TRPV4 Forms a Novel Ca2+ Signaling Complex With Ryanodine Receptors and BKCa Channels

TRPV4 Forms a Novel Ca2 Signaling Complex WithRyanodine Receptors and BKCa ChannelsScott Earley, Thomas J. Heppner, Mark T. Nelson, Joseph E. Brayden

Abstract—Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure andflow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloidtransient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth musclecells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currentswith properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4-phorbol12,13-didecanoate (4-PDD) (5 mol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeico-satrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12EET increased the frequency of unitary Ca2 release events (Ca2 sparks) via ryanodine receptors located on thesarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2 sparks activated nearby sarcolemmallarge-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 EET–induced increases in Ca2 spark andSTOC frequency were inhibited by lowering external Ca2 from 2 mmol/L to 10 mol/L but not by voltage-dependentCa2 channel inhibitors, suggesting that these responses require extracellular Ca2 influx via channels other thanvoltage-dependent Ca2 channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteriesprevented 11,12 EET–induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 formsa novel Ca2 signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolar-ization and arterial dilation via Ca2-induced Ca2 release in response to an endothelial-derived factor. (Circ Res.2005;97:1270-1279.)

Key Words: Ca2 sparks Ca2 transients eicosanoids ion channels ryanodine receptor vascular smooth muscle vasodilation

Diffusible factors produced by the vascular endotheliumare vital for the regulation of smooth muscle membrane

potential, arterial tone, blood pressure, and blood flow.Epoxyeicosatrienoic acids (EETs), the cytochrome P450epoxygenase products of arachidonic acid,1 cause vasodila-tion and account for endothelium-derived hyperpolarizingfactor (EDHF) activity in some vascular beds.2,3 Criticalcomponents of signaling pathways activated by these com-pounds are not fully understood.1 Although evidence support-ive of a receptor-dependent smooth muscle hyperpolarizationmechanism has been presented,4 the molecular identity of the“EET receptor” has not been reported. Vanilloid transientreceptor potential (TRPV) channels are important sensors ofbiochemical and physiological stimuli.5 Therefore, we testedthe hypothesis that TRPV channels are involved in transduc-ing endothelial signals to underlying vascular smooth muscle.EETs promote Ca2 influx in HEK cells overexpressingTRPV46 and increase intracellular [Ca2] in cultured smoothmuscle cells.7 In addition, activation of TRPV4 with thephorbol compound 4-phorbol 12,13-didecanoate8 (4-PDD)

increases intracellular [Ca2] in airway smooth muscle.9 Anelevation in intracellular [Ca2] is usually associated withvasoconstriction. Thus, these reports pose a paradox: Howdoes EET-induced Ca2 influx cause vasodilation? A numberof studies suggest involvement of large-conductance Ca2-ac-tivated K (BKCa) channels in EET-induced smooth musclehyperpolarization and vasodilation.1,2 Under physiologicalconditions, BKCa channels in smooth muscle cells are acti-vated by elementary Ca2-release events (Ca2 sparks) viaryanodine receptors (RyRs) located on the sarcoplasmicreticulum (SR). Ca2 sparks activate nearby BKCa channels tocause transient membrane hyperpolarization and vasodila-tion.10 Thus, EET-induced dilation could be explained byCa2 sparks activating BKCa channels. We further hypothe-sized that Ca2 influx through TRPV4 preferentially stimu-lates RyRs in the SR, generating Ca2 sparks that signaladjacent BKCa channels to open and cause membrane hyper-polarization and vasodilation.10 Consistent with this possibil-ity, the open probability (NPo) of SR RyRs is increased byboth elevated cytoplasmic [Ca2]11 and enhanced SR Ca2

Original received August 2, 2005; revision received September 21, 2005; accepted October 20, 2005.From the Department of Pharmacology, University of Vermont College of Medicine, Burlington.Correspondence to Scott Earley, PhD, Department of Pharmacology, University of Vermont College of Medicine, 89 Beaumont Ave, Burlington, VT

05405. E-mail [email protected]© 2005 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000194321.60300.d6

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loading,12 which augments Ca2 spark activity in arterialsmooth muscle. Thus, Ca2 influx via TRPV4 could stimulateSR RyRs, either directly or through increased SR Ca2

loading, or both, to generate Ca2 sparks and enhance BKCa

channel activity, thereby causing smooth muscle hyperpolar-ization and relaxation. We propose that TRPV4 forms a novelCa2 signaling complex with RyRs and BKCa channels thatelicits smooth muscle hyperpolarization and arterial dilationvia Ca2-induced Ca2 release13,14 (CICR).

Materials and MethodsCerebral and cerebellar arteries used for these studies were isolatedfrom male Sprague–Dawley rats (250 to 350 g; Charles RiverLaboratories, St Constant, Quebec, Canada). All animal use proce-dures were in accordance with institutional guidelines and approvedby the Institutional Animal Care and Use Committee of the Univer-sity of Vermont. Total RNA was extracted from whole brain, isolatedcerebral arteries, and freshly dispersed cerebral artery smooth musclecells, and RT-PCR was used to determine whether TRPV4 mRNAwas present in these tissues. Forward and reverse primers specific forTRPV4 were TRPV4F (5-GGGCAGCTCCCCAAAGTAGAA-3)and TRPV4R (5-GTGCCTGGGCCCAAGAAA-3). These primersyield a 578-bp PCR product.

Whole-cell patch-clamp experiments were used to determinewhether ion channels with biophysical properties similar to TRPV4were present in freshly isolated cerebral artery myocytes. Currentswere recorded during voltage ramps between 120 and 80 mVbefore and after administration of 4-PDD or 11,12 EET and werenormalized to membrane capacitance (pA/pF). Isolated smoothmuscle cells were loaded with the rapid Ca2 indicator dye fluo-4(Molecular Probes), and the effects of 11,12 EET on Ca2 sparkfrequency were evaluated using high-speed (30 to 60 frames/s)laser-scanning confocal microscopy.15,16 Cerebral myocytes werealso patch clamped in the perforated-patch configuration to examinethe effects of 11,12 EET on the frequency of transient macroscopicBKCa currents (“spontaneous transient outward currents” [STOCs]).

Downregulation of TRPV4 expression in isolated cerebral arterieswas accomplished with antisense oligonucleotides. The sequence ofthe TRPV4 antisense oligonucleotide used for these studies was5-CATCACCAGGATCTGCCATACTG-317 (Operon Biotechnol-ogies Inc). The complementary sequence was used as the sense(control) oligonucleotide. Sense and antisense oligonucleotides wereintroduced into intact cerebral arteries using a reversible permeabi-lization procedure.18 Following reversal of permeabilization, arterieswere organ cultured for 2 to 3 days in DMEM/F-12 medium withoutserum to allow for TRPV4 downregulation. Semiquantitative RT-PCR was used to evaluate the effects of antisense treatment onTRPV4 mRNA levels. Smooth muscle cells isolated from culturedarteries were patch clamped in whole-cell or perforated-patch con-

Figure 1. Functional TRPV4 channels arepresent in rat cerebral artery smooth mus-cle. a, Conventional whole-cell patch-clamp recording showing activation of anoutwardly rectifying current by 4-PDD(5 mol/L) in freshly isolated cerebralartery smooth muscle cells. Inset, RT-PCRdemonstrating that mRNA encodingTRPV4 is present in rat brain, whole cere-bral arteries (CA), and isolated cerebralartery smooth muscle cells (SMC). NT indi-cates no template control. b, Summarydata for 4-PDD–induced (difference) cur-rents (n3). c, Whole-cell current activatedby 11,12 EET (300 nmol/L) in cerebralartery smooth muscle cells. d, Whole-cellrecording showing the effects of the TRPVinhibitor RR (1 mol/L) on 11,12 EET–in-duced currents. Currents are shown underbasal conditions (gray), following RRadministration (red), and following 11,12EET (300 nmol/L) administration (black). e,Summary data for 11,12 EET–induced (dif-ference) currents at 80 and 80 mV rec-orded under control conditions or in thepresence of RR. *P0.05 vs control (80mV), #P0.05 vs control (80 mV). n6for control cells; n4 for RR-treated cells.f, Time course of outward whole-cell cur-rent activated by 11,12 EET under controlconditions or in the presence of RR. g,Whole-cell recording of 11,12 EET–in-duced currents with NMDG substituted forcations in the bathing solution. h, Sum-mary data for 11,12 EET–induced (differ-ence) currents in NMDG bathing solution(n3). All data are meanSE.

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figuration to investigate the effects of TRPV4 antisense on 11,12EET–induced cation currents and transient BKCa channel activity. Inadditional experiments, sense- and antisense-treated arteries weremounted in an arteriograph, endothelial cells were removed, and theeffects of TRPV4 downregulation on 11,12 EET–induced increasesin Ca2 spark activity, smooth muscle cell hyperpolarization, andvasodilation were evaluated.

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

ResultsTRPV4-Like Channels Are Present in CerebralArtery Smooth MuscleMessage encoding TRPV4 was found in rat brain, cerebralarteries, and isolated cerebral artery smooth muscle cells(Figure 1a, inset). PCR products were not detected whentemplate cDNA was omitted from the PCR (Figure 1a) orwhen reverse-transcription reactions were performed in theabsence of reverse transcriptase (not shown). Because anumber of transient receptor potential channels, includingTRPC1,19 TRPC3,19–21 TRPC4,19 TRPC6,19,21 and TRPM4,22

are also present in cerebral artery myocytes, conventionalwhole-cell patch-clamp experiments were performed to de-termine whether ion channels with biophysical propertiessimilar to those of cloned TRPV4 channels were present inthese cells. The TRPV4 agonist 4-PDD (5 mol/L) acti-vated an outwardly rectifying current in these cells (Figure 1aand 1b). 11,12 EET (300 nmol/L) also rapidly (1 minute)evoked a current with similar current–voltage characteristics(Figure 1c). Activation of whole-cell currents by 11,12 EET(300 nmol/L) was blocked by prior administration of theTRPV channel inhibitor ruthenium red (RR) (1 mol/L)(Figure 1d and 1e). The rapid activation and inactivation of11,12 EET–evoked currents in cerebral myocytes (Figure 1f)was similar to the reported time course of EET–induced Ca2

influx and whole-cell currents in cultured cells overexpress-ing 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 extra-cellular solution (Figure 1g and 1h). Thus, properties of thiscurrent in cerebral myocytes, such as current–voltage rela-tionships, activation by 4-PDD and 11,12 EET, and blockby RR, are consistent with previously described characteris-tics of cloned TRPV4 channels6,8,23 and, combined withexpression of TRPV4 mRNA in these cells, strongly suggeststhat functional TRPV4 channels are present in cerebral arterysmooth muscle.

EETs Increase Ca2 Spark and Transient BKCa

Activity in Cerebral MyocytesEETs, which activate TRPV4 channels,6 have also beenshown to induce membrane potential hyperpolarization andrelaxation of arteries, which is prevented by blocking BKCa

channels.2 We, therefore, examined the effects of EETs onintracellular Ca2 dynamics in cerebral artery smooth musclecells loaded with the rapid Ca2 indicator dye fluo-4. 11,12EET (300 nmol/L) increased Ca2 spark frequency nearly2-fold in freshly isolated cerebral myocytes (Figure 2a and2b). Previous reports demonstrate that Ca2 sparks stimulatenearby sarcolemmal BKCa channels to cause transient K

currents (STOCs).10 11,12 EET (100 nmol/L) increased thefrequency of STOCs in voltage-clamped (0 mV) arterialsmooth muscle cells (Figure 2c and 2d). 11,12 EET (300nmol/L) also significantly (P0.05) increased STOC fre-quency (0.310.01 versus 0.730.14 Hz; n3) for cellsvoltage clamped at 40 mV, a membrane potential in thephysiological range for smooth muscle cells in pressurizedcerebral arteries. STOC activity was not altered by the vehiclefor 11,12 EET (DMSO, 0.1%) (Figure 2d) or by the metabolicprecursor of 11,12 EET, arachidonic acid (1 mol/L;101.427% of control; n5). STOC activity in the presenceof 11,12 EET was greatly diminished by ryanodine(5 mol/L; 0.550.08 versus 0.120.07 Hz; n3) and the

Figure 2. 11,12 EET–induced increasesin Ca2 spark frequency and STOC fre-quency in cerebral myocytes. a, Frac-tional change in [Ca2] (F/Fo) for controland 11,12 EET–treated cells. Inset,Pseudocolor image of a Ca2 spark in acerebral artery myocyte. b, Effects of11,12 EET on Ca2 spark frequency.*P0.05 vs control. n16 for control;n13 for 11,12 EET. Spark amplitudesdid not differ. c, Perforated patch–clamprecording showing the effects of 11,12EET (100 nmol/L) on STOC frequency incerebral artery smooth muscle cells. d,Effects of 11,12 EET (100 nmol/L) onSTOC frequency. *P0.05 vs all othergroups. n9 for all groups.

1272 Circulation Research December 9/23, 2005



specific BKCa channel blocker iberiotoxin (100 nmol/L;0.330.11 versus 0.050.02 Hz; n3). In addition, 11,12EET (300 nmol/L) did not increase the open probability (NPo)of BKCa channels in membrane patches obtained from cere-bral artery myocytes (online Figure I). These findingsstrongly support the concept that EETs signal RyRs toincrease Ca2 sparks, which in turn activate BKCa channels.

EET–Induced Increases in Ca2 Sparks andTransient BKCa Activity Requires Ca2 InfluxTRPV4 channels are Ca2 permeable,23 consistent with thepossibility that Ca2 entry through this channel could triggerCa2 sparks via CICR. We, therefore, examined the effects ofreduced extracellular Ca2 on EET-induced increases in Ca2

spark activity in arterial myocytes. In the presence of anL-type voltage-dependent Ca2 channel (VDCC) blocker(30 mol/L diltiazem), 11,12 EET (300 nmol/L) significantly(P0.05) elevated Ca2 spark frequency when external[Ca2] was maintained at physiological levels (2 mmol/L)(Figure 3a). In contrast, when extracellular [Ca2] was re-duced to 10 mol/L in the presence of diltiazem, Ca2 sparkfrequency did not differ between control and 11,12 EET–treated cells (Figure 3a). 11,12 EET (300 nmol/L) also

increased STOC frequency when external [Ca2] was2 mmol/L and VDCCs were blocked (1 mol/L nisoldipine),whereas when bath [Ca2] was reduced to 10 mol/L whileVDCC inhibition was maintained, STOC activity was notaltered by 11,12 EET administration (Figure 3b and 3c).These findings demonstrate that EET-induced increases inCa2 spark and STOC frequency are dependent on Ca2 entryvia a non-VDCC channel.

EET–Induced Increases in Transient BKCa ActivityAre TRPV4 DependentTRPV4 channels are activated by EETs,6 consistent with thepossibility that these channels could mediate EET-inducedhyperpolarization in cerebral artery smooth muscle cells. TheTRPV antagonist RR (1 mol/L), applied externally, blocked11,12 EET–induced (300 nmol/L) increases in STOC fre-quency without altering basal STOC activity (Figure 4a and4b). In addition, RR (1 mol/L) reversed 11,12 EET–inducedincreases in STOC frequency to control levels (Figure 4c and4d). The TRPV4 agonist 4-PDD (5 mol/L) also elevatedSTOC frequency in cerebral myocytes, and this response wasinhibited by RR (1 mol/L) (Figure 4e and 4f).

To examine further the relationship between EET-induced,TRPV4-mediated Ca2 influx and smooth muscle hyperpo-

Figure 3. EET-induced increases in Ca2 spark and STOC frequency require extracellular Ca2. a, Effect of normal and reduced extra-cellular [Ca2] on 11,12 EET–induced (300 nmol/L) increases in Ca2 spark frequency in the presence of the VDCC blocker diltiazem(30 mol/L).*P0.05 vs all other groups. n8 to 16. b, Perforated patch–clamp recording showing the effects of normal and reducedexternal [Ca2] on 11,12 EET–induced increases in STOC frequency in the presence of the VDCC blocker nisoldipine (1 mol/L). c,Effect of normal and reduced extracellular [Ca2] on 11,12 EET–induced (300 nmol/L) increases in STOC frequency during VDCCinhibition.*P0.05 vs all other groups, #P0.05 vs 11,12 EET–treated cells in normal external [Ca2] solution. n5 for all groups. Dataare meanSE.




larization, TRPV4 expression was suppressed in intact ves-sels using antisense oligonucleotides. Cerebral arteries werepermeabilized,18 were exposed to TRPV4 sense or antisenseoligonucleotides and, following reversal of permeabilization,were organ cultured for 2 to 3 days to allow TRPV4downregulation.21,22 Semiquantitative RT-PCR was used to

examine the efficacy of these procedures, and TRPV4 mRNAlevels were found to be diminished in antisense- versussense-treated arteries (Figure 5a, inset).

11,12 EET (300 nmol/L) activated an outwardly rectifyingcurrent in smooth muscle cells from sense-treated vessels thathad a similar current–voltage relationship, current density,

Figure 4. The TRPV antagonist RR blocks 11,12 EET– and 4-PDD–induced increases in STOC frequency. a, Example patch-clamprecording demonstrating that RR (1 mol/L) blocks 11,12 EET–induced increases in STOC frequency. b, Summary data (n4 for eachgroup). There were no significant differences. c, Example patch-clamp recording demonstrating that RR (1 mol/L) reverses 11,12 EET-induced (300 nmol/L) increases in STOC frequency. d, Summary data (n5 for each group). *P0.05 vs all other groups. e, Examplepatch-clamp recording demonstrating that the TRPV4 agonist 4-PDD (5 mol/L) increases STOC frequency. This response wasblocked by RR (1 mol/L). f, Summary data (n4 to 5 for each group). *P0.05 vs all other groups. Data are meanSE.




time course, and reversal potential as 11,12 EET– and4-PDD–induced currents in cerebral myocytes from freshlyisolated arteries (Figure 5a and 5e). However, 11,12 EET–activated currents were significantly reduced in smooth mus-cle cells from antisense-treated vessels (Figure 5b through5d).

To further elucidate the consequences of TRPV4 activa-tion, 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-treatedcerebral arteries (Figure 6a and 6b), whereas Ca2 sparkfrequency in antisense-treated vessels was not altered by11,12 EET administration (Figure 6a and 6b). Consistent withthese observations, 11,12 EET (300 nmol/L) elevated STOCfrequency in cerebral myocytes isolated from sense-treated,

Figure 5. TRPV4 downregulation attenuates 11,12 EET–activated whole-cell currents. a, Whole-cell currents activated by 11,12 EET(300 nmol/L) in smooth muscle cells isolated from TRPV4 sense-treated cerebral arteries. Inset, Semiquantitative RT-PCR demonstrat-ing the effects of antisense treatment on TRPV4 expression in cerebral arteries. AS indicates antisense; NT, no template control. b,Whole-cell currents activated by 11,12 EET (300 nmol/L) in smooth muscle cells isolated from TRPV4 antisense-treated cerebral arter-ies. c, Difference currents before and after 11,12 EET administration for smooth muscle cells from TRPV4 sense and antisense-treatedarteries. d, Summary data showing the effect of TRPV4 downregulation on 11,12 EET–induced (difference) currents at 80 and 80mV. *P0.05 vs sense-treated arteries (80 mV), # P0.05 vs sense-treated arteries (80 mV). n5 for both groups. e, Time course of11,12 EET–induced current activation in TRPV4 sense- and antisense-treated arteries.




but not antisense-treated, arteries (Figure 6c and 6d). Thesefindings demonstrate that TRPV4 is a critical mediator of11,12 EET–induced increases in Ca2 spark–activated tran-sient BKCa currents in cerebral artery smooth muscle.

TRPV4 Mediates EET–Induced Smooth MuscleHyperpolarization and VasodilationAn elevation of Ca2 spark frequency can dilate cerebralarteries through activation of BKCa channels. Therefore, therelationships between TRPV4 downregulation, EET-inducedchanges in Ca2 spark frequency, and alterations in smoothmuscle membrane potential and vasomotor responses wereexamined. 11,12 EET (300 nmol/L) caused an approximately10 mV smooth muscle membrane potential hyperpolarizationin pressurized (60 mm Hg), TRPV4 sense-treated arteries(Figure 7a and 7b). In contrast, membrane potential ofcerebral myocytes in antisense-treated vessels was not signif-icantly altered by 11,12 EET (Figure 7a and 7b). TRPV4sense- and antisense-treated vessels constricted to the sameextent in response to a depolarizing concentration of KCl(60 mmol/L) or pressure (60 mm Hg), suggesting that mem-brane depolarization–induced Ca2 influx through VDCCs

was unaffected. Vasodilation in response to the KATP channelagonist pinacidil (10 mol/L) also did not differ betweengroups (sense, 83.33.7% of passive diameter; antisense,86.75.5%; n3 for each group). In contrast, 11,12 EET–induced vasodilator responses were significantly inhibited byTRPV4 downregulation (Figure 7c and 7d), whereas TRPV4sense-treated arteries dilated to an extent similar to that offreshly isolated arteries (Figure 7c and 7d). In the presence ofryanodine (10 mol/L), freshly isolated cerebral arteriesexhibited only slight, statistically insignificant dilation(6.92.5 m; n3) in response to 11,12 EET (300 nmol/L),providing further evidence for a Ca2 spark–dependent mech-anism in this response. These findings demonstrate a role forTRPV4 and RyRs in EET-induced smooth muscle hyperpo-larization and vasodilation.

DiscussionThe major findings of this study are as follows: (1) functionalTRPV4 ion channels are present in cerebral artery smoothmuscle; (2) activation of TRPV4 in cerebral myocytes with11,12 EET elevates Ca2 spark and transient BKCa channel

Figure 6. TRPV4 downregulation blocks 11,12 EET–induced increases in Ca2 spark and STOC frequency. a, Fractional change in[Ca2] (F/Fo) before and after administration of 11,12 EET (300 nmol/L) for TRPV4 sense- and antisense-treated vessels. b, Effect of11,12 EET (300 nmol/L) on Ca2 spark frequency in TRPV4 sense and antisense-treated arteries. Data are Ca2 sparks per spark sitebefore and after 11,12 EET administration. *P0.05 vs sense control. n9 (sense) or n6 (antisense). c, Perforated patch–clamp re-cordings showing the effects of 11,12 EET (300 nmol/L) on STOCs in smooth muscle cells isolated from TRPV4 sense- and antisense-treated arteries. d, Effect of 11,12 EET (300 nmol/L) on STOC frequency for TRPV4 sense- and antisense-treated arteries. *P0.05 vscontrol sense-treated arteries. n5 for both groups. Data are meanSE.




activity; (3) increases in Ca2 spark and transient BKCa

channel activity elicited by 11,12 EET are unaffected byinhibition of VDCCs but are absent when extracellular [Ca2]is lowered; and (4) antisense-mediated TRPV4 downregula-tion in isolated cerebral arteries blocks EET-induced in-creases in Ca2 spark and transient BKCa channel activity aswell as membrane potential hyperpolarization and vasodila-tion. Thus, our findings show that in cerebral artery myo-cytes, Ca2 influx through TRPV4, stimulated by 11,12 EET,6

increases Ca2 spark frequency and BKCa channel activity,resulting in cerebral myocyte hyperpolarization. Consistentwith an important functional role for this pathway, suppres-sion of TRPV4 expression in intact cerebral arteries prevents11,12 EET–induced smooth muscle hyperpolarization andvasodilation.

Cytochrome P450 epoxygenase products are recognized aspotent vasodilatory factors produced by the vascular endo-thelium.3,24 Elucidation of the intracellular signaling path-

ways responsible for smooth muscle cell hyperpolarization bythese compounds has been the subject of considerable effort.An important role for K channel activation in hyperpolariz-ing and vasodilatory response has been demonstrated in anumber of previous reports. Although evidence suggestingactivation of KATP channels by EETs has been presented,25

most studies support a role for BKCa channels in EET-inducedhyperpolarization. The simplest explanation for the role ofthis channel in vascular responses is direct activation of BKCa

by EETs. Although data supporting this mechanism havebeen reported,26 our findings (online Figure I) are in agree-ment with a number of previous reports4,27,28 and demonstrateno direct effect of 11,12 EET on BKCa NPo in isolatedmembrane patches. These data suggest that, rather than actingdirectly on the channel, EETs activate intracellular signalingpathways that ultimately cause smooth muscle hyperpolariza-tion via BKCa channels. Recently reported findings thatTRPV4-dependent currents are activated by EETs6 prompted

Figure 7. TRPV4 downregulation blocks 11,12 EET–induced smooth muscle hyperpolarization and vasodilation. a, Recordings ofsmooth muscle cell membrane potential (Em) following 11,12 EET administration for TRPV4 sense- and antisense-treated arteries pres-surized to 60 mm Hg. b, Effect of 11,12 EET on Em in sense- and antisense-treated vessels. *P0.05 vs sense control, #P0.05 vssense 11,12 EET treated. n6 (sense) or n4 (antisense). c, 11,12 EET–induced vasodilatory responses of TRPV4 sense- andantisense-treated cerebral arteries. Vessels were pressurized to 60 mm Hg and allowed to develop myogenic tone before 11,12 EETadministration. d, Vasodilation (in micrometers) as a function of 11,12 EET concentration for untreated and TRPV4 sense- andantisense-treated arteries. *P0.05 vs untreated and sense-treated arteries. n5 for all groups. Data are meanSE.




our investigation of a potential role for this channel invascular control.

We show here that stimulation of Ca2 influx via TRPV4by an endothelium-derived factor (11,12 EET) can alter localCa2 signaling, generating membrane potential hyperpolar-ization and vasodilation. This is the first reported example ofCa2 influx–induced smooth muscle cell membrane hyperpo-larization. We propose that, similar to CICR13,14 mechanismsthat amplify Ca2 influx through VDCC to cause cardiacmyocyte contraction,29 TRPV4-dependent Ca2 signals areamplified by opening SR RyRs11 and serve to increase Ca2

spark frequency. TRPV4-dependent Ca2 influx could poten-tially increase SR Ca2 loading, which would also result inelevated Ca2 spark activity.12 However, 11,12 EET did notincrease Ca2 spark amplitude, arguing against increased SRCa2 loading. 11,12 EET could also activate Ca2 sparkactivity by acting directly on RyRs. However, it is unlikelythat TRPV4 downregulation would influence this response.We propose that TRPV4-dependent Ca2 signals ultimatelyresult in smooth muscle hyperpolarization and vasodilationthrough activation of Ca2 sparks and BKCa-dependent tran-sient membrane hyperpolarization (Figure 8). The hypothe-sized signaling pathway predicts close coupling betweenTRPV4 channels and the SR to allow local TRPV4-mediatedCa2 influx to stimulate nearby RyRs. Although direct acti-vation of BKCa channels by TRPV4-dependent Ca2 influx ispossible, BKCa activation by this mechanism would be sus-tained, rather than transient in nature. For TRPV4-mediatedCa2 influx to directly cause increases in transient BKCa

activity (STOCs), TRPV4 channels would need to permitCa2 entry that has the amplitude and time course of a Ca2

spark through a RyR. There is no evidence that TRPV4channels operate in this manner; thus, our findings areconsistent with TRPV4-dependent currents modulating RyRactivity. Significantly, our findings address previously unre-solved issues concerning EET-induced responses in the vas-culature and suggest that TRPV4 may act as an extracellularreceptor for 11,12 EET in arterial smooth muscle cells.

Transient receptor potential channels are ubiquitously ex-pressed,5 and dynamic Ca2 release events influence the

properties of a number of tissues, including sensory neu-rons,30 pancreatic cells,31 and cardiac myocytes,29 as well asmany types of smooth muscle.32 Thus, in addition to provid-ing novel information regarding regulation of vascular tone,our observations suggest the possibility that TRPV4 (andperhaps other TRPV channels) can alter the function of manytypes of excitable cells by modulating complex Ca2 events.Furthermore, as TRPV channels are activated by a number ofstimuli, such as changes in osmolarity,23 temperature (in thephysiological range),33 and fatty acids,34 this mechanism mayconstitute a fundamental way in which environmental factorsinfluence excitable cells through a local Ca2 signalingmechanism involving CICR.

AcknowledgmentsThis work was supported by NIH grant F32HL075995 and AmericanHeart Association Grant 0535226N (to S.E.); NIH grantsRO1HL44455, RO1HL63722, RO1DK053832, and RO1DK065947(to M.T.N.); NIH grant RO1HL58231 (to J.E.B.); and the TotmanMedical Trust. The Noran confocal microscope used for thesestudies is housed in the Neuroscience Centers of Biomedical Re-search Excellence (COBRE) Imaging/Physiology core facility (NIHgrant P20 RR16435 from the COBRE Program of the NationalCenter for Research Resources) and supported by a National ScienceFoundation grant. We thank Katherine Lutz and Emily R. Levy fortechnical assistance; Adrian Bonev, PhD, for insightful commentsand design of custom software for the analysis of dynamic Ca2

events; and Stephen V. Straub, PhD, for comments onthe manuscript.

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Figure 8. Proposed mechanism for TRPV4-dependent, EET-induced smooth muscle hyperpolarization.




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1

1

EXPANDED MATERIALS and METHODS

Animals

Male Sprague-Dawley rats (300-400 g; Charles River Laboratories; St.

Constant, Quebec, Canada) were used for these studies. Animals were deeply

anesthetized with pentobarbital sodium (50 mg. i.p.) and euthanized by

exsanguination according to a protocol approved by the Institutional Animal

Care and Use Committee (IACUC) of the University of Vermont. Brains were

isolated in ice-cold MOPS-buffered saline [3 mmol/L MOPS (pH 7.4), 145

mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, 2.5 mmol/L CaCl2, 1 mmol/L

KH2PO4, 0.02 mmol/L EDTA, 2 mmol/L pyruvate, 5 mmol/L 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

prior to further manipulation.

Cerebral Artery Smooth Muscle Cell Preparation

To isolate VSM cells, vessels were cut into 2-mm segments, and placed

in the following cell isolation solution (in mmol/L): 60 NaCl, 80 Na glutamate,

5 KCl, 2 MgCl2, 10 glucose, and 10 HEPES; pH 7.2. Arterial segments were

initially incubated at 37°C in 0.3 mg/ml papain (Worthington) and 0.3 mg/ml

dithioerythritol for 15 min, followed by 15 min incubation in 0.67 mg/ml type F

collagenase (Sigma), 0.33 mg/ml type H collagenase (Sigma), and 100 µmol/L

CaCl2. The digested segments were then washed three times in ice-cold

isolation solution and triturated to release VSM cells. Cells were stored on ice in

isolation solution for use the same day.

2

2

RNA Preparation and RT-PCR

RNA was prepared from whole rat brain, cerebral artery homogenates,

and isolated smooth muscle cells. Smooth muscle cells (~1000) were visually

identified under phase contrast microscopy (600X) and harvested using a

micropipette directed by a micromanipulator. Following total RNA extraction

(RNeasy mini kit; Qiagen Inc.), first strand cDNA was synthesized using the

superscript reverse transcriptase (RT) kit (Qiagen Inc). 5 µl of each first strand

cDNA reaction was subsequently placed in a PCR reaction solution (50 µl)

containing forward and reverse primers, dNTPs, 2 x reaction buffer “J”

(Epicenter) and 2.5 U Fail-safe DNA polymerase (Epicenter). PCR was

performed for ~40 cycles of 94oC for 60 s, 58oC for 90 s, and 72oC for 60 s.

Primer sequences were 5’-GGGCAGCTCCCCAAAGTAGAA-3’ (forward) and

5’-GTGCCTGGGCCCAAGAAA-3’ (reverse). Controls consisted of PCR

reactions that did not contain template cDNA (No template, NT) and reactions

that contained template from RT reactions that were performed in the absence

of RT (no RT). Furthermore, PCR primers were designed to span intron exon

boundaries in the TRPV4 gene to minimize the possibility of amplification of

contaminating genomic DNA. Reaction products were resolved on 1.0%

agarose gels.

Ca2+ Imaging

Ca2+ sparks were recorded according to previously described methods1.

Briefly, dispersed cells and pressurized cerebral arteries were loaded with the

fast Ca2+ sensitive dye fluo-4 and were imaged with a laser scanning confocal

3

3

system (OZ; Noran Instruments) mounted in an Diaphot inverted microscope

with a 60× water immersion objective. Dispersed arterial myocytes were studied

in a bath solution consisting of (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10

glucose, and 10 HEPES pH 7.4 at room temperature. Isolated vessels were

superfused and pressurized (60 mmHg) with the same solution warmed to

37ºC. Images were acquired at a rate of 30/sec for dispersed cell experiments

and 60/sec for pressurized vessel experiments. Ca2+ spark recordings were

analyzed with the aid of a custom software application.

Patch-Clamp Experiments

Patch clamp experiments were performed using an Axopatch 200B

amplifier equipped with a CV203BU headstage (Axon Instruments). Recording

electrodes (resistance, ~6 MΩ) were pulled from borosilicate glass (1.5 mm OD,

1.17 mm ID; Sutter Instrument, Novato, CA) and coated with wax to reduce

capacitance. Currents were filtered at 1 kHz, digitized at 40 kHz, and stored for

subsequent analysis. pCLAMP version 9.0, and Clampfit version 9.0 (Axon

Instruments) were used for data acquisition and analysis. For whole-cell

recordings, the bathing solution contained (in mM): 142 NaCl, 10 Glucose, 10

HEPES, 2 CaCl2, 6 KCl, 1 MgCl2, pH 7.4. The pipette solution was: 100 Na-

glutamate, 20 NaCl, 1 MgCl2, 1 EGTA, 10 HEPES, 4 Na2ATP, pH 7.2. Currents

were recorded during voltage ramps from -120 to +80 mV. Ramps were

repeated every 15 sec for at least 1 min prior to drug administration, and, after

administration, for every 15 sec until maximal current activation. Cells were held

at 0 mV between voltage ramps. To examine the effects of ruthenium red on

4

4

11,12 EET-induced currents, whole cell currents were first recorded under basal

conditions and RR (1 µM) was administered. Currents were first recorded in the

presence of RR, and then 11,12 EET (300 nM) was administered in the

presence of RR. Ramp currents were recorded every 15 seconds for at least 3

min. Additional experiments were performed in which NMDG was substituted for

all monovalent cations in the bathing solution. For all experiments, currents

were normalized to membrane capacitance and liquid junction potential offset in

pipette potential was subtracted during analysis.

For perforated patch recordings of transient BKCa currents (STOCs),

the bathing solution contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10

glucose and 10 HEPES pH 7.4 and the pipette solution contained: 110 K-

aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 5 EGTA, 10 HEPES pH 7.2 and 200-

250 µg/ml amphotericin B. STOCs were recorded at a holding potential of 0 mV.

STOCs were defined as transient current events that were greater than three

times the expected unitary current for BKCa channels at the membrane potential

for which the recordings were obtained. For a membrane potential of 0 mV, the

threshold current was set at 24 pA. All events greater than the threshold current

were counted and STOC frequency was calculated by dividing the number of

events by the duration of the recording. All recordings were at least 1 minute in

duration. All patch clamp experiments were performed at room temperature

(~22ºC).

5

5

TRPV4 Antisense Procedures

The sequence of the antisense oligonucleotide used for these studies

(previously shown to suppress TRPV4 expression2) was 5’-

CATCACCAGGATCTGCCATACTG-3’. The last three bases on the 5’ and 3’

ends of the oligonucleotide were phosphorothioated to limit degradation by

cellular nucleases. Oligonucleotides were dissolved at a concentration of 2 mM

in nuclease-free water and were introduced into intact cerebral arteries using a

reversible permeabilization procedure as previously described3,4. Arteries were

first incubated for 20 minutes at 4oC in (in mM): KCl,120; MgCl2, 2; EGTA, 10;

Na2ATP, 5; TES, 20; pH 6.8. These vessels were then placed in a similar

solution containing oligonucleotides (200 µM) for 90 minutes at 4oC and then in

a similar oligonucleotides –containing solution with elevated MgCl2 (10 mM).

Permeabilization was reversed by placing the arteries in MOPS buffered

solution containing (in mM): NaCl, 140, KCl, 5, MgCl2, 10, glucose, 5, MOPS, 2;

pH 7.1, 22oC. The [Ca2+] of this solution was then gradually (~45 min) increased

from nominally Ca2+-free to 0.01, 0.1, and 1.8 mM. Arteries were organ cultured

in D-MEM/F-12 media supplemented with L-glutamine (2 mM), penicillin (50

units/ml) and streptomycin (50 ug/ml) in a humidified incubator. CO2 was

maintained at 5%.

Isolated Vessel Experiments

Isolated vessel procedures were performed essentially as previously

described3. Briefly, arterial segments were cleaned and transferred to a vessel

chamber. The proximal end of the vessel was cannulated with a glass

6

6

micropipette and secured, the lumen was gently rinsed, and the distal end of the

vessel cannulated and secured. The endothelium was removed by passage of

an air bubble through the lumen. Vessels were pressurized to 60 mmHg with

physiological saline solution (PSS) and superfused with warmed (37°C) PSS

aerated with a normoxic gas mixture (21% O2, 5% CO2, balance N2) and

allowed to develop spontaneous myogenic tone. Inner diameter was

continuously monitored using video microscopy and edge-detection software

(Ionoptix) as increasing concentrations of 11,12 EET (Cayman Chemical) were

added to the superfusion solution.

Smooth Muscle Cell Membrane Potential

For measurement of smooth muscle cell membrane potential, cerebral

arteries were isolated and pressurized, and VSM cells were impaled through the

abluminal wall with glass intracellular microelectrodes (tip resistance 100-200

MΩ). 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 approximately 0 mV after the electrode was retracted from the

cell.

7

7

Calculations and Statistics

All data are mean ± SE. Values of n refer to number of cells for

experiments involving isolated cerebral myocytes or number of animals for

experiments employing isolated arteries. Comparisons involving two

experimental groups were made by unpaired Student’s t-tests. Comparisons

involving multiple groups were made by one-way or two-way ANOVA or

repeated measures two-way ANOVA, as appropriate, followed by Student-

Newman-Keuls post hoc test. A level of P ≤ 0.05 was accepted as statistically

significant for all experiments.

REFERENCES

1. Perez GJ, Bonev AD, Patlak JB, Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol. 1999;113:229-38.

2. Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, Levine JD. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron. 2003;39:497-511.

3. Earley S, Waldron BJ, Brayden JE. Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ Res. 2004;95:922-9.

4. Welsh DG, Nelson MT, Eckman DM, Brayden JE. Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure. J Physiol. 2000;527 Pt 1:139-48.

Figure S1: a: Single BKCa channel recordings of membrane patches from cerebral artery smooth muscle cells before and after 11,12 EET (300 nM) was administered. C = closed state of the channel. b: Channel open probability (NPo) as a function of membrane potential (VM) for untreated cells and cells treated with 11,12

EET (300 nM). n = 5-7 at each membrane potential. There were no significant differences.

Scott Earley, Thomas J. Heppner, Mark T. Nelson and Joseph E. BraydenChannels

Ca Signaling Complex With Ryanodine Receptors and BK2+TRPV4 Forms a Novel Ca

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TRPV4 Forms a Novel Ca2+ Signaling Complex With Ryanodine Receptors and BKCa Channels

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Transcript of TRPV4 Forms a Novel Ca2+ Signaling Complex With Ryanodine Receptors and BKCa Channels