EETeCinduced (300 nmol/L) increases in STOC frequency without altering basal STOC activity (Figure 4a and 4b). In addition, RR (1 eol/L) reversed 11,12 EETeCinduced increases in STOC frequency to control levels (Figure 4c and 4d). The TRPV4 agonist 4-PDD (5 eol/L) also elevated STOC frequency in cerebral myocytes, and this response was inhibited by RR (1 eol/L) (Figure 4e and 4f).
To examine further the relationship between EET-induced, TRPV4-mediated Ca2+ influx and smooth muscle hyperpolarization, TRPV4 expression was suppressed in intact vessels using antisense oligonucleotides. Cerebral arteries were permeabilized,18 were exposed to TRPV4 sense or antisense oligonucleotides and, following reversal of permeabilization, were organ cultured for 2 to 3 days to allow TRPV4 downregulation.21,22 Semiquantitative RT-PCR was used to examine the efficacy of these procedures, and TRPV4 mRNA levels were found to be diminished in antisense- versus sense-treated arteries (Figure 5a, inset).
11,12 EET (300 nmol/L) activated an outwardly rectifying current in smooth muscle cells from sense-treated vessels that had a similar currenteCvoltage relationship, current density, time course, and reversal potential as 11,12 EETeC and 4-PDDeCinduced currents in cerebral myocytes from freshly isolated arteries (Figure 5a and 5e). However, 11,12 EETeCactivated currents were significantly reduced in smooth muscle cells from antisense-treated vessels (Figure 5b through 5d).
To further elucidate the consequences of TRPV4 activation, the effects of 11,12 EET on Ca2+ sparks were measured in isolated, pressurized cerebral arteries loaded with the Ca2+ indicator dye fluo-4. 11,12 EET (300 nmol/L) increased Ca2+ spark frequency in pressurized (60 mm Hg) sense-treated cerebral arteries (Figure 6a and 6b), whereas Ca2+ spark frequency in antisense-treated vessels was not altered by 11,12 EET administration (Figure 6a and 6b). Consistent with these observations, 11,12 EET (300 nmol/L) elevated STOC frequency in cerebral myocytes isolated from sense-treated, but not antisense-treated, arteries (Figure 6c and 6d). These findings demonstrate that TRPV4 is a critical mediator of 11,12 EETeCinduced increases in Ca2+ sparkeCactivated transient BKCa currents in cerebral artery smooth muscle.
TRPV4 Mediates EETeCInduced Smooth Muscle Hyperpolarization and Vasodilation
An elevation of Ca2+ spark frequency can dilate cerebral arteries through activation of BKCa channels. Therefore, the relationships between TRPV4 downregulation, EET-induced changes in Ca2+ spark frequency, and alterations in smooth muscle membrane potential and vasomotor responses were examined. 11,12 EET (300 nmol/L) caused an approximately 10 mV smooth muscle membrane potential hyperpolarization in pressurized (60 mm Hg), TRPV4 sense-treated arteries (Figure 7a and 7b). In contrast, membrane potential of cerebral myocytes in antisense-treated vessels was not significantly altered by 11,12 EET (Figure 7a and 7b). TRPV4 sense- and antisense-treated vessels constricted to the same extent in response to a depolarizing concentration of KCl (60 mmol/L) or pressure (60 mm Hg), suggesting that membrane depolarizationeCinduced Ca2+ influx through VDCCs was unaffected. Vasodilation in response to the KATP channel agonist pinacidil (10 eol/L) also did not differ between groups (sense, 83.3±3.7% of passive diameter; antisense, 86.7±5.5%; n=3 for each group). In contrast, 11,12 EETeCinduced vasodilator responses were significantly inhibited by TRPV4 downregulation (Figure 7c and 7d), whereas TRPV4 sense-treated arteries dilated to an extent similar to that of freshly isolated arteries (Figure 7c and 7d). In the presence of ryanodine (10 eol/L), freshly isolated cerebral arteries exhibited only slight, statistically insignificant dilation (6.9±2.5 e; n=3) in response to 11,12 EET (300 nmol/L), providing further evidence for a Ca2+ sparkeCdependent mechanism in this response. These findings demonstrate a role for TRPV4 and RyRs in EET-induced smooth muscle hyperpolarization and vasodilation.
Discussion
The major findings of this study are as follows: (1) functional TRPV4 ion channels are present in cerebral artery smooth muscle; (2) activation of TRPV4 in cerebral myocytes with 11,12 EET elevates Ca2+ spark and transient BKCa channel activity; (3) increases in Ca2+ spark and transient BKCa channel activity elicited by 11,12 EET are unaffected by inhibition of VDCCs but are absent when extracellular [Ca2+] is lowered; and (4) antisense-mediated TRPV4 downregulation in isolated cerebral arteries blocks EET-induced increases in Ca2+ spark and transient BKCa channel activity as well as membrane potential hyperpolarization and vasodilation. Thus, our findings show that in cerebral artery myocytes, Ca2+ influx through TRPV4, stimulated by 11,12 EET,6 increases Ca2+ spark frequency and BKCa channel activity, resulting in cerebral myocyte hyperpolarization. Consistent with an important functional role for this pathway, suppression of TRPV4 expression in intact cerebral arteries prevents 11,12 EETeCinduced smooth muscle hyperpolarization and vasodilation.
Cytochrome P450 epoxygenase products are recognized as potent vasodilatory factors produced by the vascular endothelium.3,24 Elucidation of the intracellular signaling pathways responsible for smooth muscle cell hyperpolarization by these compounds has been the subject of considerable effort. An important role for K+ channel activation in hyperpolarizing and vasodilatory response has been demonstrated in a number of previous reports. Although evidence suggesting activation of KATP channels by EETs has been presented,25 most studies support a role for BKCa channels in EET-induced hyperpolarization. The simplest explanation for the role of this channel in vascular responses is direct activation of BKCa by EETs. Although data supporting this mechanism have been reported,26 our findings (online Figure I) are in agreement with a number of previous reports4,27,28 and demonstrate no direct effect of 11,12 EET on BKCa NPo in isolated membrane patches. These data suggest that, rather than acting directly on the channel, EETs activate intracellular signaling pathways that ultimately cause smooth muscle hyperpolarization via BKCa channels. Recently reported findings that TRPV4-dependent currents are activated by EETs6 prompted our investigation of a potential role for this channel in vascular control.
We show here that stimulation of Ca2+ influx via TRPV4 by an endothelium-derived factor (11,12 EET) can alter local Ca2+ signaling, generating membrane potential hyperpolarization and vasodilation. This is the first reported example of Ca2+ influxeCinduced smooth muscle cell membrane hyperpolarization. We propose that, similar to CICR13,14 mechanisms that amplify Ca2+ influx through VDCC to cause cardiac myocyte contraction,29 TRPV4-dependent Ca2+ signals are amplified by opening SR RyRs11 and serve to increase Ca2+ spark frequency. TRPV4-dependent Ca2+ influx could potentially increase SR Ca2+ loading, which would also result in elevated Ca2+ spark activity.12 However, 11,12 EET did not increase Ca2+ spark amplitude, arguing against increased SR Ca2+ loading. 11,12 EET could also activate Ca2+ spark activity by acting directly on RyRs. However, it is unlikely that TRPV4 downregulation would influence this response. We propose that TRPV4-dependent Ca2+ signals ultimately result in smooth muscle hyperpolarization and vasodilation through activation of Ca2+ sparks and BKCa-dependent transient membrane hyperpolarization (Figure 8). The hypothesized signaling pathway predicts close coupling between TRPV4 channels and the SR to allow local TRPV4-mediated Ca2+ influx to stimulate nearby RyRs. Although direct activation of BKCa channels by TRPV4-dependent Ca2+ influx is possible, BKCa activation by this mechanism would be sustained, rather than transient in nature. For TRPV4-mediated Ca2+ influx to directly cause increases in transient BKCa activity (STOCs), TRPV4 channels would need to permit Ca2+ entry that has the amplitude and time course of a Ca2+ spark through a RyR. There is no evidence that TRPV4 channels operate in this manner; thus, our findings are consistent with TRPV4-dependent currents modulating RyR activity. Significantly, our findings address previously unresolved issues concerning EET-induced responses in the vasculature and suggest that TRPV4 may act as an extracellular receptor for 11,12 EET in arterial smooth muscle cells.
Transient receptor potential channels are ubiquitously expressed,5 and dynamic Ca2+ release events influence the properties of a number of tissues, including sensory neurons,30 pancreatic cells,31 and cardiac myocytes,29 as well as many types of smooth muscle.32 Thus, in addition to providing novel information regarding regulation of vascular tone, our observations suggest the possibility that TRPV4 (and perhaps other TRPV channels) can alter the function of many types of excitable cells by modulating complex Ca2+ events. Furthermore, as TRPV channels are activated by a number of stimuli, such as changes in osmolarity,23 temperature (in the physiological range),33 and fatty acids,34 this mechanism may constitute a fundamental way in which environmental factors influence excitable cells through a local Ca2+ signaling mechanism involving CICR.
Acknowledgments
This work was supported by NIH grant F32HL075995 and American Heart Association Grant 0535226N (to S.E.); NIH grants RO1HL44455, RO1HL63722, RO1DK053832, and RO1DK065947 (to M.T.N.); NIH grant RO1HL58231 (to J.E.B.); and the Totman Medical Trust. The Noran confocal microscope used for these studies is housed in the Neuroscience Centers of Biomedical Research Excellence (COBRE) Imaging/Physiology core facility (NIH grant P20 RR16435 from the COBRE Program of the National Center for Research Resources) and supported by a National Science Foundation grant. We thank Katherine Lutz and Emily R. Levy for technical assistance; Adrian Bonev, PhD, for insightful comments and design of custom software for the analysis of dynamic Ca2+ events; and Stephen V. Straub, PhD, for comments on the manuscript.
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Abstract
Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4-phorbol 12,13-didecanoate (4-PDD) (5 eol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EETeCinduced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 eol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EETeCinduced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.
Key Words: Ca2+ sparks Ca2+ transients eicosanoids ion channels ryanodine receptor vascular smooth muscle vasodilation
Introduction
Diffusible factors produced by the vascular endothelium are vital for the regulation of smooth muscle membrane potential, arterial tone, blood pressure, and blood flow. Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase products of arachidonic acid,1 cause vasodilation and account for endothelium-derived hyperpolarizing factor (EDHF) activity in some vascular beds.2,3 Critical components of signaling pathways activated by these compounds are not fully understood.1 Although evidence supportive of a receptor-dependent smooth muscle hyperpolarization mechanism has been presented,4 the molecular identity of the "EET receptor" has not been reported. Vanilloid transient receptor potential (TRPV) channels are important sensors of biochemical and physiological stimuli.5 Therefore, we tested the hypothesis that TRPV channels are involved in transducing endothelial signals to underlying vascular smooth muscle. EETs promote Ca2+ influx in HEK cells overexpressing TRPV46 and increase intracellular [Ca2+] in cultured smooth muscle cells.7 In addition, activation of TRPV4 with the phorbol compound 4-phorbol 12,13-didecanoate8 (4-PDD) increases intracellular [Ca2+] in airway smooth muscle.9 An elevation in intracellular [Ca2+] is usually associated with vasoconstriction. Thus, these reports pose a paradox: How does EET-induced Ca2+ influx cause vasodilation A number of studies suggest involvement of large-conductance Ca2+-activated K+ (BKCa) channels in EET-induced smooth muscle hyperpolarization and vasodilation.1,2 Under physiological conditions, BKCa channels in smooth muscle cells are activated by elementary Ca2+-release events (Ca2+ sparks) via ryanodine receptors (RyRs) located on the sarcoplasmic reticulum (SR). Ca2+ sparks activate nearby BKCa channels to cause transient membrane hyperpolarization and vasodilation.10 Thus, EET-induced dilation could be explained by Ca2+ sparks activating BKCa channels. We further hypothesized that Ca2+ influx through TRPV4 preferentially stimulates RyRs in the SR, generating Ca2+ sparks that signal adjacent BKCa channels to open and cause membrane hyperpolarization and vasodilation.10 Consistent with this possibility, the open probability (NPo) of SR RyRs is increased by both elevated cytoplasmic [Ca2+]11 and enhanced SR Ca2+ loading,12 which augments Ca2+ spark activity in arterial smooth muscle. Thus, Ca2+ influx via TRPV4 could stimulate SR RyRs, either directly or through increased SR Ca2+ loading, or both, to generate Ca2+ sparks and enhance BKCa channel activity, thereby causing smooth muscle hyperpolarization and relaxation. We propose that TRPV4 forms a novel Ca2+ signaling complex with RyRs and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release13,14 (CICR).
Materials and Methods
Cerebral and cerebellar arteries used for these studies were isolated from male SpragueeCDawley 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. Total RNA was extracted from whole brain, isolated cerebral arteries, and freshly dispersed cerebral artery smooth muscle cells, and RT-PCR was used to determine whether TRPV4 mRNA was present in these tissues. Forward and reverse primers specific for TRPV4 were TRPV4F (5'-GGGCAGCTCCCCAAAGTAGAA-3') and TRPV4R (5'-GTGCCTGGGCCCAAGAAA-3'). These primers yield a 578-bp PCR product.
Whole-cell patch-clamp experiments were used to determine whether ion channels with biophysical properties similar to TRPV4 were present in freshly isolated cerebral artery myocytes. Currents were recorded during voltage ramps between eC120 and +80 mV before and after administration of 4-PDD or 11,12 EET and were normalized to membrane capacitance (pA/pF). Isolated smooth muscle cells were loaded with the rapid Ca2+ indicator dye fluo-4 (Molecular Probes), and the effects of 11,12 EET on Ca2+ spark frequency were evaluated using high-speed (30 to 60 frames/s) laser-scanning confocal microscopy.15,16 Cerebral myocytes were also patch clamped in the perforated-patch configuration to examine the effects of 11,12 EET on the frequency of transient macroscopic BKCa currents ("spontaneous transient outward currents" [STOCs]).
Downregulation of TRPV4 expression in isolated cerebral arteries was accomplished with antisense oligonucleotides. The sequence of the TRPV4 antisense oligonucleotide used for these studies was 5'-CATCACCAGGATCTGCCATACTG-3'17 (Operon Biotechnologies Inc). The complementary sequence was used as the sense (control) oligonucleotide. Sense and antisense oligonucleotides were introduced into intact cerebral arteries using a reversible permeabilization procedure.18 Following reversal of permeabilization, arteries were organ cultured for 2 to 3 days in DMEM/F-12 medium without serum to allow for TRPV4 downregulation. Semiquantitative RT-PCR was used to evaluate the effects of antisense treatment on TRPV4 mRNA levels. Smooth muscle cells isolated from cultured arteries were patch clamped in whole-cell or perforated-patch configuration to investigate the effects of TRPV4 antisense on 11,12 EETeCinduced cation currents and transient BKCa channel activity. In additional experiments, sense- and antisense-treated arteries were mounted in an arteriograph, endothelial cells were removed, and the effects of TRPV4 downregulation on 11,12 EETeCinduced increases in Ca2+ spark activity, smooth muscle cell hyperpolarization, and vasodilation were evaluated.
An expanded Materials and Methods section can be found in the online supplement available at http://circres.ahajournals.org.
Results
TRPV4-Like Channels Are Present in Cerebral Artery Smooth Muscle
Message encoding TRPV4 was found in rat brain, cerebral arteries, and isolated cerebral artery smooth muscle cells (Figure 1a, inset). PCR products were not detected when template cDNA was omitted from the PCR (Figure 1a) or when reverse-transcription reactions were performed in the absence of reverse transcriptase (not shown). Because a number of transient receptor potential channels, including TRPC1,19 TRPC3,19eC21 TRPC4,19 TRPC6,19,21 and TRPM4,22 are also present in cerebral artery myocytes, conventional whole-cell patch-clamp experiments were performed to determine whether ion channels with biophysical properties similar to those of cloned TRPV4 channels were present in these cells. The TRPV4 agonist 4-PDD (5 eol/L) activated an outwardly rectifying current in these cells (Figure 1a and 1b). 11,12 EET (300 nmol/L) also rapidly (1 minute) evoked a current with similar currenteCvoltage characteristics (Figure 1c). Activation of whole-cell currents by 11,12 EET (300 nmol/L) was blocked by prior administration of the TRPV channel inhibitor ruthenium red (RR) (1 eol/L) (Figure 1d and 1e). The rapid activation and inactivation of 11,12 EETeCevoked currents in cerebral myocytes (Figure 1f) was similar to the reported time course of EETeCinduced Ca2+ influx and whole-cell currents in cultured cells overexpressing TRPV4.6 Activation of inward, but not outward, currents by 11,12 EET (300 nmol/L) was absent when N-methyl-D-glucamine (NMDG) was substituted for cations in the extracellular solution (Figure 1g and 1h). Thus, properties of this current in cerebral myocytes, such as currenteCvoltage relationships, activation by 4-PDD and 11,12 EET, and block by RR, are consistent with previously described characteristics of cloned TRPV4 channels6,8,23 and, combined with expression of TRPV4 mRNA in these cells, strongly suggests that functional TRPV4 channels are present in cerebral artery smooth muscle.
EETs Increase Ca2+ Spark and Transient BKCa Activity in Cerebral Myocytes
EETs, which activate TRPV4 channels,6 have also been shown to induce membrane potential hyperpolarization and relaxation of arteries, which is prevented by blocking BKCa channels.2 We, therefore, examined the effects of EETs on intracellular Ca2+ dynamics in cerebral artery smooth muscle cells loaded with the rapid Ca2+ indicator dye fluo-4. 11,12 EET (300 nmol/L) increased Ca2+ spark frequency nearly 2-fold in freshly isolated cerebral myocytes (Figure 2a and 2b). Previous reports demonstrate that Ca2+ sparks stimulate nearby sarcolemmal BKCa channels to cause transient K+ currents (STOCs).10 11,12 EET (100 nmol/L) increased the frequency of STOCs in voltage-clamped (0 mV) arterial smooth muscle cells (Figure 2c and 2d). 11,12 EET (300 nmol/L) also significantly (P<0.05) increased STOC frequency (0.31±0.01 versus 0.73±0.14 Hz; n=3) for cells voltage clamped at eC40 mV, a membrane potential in the physiological range for smooth muscle cells in pressurized cerebral arteries. STOC activity was not altered by the vehicle for 11,12 EET (DMSO, 0.1%) (Figure 2d) or by the metabolic precursor of 11,12 EET, arachidonic acid (1 eol/L; 101.4±27% of control; n=5). STOC activity in the presence of 11,12 EET was greatly diminished by ryanodine (5 eol/L; 0.55±0.08 versus 0.12±0.07 Hz; n=3) and the specific BKCa channel blocker iberiotoxin (100 nmol/L; 0.33±0.11 versus 0.05±0.02 Hz; n=3). In addition, 11,12 EET (300 nmol/L) did not increase the open probability (NPo) of BKCa channels in membrane patches obtained from cerebral artery myocytes (online Figure I). These findings strongly support the concept that EETs signal RyRs to increase Ca2+ sparks, which in turn activate BKCa channels.
EETeCInduced Increases in Ca2+ Sparks and Transient BKCa Activity Requires Ca2+ Influx
TRPV4 channels are Ca2+ permeable,23 consistent with the possibility that Ca2+ entry through this channel could trigger Ca2+ sparks via CICR. We, therefore, examined the effects of reduced extracellular Ca2+ on EET-induced increases in Ca2+ spark activity in arterial myocytes. In the presence of an L-type voltage-dependent Ca2+ channel (VDCC) blocker (30 eol/L diltiazem), 11,12 EET (300 nmol/L) significantly (P<0.05) elevated Ca2+ spark frequency when external [Ca2+] was maintained at physiological levels (2 mmol/L) (Figure 3a). In contrast, when extracellular [Ca2+] was reduced to 10 eol/L in the presence of diltiazem, Ca2+ spark frequency did not differ between control and 11,12 EETeCtreated cells (Figure 3a). 11,12 EET (300 nmol/L) also increased STOC frequency when external [Ca2+] was 2 mmol/L and VDCCs were blocked (1 eol/L nisoldipine), whereas when bath [Ca2+] was reduced to 10 eol/L while VDCC inhibition was maintained, STOC activity was not altered by 11,12 EET administration (Figure 3b and 3c). These findings demonstrate that EET-induced increases in Ca2+ spark and STOC frequency are dependent on Ca2+ entry via a non-VDCC channel.
EETeCInduced Increases in Transient BKCa Activity Are TRPV4 Dependent
TRPV4 channels are activated by EETs,6 consistent with the possibility that these channels could mediate EET-induced hyperpolarization in cerebral artery smooth muscle cells. The TRPV antagonist RR (1 eol/L), applied externally, blocked 11,12 EETeCinduced (300 nmol/L) increases in STOC frequency without altering basal STOC activity (Figure 4a and 4b). In addition, RR (1 eol/L) reversed 11,12 EETeCinduced increases in STOC frequency to control levels (Figure 4c and 4d). The TRPV4 agonist 4-PDD (5 eol/L) also elevated STOC frequency in cerebral myocytes, and this response was inhibited by RR (1 eol/L) (Figure 4e and 4f).
To examine further the relationship between EET-induced, TRPV4-mediated Ca2+ influx and smooth muscle hyperpolarization, TRPV4 expression was suppressed in intact vessels using antisense oligonucleotides. Cerebral arteries were permeabilized,18 were exposed to TRPV4 sense or antisense oligonucleotides and, following reversal of permeabilization, were organ cultured for 2 to 3 days to allow TRPV4 downregulation.21,22 Semiquantitative RT-PCR was used to examine the efficacy of these procedures, and TRPV4 mRNA levels were found to be diminished in antisense- versus sense-treated arteries (Figure 5a, inset).
11,12 EET (300 nmol/L) activated an outwardly rectifying current in smooth muscle cells from sense-treated vessels that had a similar currenteCvoltage relationship, current density, time course, and reversal potential as 11,12 EETeC and 4-PDDeCinduced currents in cerebral myocytes from freshly isolated arteries (Figure 5a and 5e). However, 11,12 EETeCactivated currents were significantly reduced in smooth muscle cells from antisense-treated vessels (Figure 5b through 5d).
To further elucidate the consequences of TRPV4 activation, the effects of 11,12 EET on Ca2+ sparks were measured in isolated, pressurized cerebral arteries loaded with the Ca2+ indicator dye fluo-4. 11,12 EET (300 nmol/L) increased Ca2+ spark frequency in pressurized (60 mm Hg) sense-treated cerebral arteries (Figure 6a and 6b), whereas Ca2+ spark frequency in antisense-treated vessels was not altered by 11,12 EET administration (Figure 6a and 6b). Consistent with these observations, 11,12 EET (300 nmol/L) elevated STOC frequency in cerebral myocytes isolated from sense-treated, but not antisense-treated, arteries (Figure 6c and 6d). These findings demonstrate that TRPV4 is a critical mediator of 11,12 EETeCinduced increases in Ca2+ sparkeCactivated transient BKCa currents in cerebral artery smooth muscle.
TRPV4 Mediates EETeCInduced Smooth Muscle Hyperpolarization and Vasodilation
An elevation of Ca2+ spark frequency can dilate cerebral arteries through activation of BKCa channels. Therefore, the relationships between TRPV4 downregulation, EET-induced changes in Ca2+ spark frequency, and alterations in smooth muscle membrane potential and vasomotor responses were examined. 11,12 EET (300 nmol/L) caused an approximately 10 mV smooth muscle membrane potential hyperpolarization in pressurized (60 mm Hg), TRPV4 sense-treated arteries (Figure 7a and 7b). In contrast, membrane potential of cerebral myocytes in antisense-treated vessels was not significantly altered by 11,12 EET (Figure 7a and 7b). TRPV4 sense- and antisense-treated vessels constricted to the same extent in response to a depolarizing concentration of KCl (60 mmol/L) or pressure (60 mm Hg), suggesting that membrane depolarizationeCinduced Ca2+ influx through VDCCs was unaffected. Vasodilation in response to the KATP channel agonist pinacidil (10 eol/L) also did not differ between groups (sense, 83.3±3.7% of passive diameter; antisense, 86.7±5.5%; n=3 for each group). In contrast, 11,12 EETeCinduced vasodilator responses were significantly inhibited by TRPV4 downregulation (Figure 7c and 7d), whereas TRPV4 sense-treated arteries dilated to an extent similar to that of freshly isolated arteries (Figure 7c and 7d). In the presence of ryanodine (10 eol/L), freshly isolated cerebral arteries exhibited only slight, statistically insignificant dilation (6.9±2.5 e; n=3) in response to 11,12 EET (300 nmol/L), providing further evidence for a Ca2+ sparkeCdependent mechanism in this response. These findings demonstrate a role for TRPV4 and RyRs in EET-induced smooth muscle hyperpolarization and vasodilation.
Discussion
The major findings of this study are as follows: (1) functional TRPV4 ion channels are present in cerebral artery smooth muscle; (2) activation of TRPV4 in cerebral myocytes with 11,12 EET elevates Ca2+ spark and transient BKCa channel activity; (3) increases in Ca2+ spark and transient BKCa channel activity elicited by 11,12 EET are unaffected by inhibition of VDCCs but are absent when extracellular [Ca2+] is lowered; and (4) antisense-mediated TRPV4 downregulation in isolated cerebral arteries blocks EET-induced increases in Ca2+ spark and transient BKCa channel activity as well as membrane potential hyperpolarization and vasodilation. Thus, our findings show that in cerebral artery myocytes, Ca2+ influx through TRPV4, stimulated by 11,12 EET,6 increases Ca2+ spark frequency and BKCa channel activity, resulting in cerebral myocyte hyperpolarization. Consistent with an important functional role for this pathway, suppression of TRPV4 expression in intact cerebral arteries prevents 11,12 EETeCinduced smooth muscle hyperpolarization and vasodilation.
Cytochrome P450 epoxygenase products are recognized as potent vasodilatory factors produced by the vascular endothelium.3,24 Elucidation of the intracellular signaling pathways responsible for smooth muscle cell hyperpolarization by these compounds has been the subject of considerable effort. An important role for K+ channel activation in hyperpolarizing and vasodilatory response has been demonstrated in a number of previous reports. Although evidence suggesting activation of KATP channels by EETs has been presented,25 most studies support a role for BKCa channels in EET-induced hyperpolarization. The simplest explanation for the role of this channel in vascular responses is direct activation of BKCa by EETs. Although data supporting this mechanism have been reported,26 our findings (online Figure I) are in agreement with a number of previous reports4,27,28 and demonstrate no direct effect of 11,12 EET on BKCa NPo in isolated membrane patches. These data suggest that, rather than acting directly on the channel, EETs activate intracellular signaling pathways that ultimately cause smooth muscle hyperpolarization via BKCa channels. Recently reported findings that TRPV4-dependent currents are activated by EETs6 prompted our investigation of a potential role for this channel in vascular control.
We show here that stimulation of Ca2+ influx via TRPV4 by an endothelium-derived factor (11,12 EET) can alter local Ca2+ signaling, generating membrane potential hyperpolarization and vasodilation. This is the first reported example of Ca2+ influxeCinduced smooth muscle cell membrane hyperpolarization. We propose that, similar to CICR13,14 mechanisms that amplify Ca2+ influx through VDCC to cause cardiac myocyte contraction,29 TRPV4-dependent Ca2+ signals are amplified by opening SR RyRs11 and serve to increase Ca2+ spark frequency. TRPV4-dependent Ca2+ influx could potentially increase SR Ca2+ loading, which would also result in elevated Ca2+ spark activity.12 However, 11,12 EET did not increase Ca2+ spark amplitude, arguing against increased SR Ca2+ loading. 11,12 EET could also activate Ca2+ spark activity by acting directly on RyRs. However, it is unlikely that TRPV4 downregulation would influence this response. We propose that TRPV4-dependent Ca2+ signals ultimately result in smooth muscle hyperpolarization and vasodilation through activation of Ca2+ sparks and BKCa-dependent transient membrane hyperpolarization (Figure 8). The hypothesized signaling pathway predicts close coupling between TRPV4 channels and the SR to allow local TRPV4-mediated Ca2+ influx to stimulate nearby RyRs. Although direct activation of BKCa channels by TRPV4-dependent Ca2+ influx is possible, BKCa activation by this mechanism would be sustained, rather than transient in nature. For TRPV4-mediated Ca2+ influx to directly cause increases in transient BKCa activity (STOCs), TRPV4 channels would need to permit Ca2+ entry that has the amplitude and time course of a Ca2+ spark through a RyR. There is no evidence that TRPV4 channels operate in this manner; thus, our findings are consistent with TRPV4-dependent currents modulating RyR activity. Significantly, our findings address previously unresolved issues concerning EET-induced responses in the vasculature and suggest that TRPV4 may act as an extracellular receptor for 11,12 EET in arterial smooth muscle cells.
Transient receptor potential channels are ubiquitously expressed,5 and dynamic Ca2+ release events influence the properties of a number of tissues, including sensory neurons,30 pancreatic cells,31 and cardiac myocytes,29 as well as many types of smooth muscle.32 Thus, in addition to providing novel information regarding regulation of vascular tone, our observations suggest the possibility that TRPV4 (and perhaps other TRPV channels) can alter the function of many types of excitable cells by modulating complex Ca2+ events. Furthermore, as TRPV channels are activated by a number of stimuli, such as changes in osmolarity,23 temperature (in the physiological range),33 and fatty acids,34 this mechanism may constitute a fundamental way in which environmental factors influence excitable cells through a local Ca2+ signaling mechanism involving CICR.
Acknowledgments
This work was supported by NIH grant F32HL075995 and American Heart Association Grant 0535226N (to S.E.); NIH grants RO1HL44455, RO1HL63722, RO1DK053832, and RO1DK065947 (to M.T.N.); NIH grant RO1HL58231 (to J.E.B.); and the Totman Medical Trust. The Noran confocal microscope used for these studies is housed in the Neuroscience Centers of Biomedical Research Excellence (COBRE) Imaging/Physiology core facility (NIH grant P20 RR16435 from the COBRE Program of the National Center for Research Resources) and supported by a National Science Foundation grant. We thank Katherine Lutz and Emily R. Levy for technical assistance; Adrian Bonev, PhD, for insightful comments and design of custom software for the analysis of dynamic Ca2+ events; and Stephen V. Straub, PhD, for comments on the manuscript.
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