Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries
Background Recent studies demonstrate that spatially restricted, local Ca2+ signals are key regulators of endothelium‐dependent vasodilation in systemic circulation. There are drastic functional differences between pulmonary arteries (PAs) and systemic arteries, but the local Ca2+ signals that control endothelium‐dependent vasodilation of PAs are not known. Localized, unitary Ca2+ influx events through transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, regulate endothelium‐dependent vasodilation in resistance‐sized mesenteric arteries via activation of Ca2+‐dependent K+ channels. The objective of this study was to determine the unique functional roles, signaling targets, and endogenous regulators of TRPV4 sparklets in resistance‐sized PAs.
Methods and Results Using confocal imaging, custom image analysis, and pressure myography in fourth‐order PAs in conjunction with knockout mouse models, we report a novel Ca2+ signaling mechanism that regulates endothelium‐dependent vasodilation in resistance‐sized PAs. TRPV4 sparklets exhibit distinct spatial localization in PAs when compared with mesenteric arteries, and preferentially activate endothelial nitric oxide synthase (eNOS). Nitric oxide released by TRPV4‐endothelial nitric oxide synthase signaling not only promotes vasodilation, but also initiates a guanylyl cyclase‐protein kinase G‐dependent negative feedback loop that inhibits cooperative openings of TRPV4 channels, thus limiting sparklet activity. Moreover, we discovered that adenosine triphosphate dilates PAs through a P2 purinergic receptor‐dependent activation of TRPV4 sparklets.
Conclusions Our results reveal a spatially distinct TRPV4‐endothelial nitric oxide synthase signaling mechanism and its novel endogenous regulators in resistance‐sized PAs.
- calcium channel
- calcium signaling
- endothelial nitric oxide synthase
- pulmonary artery
- signaling pathways
- transient receptor potential vanilloid 4 channel
- vascular endothelial function
What Is New?
Our studies reveal a novel transient receptor potential vanilloid (TRPV4) channel‐dependent Ca2+ signaling mechanism that regulates endothelial function in small pulmonary arteries.
Unitary Ca2+ influx signals through TRPV4 channels (TRPV4 sparklets) activate endothelial nitric oxide synthase in small pulmonary arteries. NO released by TRPV4‐ endothelial nitric oxide synthase signaling initiates guanylyl cyclase (GC)‐protein kinase G (PKG) signaling in the endothelium that limits TRPV4 channel cooperativity and serves as a negative feedback signal to regulate TRPV4 channel function.
Furthermore, ATP dilates small pulmonary arteries predominantly via activation of TRPV4‐endothelial nitric oxide synthase signaling.
What Are the Clinical Implications?
Abnormalities in TRPV4‐dependent endothelial Ca2+ signaling may be a potential pathological mechanism that contributes to endothelial dysfunction in pulmonary vascular disorders.
Individual elements in the TRPV4‐dependent endothelial Ca2+ signaling pathway may offer novel therapeutic targets for treating pulmonary hypertension and other pulmonary vascular disorders.
Endothelial cells (ECs) from small, resistance‐sized arteries are key controllers of vascular resistance. Recent studies in systemic arteries—including mesenteric, cremaster, and cerebral arteries—have demonstrated that endothelium‐dependent vasodilation is controlled by local, spatially restricted increases in endothelial Ca2+ while the whole‐cell Ca2+ level remains unchanged.1, 2, 3, 4, 5 In contrast to systemic arteries, pulmonary arteries (PAs) comprise a low‐pressure, high‐flow circulation. Moreover, systemic and pulmonary vascular resistances can be regulated independently.6 The distinct signaling mechanisms that regulate endothelial function in small PAs are poorly understood. Unraveling the local Ca2+ signals in small PAs may be necessary for understanding endothelial regulation of pulmonary vascular resistance under normal and disease conditions.
In mesenteric, cremaster, and cerebral arteries, transient receptor potential vanilloid 4 (TRPV4) channels are key regulators of endothelium‐dependent vasodilation.1, 4, 7 In mesenteric arteries, local, unitary Ca2+ influx events through endothelial TRPV4 channels, termed “TRPV4 sparklets,” activated endothelial IK/SK (Ca2+‐activated intermediate and small conductance K+) channels to cause vasodilation.4, 5 The biophysical properties, signaling targets, and endogenous regulators of TRPV4 sparklets in small PAs remain unknown. Endothelium‐derived nitric oxide (NO) is thought to be the predominant vasodilator in the pulmonary circulation.8, 9, 10, 11, 12 Increase in global Ca2+ has long been associated with activation of endothelial nitric oxide synthase (eNOS). Whether a specific local Ca2+ signal can activate eNOS has not been elucidated. In this study, we explored the possibility that local, unitary Ca2+ influx through TRPV4 channels activates eNOS in small PAs, a signaling mechanism different from small mesenteric/cremaster/cerebral arteries.1, 4, 7
NO can alter the activity of several ion channels by S‐nitrosylation or activation of guanylyl cyclase‐protein kinase G (GC‐PKG) signaling.13, 14, 15, 16, 17 In cultured cells, S‐nitrosylation of TRPV4 channels increased the channel function,18 and activation of GC‐PKG signaling reduced the channel activity.19 Therefore in PAs, TRPV4‐eNOS signaling could activate a bidirectional regulation, where TRPV4 channels promote NO release, which in turn regulates TRPV4 channel function. Ca2+‐dependent cooperative opening of TRPV4 channels in a cluster has emerged as a key mechanism for modulating TRPV4 channel function.4, 5, 20, 21 We postulated that TRPV4‐induced NO release modulates TRPV4 channel activity by altering cooperative openings of TRPV4 channels.
Discovering the endogenous activators of TRPV4 channels in small PAs is central to deciphering the unique physiological roles of TRPV4 channels in pulmonary circulation. In this regard, purinergic receptor agonist ATP has been shown to increase endothelial Ca2+ and cause vasodilation in large, conduit PAs.22, 23, 24 ATP can be released into the circulation by ECs, smooth muscle cells (SMCs), and red blood cells.25, 26 Circulatory ATP may, therefore, serve as an important regulator of pulmonary vascular resistance. We hypothesized that ATP is a novel, endogenous activator of TRPV4 channels in small PAs.
In the current study we provide the first evidence that local Ca2+ signals–TRPV4 sparklets–regulate baseline and induced eNOS activity to dilate small PAs. Moreover, we report ATP as a novel endogenous activator of TRPV4 channels that promotes TRPV4‐eNOS signaling through P2 purinergic receptors. NO is another endogenous regulator of TRPV4 channels that limits TRPV4 channel activity by disrupting the coupling among TRPV4 channels via activation of endothelial GC‐PKG mechanism. These results reveal distinct, novel local Ca2+ signaling mechanisms that regulate endothelial function in small PAs.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Drugs and Chemical Compounds
Apamin, cyclopiazonic acid (CPA), GSK2193874, GSK1016790A, HC067047, NS309, 1400W, Nω‐propyl‐l‐arginine hydrochloride, ODQ, RN1747, Rp‐8‐Br‐PET‐cGMPS, Suramin, and Tram 34 were purchased from Tocris Bioscience (Minneapolis, MN). ATP and Nω‐nitro‐l‐arginine were obtained from Sigma‐Aldrich (St. Louis, MO). DAF‐FM diacetate, Fluo‐4AM (Ca2+ indicator), and EGTA‐AM (Ca2+ chelator) were purchased from Fischer Scientific (Pittsburgh, PA). Spermine NONOate and U46619 were purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals were obtained from Sigma‐Aldrich (St. Louis, MO).
All animal protocols were approved by the Animal Care and Use Committee of the University of Virginia. Male C57BL6/J, transgenic GCaMP2Cx40, TRPV4−/−, and eNOS−/− (The Jackson Laboratory, Bar Harbor, ME) mice (10–14 weeks old) were used for all the studies. GCaMP2Cx40 mice express GCaMP2, a Ca2+‐specific biosensor under the connexin 40 promoter, thereby limiting its expression to only ECs.27, 28 Mice were euthanized with pentobarbital (90 mg/kg; intraperitoneal) followed by decapitation. Third‐order mesenteric arteries (MAs; ≈100 μm in diameter), fourth‐order pulmonary arteries (PAs, ≈100–200 μm), and second‐order PAs (≈400 μm) were isolated in cold HEPES‐buffered physiological salt solution (HEPES‐physiological salt solution [PSS], in mmol/L, 10 HEPES, 134 NaCl, 6 KCl, 1 MgCl2 hexahydrate, 2 CaCl2 dihydrate, and 7 dextrose, pH adjusted to 7.4 using 1 mol/L NaOH). Data were collected from at least 3 different arteries from at least 3 mice.
Isolated PAs and MAs were cannulated on glass pipettes mounted on an arteriography chamber (The Instrumentation and Model Facility, University of Vermont, Burlington, VT) at areas lacking branching points and pressurized to physiological pressure (15 mm Hg for PA and 80 mm Hg for MA). Arteries were superfused with PSS (in mmol/L, 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgCl2 hexahydrate, 2.5 CaCl2 dihydrate, 7 dextrose, and 24 NaHCO3) at 37°C and bubbled with 20% O2/5% CO2 to maintain the pH at 7.4. All drug treatments were added to the superfusing PSS. Because PAs do not develop myogenic tone at 15 mm Hg, they were preconstricted with 100 nmol/L U46619 (a thromboxane A2 agonist). In functional studies with PAs, all other pharmacological treatments were performed in the presence of U46619. Before measurement of vascular reactivity, arteries were treated with NS309 (1 μmol/L), a direct opener of endothelial IK/SK channel, to assess endothelial health. Arteries that failed to dilate to NS309 were discarded. Endothelial denudation was performed by passing an air bubble through the artery for ≈60 s. Complete removal of the endothelial layer was verified by the absence of dilation to NS309. Changes in arterial diameter were recorded at a 60‐ms frame rate using a charge‐coupled device camera and edge‐detection software (IonOptix LLC, Westwood, MA).4, 5 All drug treatments were incubated for 10 minutes. At the end of each experiment, Ca2+‐free PSS (in mmol/L, 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgCl2 hexahydrate, 7 dextrose, 24 NaHCO3, and 5 EGTA) was applied to assess the maximum passive diameter. Percent constriction was calculated by: (1)where Diameterbefore is the diameter of the artery before a treatment and Diameterafter is the diameter after the treatment. Percent dilation was calculated by: (2)where Diameterbasal is the stable diameter before drug treatment, Diameterdilated is the diameter after drug treatment, and DiameterCa‐free is the maximum passive diameter.
PAs and MAs were surgically opened and pinned down on a Sylgard block in en face preparation for NO imaging from intact EC and SMC layers. NO levels were assessed using 5 μmol/L DAF‐FM (4‐amino‐5 methylamino‐2′,7′‐difluorofluorescenin diacetate) prepared in HEPES PSS with 0.02% pluronic acid.29 DAF‐FM forms a fluorescent triazole compound after binding to NO. En face PAs or MAs were pretreated with GSK101, l‐NG‐nitroarginine (L‐NNA), or GSK219 in HEPES‐PSS for 5 minutes at 30°C. Arteries were then incubated with DAF‐FM containing the drug under consideration for 20 minutes at 30°C in the dark. Validity of DAF‐FM as an NO indicator was tested by treating PAs with NO donor Spermine NONOate (NONOate, 3–30 μmol/L), and recording the DAF‐FM fluorescence. Spermine NONOate was selected because it has a long half‐life (≈39 minutes) and releases a controlled amount of NO in solution. The experiments using NONOate were performed in the presence of L‐NNA to eliminate the effect of endogenous release of NO in response to TRPV4 channel activation. The arteries were incubated with either HEPES‐PSS (baseline) or 30 nmol/L GSK101 in the absence or presence of 200 μmol/L L‐NNA to determine whether localized Ca2+ influx through EC TRPV4 channels contributes to NO release through NOS activation. DAF‐FM fluorescence was imaged using Andor Revolution WD (with Borealis) spinning‐disk confocal imaging system (Andor Technology, Belfast, UK) comprising an upright Nikon microscope with a ×60 water‐dipping objective (numerical aperture 1.0) and an electron‐multiplying CCD camera. DAF‐FM fluorescence was recorded using an excitation wavelength of 488 nm and emitted fluorescence was captured with a 525/36‐nm band‐pass filter. Images were obtained along the z‐axis at a slice size of 0.05 μm from the top of the ECs to the bottom of the SMCs. DAF‐FM fluorescence was analyzed using custom‐designed SparkAn software by Dr Adrian D. Bonev (University of Vermont, Burlington, VT). An outline was drawn around each EC or SMC to obtain the arbitrary fluorescence intensity of that cell. The background fluorescence was then subtracted from the recorded fluorescence. The fluorescence numbers from all the cells in a field of view were averaged to obtain single fluorescence number for that field. Relative changes in DAF‐FM fluorescence were obtained by dividing the fluorescence in the treatment group by that in the control group. Each field of view was considered as n=1, and several fields of view from at least 3 arteries from at least 3 mice were included in the final analysis.
Measurement of Ca2+ events in the native ECs from PAs was performed as previously described.4, 5 Briefly, Andor Revolution WD (with Borealis) spinning‐disk confocal imaging system described above was used to record Ca2+ influx events in en face PAs. Ca2+ events were recorded at 30 ms per image before and after 5 minutes of each treatment. The arteries were loaded with fluo‐4 AM (10 μmol/L) in the presence of pluronic acid (0.04%) at 30°C for 30 minutes for PAs and 45 minutes for MAs. The majority of experiments were carried out in the presence of CPA (20 μmol/L, a sarco‐endoplasmic reticulum (ER) Ca2+‐ATPase inhibitor) in order to eliminate the interference from Ca2+ release from intracellular stores. Ca2+ binding‐induced changes in emitted fluorescence were observed by exciting at 488 nm with a solid‐state laser and collecting emitted fluorescence using a 525/36‐nm band‐pass filter. In the studies examining whether S‐nitrosylation of TRPV4 channels mediated the effect of NO on channel activity, flash photolysis using ultraviolet light pulse of 10 ms (Andor Mosaic 3 Infinity, Andor Technology, and pE‐4000, CoolLED Ltd, Andover, UK) was conducted to dissociate the S‐NO bonds on TRPV4 channels. The experiments using NONOate were performed in the presence of L‐NNA to eliminate the effect of endogenous release of NO in response to TRPV4 channel activation. TRPV4 Ca2+ sparklets and Ca2+ release events from the ER in ECs were analyzed using custom‐designed SparkAn software. To generate fractional fluorescence (F/F0) traces, a region of interest defined by a 1.7‐μm2 (5×5 pixels) box was placed at a point corresponding to peak sparklet amplitude. Each field of view was ≈110×110 μm and covered ≈15 ECs. Representative F/F0 traces were filtered using a Gaussian filter and a cutoff corner frequency of 4 Hz. Sparklet activity, all‐points histograms, and coupling coefficients were determined as described previously using the custom‐designed SparkAn software, Clampfit, and OriginPro7.5.4, 5
Localization of Sparklets at Myoendothelial Projections
To determine the localization of sparklets at myoendothelial projections (MEPs), the arteries were incubated with 100 μmol/L Alexa Fluor 633 hydrazide. Alexa 633 hydrazide cannot penetrate into the cells, but preferentially binds to elastin‐containing internal elastic lamina (IEL), thereby illuminating the IEL1, 30 for 15 minutes following Ca2+ imaging experiment. The images were then acquired in the fields of view from which Ca2+ signals were recorded, using excitation wavelengths of 640 nm (for IEL staining) and 488 nm (for fluo‐4), and capturing the emitted fluorescence using 685/40‐ and 525/36‐nm band‐pass filters, respectively. Regions of interest (1.7 μm2) corresponding to the peak sparklet fluorescence were overlaid onto the IEL staining image. The regions of interest within 5 μm from the center of the holes in the IEL were counted as MEP sparklet sites and the remaining regions of interest were counted as non‐MEP sparklet sites, as we have done previously.5 A sparklet site displays spread of calcium away from the site of initiation. In 88% of sparklet sites, the site of sparklet initiation coincided with the peak sparklet amplitude. Therefore, the region of interest corresponding to the peak sparklet amplitude was used to analyze the sparklet localization data.
Calculation of Sparklet Activity Per Site and Sparklet Activity Per Field
Activity of TRPV4 Ca2+ sparklets was evaluated as described previously.4, 5 Area under the curve for all the events at a site was determined using trapezoidal numerical integration ([F−F0]/F0 over time, in seconds). The average number of active TRPV4 channels, as defined by NPO (NPO, where N is the number of channels at a site and PO is the open state probability of the channel), was calculated by (3)where T is the dwell time at each quantal level detected at TRPV4 sparklet sites and Ttotal is the duration of the recording. NPO was determined using Single Channel Search module of Clampfit and quantal amplitudes derived from all‐points histograms (ΔF/F0 of 0.29 for Fluo‐4–loaded PAs). NPO for all the sites in a field was averaged to obtain NPO per site. For the studies on baseline sparklet activity (without GSK101), some of the fields showed no sparklet activity at the baseline but L‐NNA still increased the sparklet activity. Therefore, we used NPO per field instead of NPO per site to estimate the effect of L‐NNA on baseline sparklet activity. All NPO per site values for all the sites in a field were summated to calculate NPO per field.
Construction of All‐Points Histograms
As described previously,4 all‐points amplitude histograms were constructed by first filtering Ca2+ images with a Kalman filter (adopted from an ImageJ plug‐in written by Christopher Philip Mauer, Northwestern University, Chicago, IL; acquisition noise variance estimate=0.05; filter gain=0.8) to assess quantal amplitudes of Ca2+ influx events (ie, equal increments of fluorescence signals over increasing numbers of TRPV4 channels). All sparklet events with at least 5 steady baseline points and a steady peak of at least 5 data points were used for the construction of all‐points histograms, whereas channel openings with either unstable baseline or shorter duration of opening (eg, fewer than 5 data points at the peak) were excluded from this analysis. The analysis was undertaken with Ca2+ images obtained from 3 to 5 fields from 3 to 5 PAs from C57BL6/J or GCaMP2 mice. F/F0 traces were exported to ClampFit10.3 for constructing all‐points histograms, which were then fit with multiple Gaussian function: (4)where F/F0, a, μ, and σ2 represent the fractional fluorescence, area, mean value, and variance of the Gaussian distribution, respectively. Statistical differences in the quantal levels of TRPV4‐mediated Ca2+ sparklets between diverse treatment regimens were evaluated based on a 95% confidence interval calculated from the mean and SE for each peak.
Determination of Coupling Coefficients
Coupling coefficients for the coupling among TRPV4 channels at a cluster were determined using a coupled Markov chain model in MATLAB, as previously described.5, 31, 32, 33, 34 [F‐F0]/F0 traces showing steady baseline (ie, at least 30 s duration) were selected for analyzing the cooperative gating of TRPV4 channels. Each TRPV4 sparklet site was examined separately. The coupled Markov chain model developed by Chung and Kennedy32 was used to simulate and fit independent records of partially coupled channels. Openings of single TRPV4 channel were identified using a single channel amplitude (ie, quantal level) of 0.29 ΔF/F0 for mouse PAs loaded with fluo‐4 and a half‐amplitude protocol in a program written in MATLAB. TRPV4 channel activity was considered as a first‐order and discrete Markov chain. The built‐in Hidden Markov parameter estimation function in MATLAB was utilized to estimate a Markovian transition matrix based on the TRPV4 sparklet data and their corresponding channel opening time course. The estimated transition matrix was modeled as a partially coupled Markov chain, suggesting the coupling coefficient (κ) values varying from 0 (no coupling or independent gating) to 1 (maximum coupling).
Immunostaining for AKAP150 and PKG in ECs of the Intact PAs and MAs
Immunostaining assay was performed as described previously.5 Briefly, mouse intact PAs and MAs cut longitudinally and pinned down on SYLGARD blocks were rapidly fixed with ice‐cold acetone for 10 minutes and then washed 3 times with PBS. The arteries were permeabilized with 0.2% Triton‐X for 30 minutes, blocked with 5% normal donkey serum (ab7475, Abcam, Cambridge, MA) for 1 hour, and incubated with a goat polyclonal AKAP150 antibody (sc‐6445, 1:250, Santa Cruz Biotechnology, Dallas, TX) or a rabbit polyclonal anti‐cGKI antibody (ab37709, 1:100, Abcam, Cambridge, MA) overnight at 4°C. After 5 washes with PBS, PAs and MAs were incubated with Alexa Fluor® 568‐conjugated donkey anti‐goat (A11057, 1:500, Life Technologies, Carlsbad, CA) or anti‐rabbit secondary antibody (A10042, 1:500, Life Technologies) for 1 hour at room temperature in a dark room. Immunostaining images were acquired using the Andor imaging system described above. Images were obtained along the z‐axis from the top of ECs to the bottom of SMCs with a slice size of 0.05 μm. Connective tissue autofluorescence was evaluated by exciting at 488 nm with a solid‐state laser and collecting emitted fluorescence with a 525/36‐nm band‐pass filter. AKAP150 and PKG immunostaining (ie, red‐pseudo‐color images) was assessed by exciting at 561 nm and collecting emitted fluorescence with a 607/36‐nm band‐pass filter. The specificity of the antibodies was tested using competing peptides and by substitution with PBS. AKAP150 or PKG‐associated staining was absent under these conditions.
The n number represents the unit of analysis, and has been specified in each figure legend. The data in this article were normally distributed; therefore, parametric statistics were performed and mean±SEM were used to describe the data set. P<0.05 was considered significant. The n numbers and P values are indicated in each figure legend. Two‐tailed, paired (for paired observations) or independent 2‐sample t test was used for comparisons between 2 groups. One‐way ANOVA with post hoc Tukey test (comparing different means) or Dunnett test (comparisons with control group), or 2‐way ANOVA with post hoc Tukey test was used for comparing 3 or more groups. Statistical analysis was performed using OriginPro7.5. For all the calculations of TRPV4 sparklet activity per site and activity per field, n value represents number of fields.4, 5 For the coupling coefficient analysis, n value represents number of sites.5 Only 1 artery from a mouse was used for 1 experimental treatment. Within an experimental treatment group, activity per site did not change significantly among different fields from the same artery (P>0.05), or among fields from different arteries as determined using 1‐way ANOVA. Moreover, the coupling coefficient values also did not change significantly among different fields from the same artery, or among fields from different arteries under the same experimental treatment group. In some cases, activity per field was calculated instead of activity per site to account for fields with no active sites, and was not significantly different among different arteries from the same experimental treatment group.
Native Endothelium From Small PAs Exhibits Unitary TRPV4 Sparklets With Distinct Spatial Properties
We recently discovered localized, unitary Ca2+ influx events through TRPV4 channels in the endothelium from small MAs, and termed them TRPV4 sparklets.4, 5 Because of the structural and functional differences between systemic and pulmonary circulations,6 we hypothesized that TRPV4 sparklets from small PAs exhibit unique biophysical properties. Local Ca2+ signals were studied in the intact endothelium from small (≈100–200 μm), fourth‐order PAs (Figure 1A, top panel). CPA (20 μmol/L, sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase inhibitor) was used to deplete intracellular stores of Ca2+ and to eliminate the interference from Ca2+ release from intracellular stores in en face PAs. In the presence of CPA alone, there were ≈2 TRPV4 sparklet sites per field of view (≈15 ECs, Figure 1A, top panel) within a recording duration of 1 minute (Figure S1A). The number of TRPV4 sparklet sites per field was increased 2‐ and 7‐fold by the selective channel agonists, GSK1016790A (GSK101; 3 nmol/L; Video S1) and RN1747 (1 μmol/L), respectively (Figure S1A and S1B). The TRPV4 sparklet activity was almost entirely inhibited by selective TRPV4 inhibitors GSK2193874 (GSK219; 100 nmol/L) and HC067047 (HC067; 1 μmol/L) (Figure 1A, middle and bottom panels; Figure S1A). The Ca2+ sparklets elicited by GSK101 and RN1747 were absent in arteries from TRPV4−/− mice (Figure 1A, bottom panel—Figure S1A). The Ca2+ sparklets elicited by GSK101 were immediately abolished as the external Ca2+ was changed from 2 to 0 mmol/L (Figure 1A, bottom panel; Figure S1C), confirming that the TRPV4 sparklets represented the influx of extracellular Ca2+ through TRPV4 channels on EC membrane. Moreover, the increase in fluorescence at a sparklet site was not accompanied by an increase in whole‐cell fluorescence (Figure S2), indicating that TRPV4 sparklets did not alter whole‐cell Ca2+ levels. The surface area encompassed by EC outlines was ≈45‐fold higher than the spatial spread of TRPV4 sparklets, further supporting the local nature of sparklets (Figure S2C).
The fractional fluorescence (F/F0) traces revealed square, discrete amplitudes of TRPV4 sparklets, reminiscent of single‐channel openings from a patch clamp experiment (Figure 1A, middle panel). Therefore, we used the following single‐channel opening criteria to determine whether Ca2+ sparklets in PAs are unitary events4, 35: (1) small recording volumes; (2) high Ca2+ permeability and single channel conductance; (3) quantal amplitudes; and (4) dependence of the sparklet amplitude on Ca2+ electrochemical gradient but not on the concentration of the agonist or inhibitor.35 Regarding the first criterion, ECs from small PAs are ≈1‐μm thick, corresponding to a recording volume of 1.2 fL. TRPV4 channels have been demonstrated to have a large single channel conductance and Ca2+ permeability,36 satisfying the second criterion. A multiple Gaussian fit to all‐points histogram of the fractional fluorescence established a stepwise increase in amplitude, with the quantal level being 0.29 ∆F/F0 for the arteries loaded with fluo‐4 (Figure 1B), and 0.19 ∆F/F0 for the arteries from GCaMP2Cx40 mice (Figure S3A, top). Increasing the extracellular Ca2+ from 2 to 10 mmol/L produced a 70% increase in the quantal level of Ca2+ sparklets (Figure S3A, middle). Reducing the electrochemical gradient for Ca2+ influx by depolarizing EC membranes using 100 mmol/L extracellular K+ decreased the amplitude of these events by 50% (Figure S3A, bottom), satisfying the third criterion. Moreover, the quantal amplitude did not change with the use of different agonists, with increasing concentration of an agonist, or with the use of TRPV4 inhibitor (Figure S3B), satisfying the fourth criterion. These results supported the concept that TRPV4 sparklets in PA endothelium represent unitary Ca2+ influx events through TRPV4 channels.
In small MAs, the majority (≈60%) of TRPV4 sparklet sites occurred at endothelial projections to SMCs or MEPs,5 where IK channels were also localized.1, 5 In small PAs, however, a small percentage of sparklet sites (25±3%; n=5 PAs) occurred at the MEPs. To confirm the spatial differences in sparklet localization between small PAs and MAs, IEL was stained with Alexa Fluor 633 (a marker for extracellular matrix)30 immediately after Ca2+ imaging experiments. Overlay of sparklet site regions of interest with IEL staining images showed that only ≈28% of the sparklet sites overlapped with MEPs in small PAs, whereas ≈65% of sparklet sites overlapped with MEPs in small MAs (Figure 1C and 1D). The total number of holes per field of view was, however, higher in PAs compared with MAs (Figure 1D). These results established the spatial differences in TRPV4 sparklet activity between small PAs and MAs. We previously showed that MEP‐localized A‐kinase anchoring protein 150 was required for localization of TRPV4 sparklets at MEPs in MAs. Consistent with the non‐MEP localization of sparklets in PAs, only ≈5% of holes in the IEL showed identifiable AKAP150 staining in PAs when compared with ≈90% of the MEPs in MAs (Figure 1E).
TRPV4 Sparklets Cause Vasodilation in Small PAs via eNOS Activation
Administration of TRPV4 channel agonist GSK101 lowered PAP (pulmonary arterial pressure) in rats.37 At the level of small PAs, however, the functional effect of endothelial TRPV4 channel activation and the Ca2+‐sensitive targets have not been identified. PAs are normally exposed to intravascular pressures of 10 to 20 mm Hg. We therefore cannulated and pressurized fourth‐order PAs at 15 mm Hg to assess the effect of TRPV4 channel activation on PA diameter. PAs were preconstricted with U46619 (100 nmol/L), a thromboxane A2 receptor agonist (32±2% constriction; n=30 PAs). NS309, a highly specific activator of endothelial IK and SK channels,38 induced dilations in PAs that were absent in endothelium‐denuded PAs, confirming the presence of functional IK and SK channels in PA endothelium (Figure 2A and 2B). PAs were then treated with GSK101 (3–30 nmol/L), which caused a concentration‐dependent vasodilation (Figure 2A and 2B). GSK101‐induced dilation was absent in endothelium‐denuded PAs, PAs from TRPV4−/− mice, and in the presence of TRPV4 channel inhibitor GSK219 (Figure 2A and 2B), confirming that endothelial TRPV4 channel activation dilated small PAs.
We previously showed that endothelial TRPV4 channels induced vasodilation predominantly via activation of IK/SK channels in small MAs constricted by intravascular pressure.4 Presence of U46619 in the superfusate did not alter the IK/SK channel‐dependent nature of TRPV4 channel‐induced vasodilation in MAs (Figure 2C and 2D). We, therefore, tested the effect of IK and SK channel inhibitors, Tram‐34 (1 μmol/L) and apamin (300 nmol/L), respectively, on TRPV4‐vasodilation in small PAs. While NS309‐induced vasodilation was reduced by 70.0±10.6% (n=5 PAs) in the presence of Tram‐34 and apamin, TRPV4 channel‐induced vasodilation was not affected by IK/SK channel inhibitors (Figure 2A and 2B), raising an interesting possibility that a distinct local Ca2+ signaling network mediated TRPV4‐induced vasodilation in small PAs. Although the activation of eNOS by localized Ca2+ signals has not been described, global increase in Ca2+ is known to activate eNOS.39 We, therefore, tested the possibility that TRPV4 sparklets activated eNOS to cause vasodilation in small PAs. Inhibition of NOS with L‐NNA constricted PAs by ≈13±1% (n=12 PAs), indicating a tonic influence of NO on basal diameter of PAs. In the presence of L‐NNA, GSK101‐induced vasodilation was abolished (Figure 2A and 2B). However, L‐NNA is a nonselective inhibitor of eNOS, inducible NOS, and neuronal NOS. To determine the relative contribution from each NOS isoform to TRPV4‐vasodilation, we used a selective inducible NOS inhibitor 1400W40 (1 μmol/L) or a selective neuronal NOS inhibitor Nω‐propyl‐l‐arginine hydrochloride41 (300 nmol/L). In the presence of either of these inhibitors, the TRPV4‐vasodilation was not altered (Figure 2A and 2B), pointing to a TRPV4‐eNOS signaling network in PAs. The role of eNOS in TRPV4‐induced vasodilation was verified by studying TRPV4 dilations in eNOS−/− mice in the presence of 1400W (inducible NOS inhibitor)40 to account for a possible compensation by inducible NOS.42 In the PAs from eNOS−/− mice, TRPV4 dilations were absent (Figure 2A and 2B), confirming that TRPV4‐vasodilation in PAs was being mediated by eNOS activation.
TRPV4 Sparklets Regulate NO Release From the Endothelium in PAs but Not in MAs
The eNOS‐dependent nature of TRPV4 sparklet‐induced vasodilation suggested a local TRPV4‐eNOS coupling in small PAs. Because NO generated in ECs can passively diffuse to the SMCs, we postulated that NO levels are increased in both EC and SMC layers following TRPV4 channel activation. NO levels in ECs and SMCs were assessed by recording DAF‐FM fluorescence. Spermine NONOate (NONOate, 3–30 μmol/L), a NO donor, caused a concentration‐dependent increase in DAF‐FM fluorescence in both EC and SMC layers (Figure S4A and S4B), demonstrating that an increase in NO level could be detected with DAF‐FM. Both ECs and SMCs showed a low level of basal DAF‐FM fluorescence (Figure 3A and 3B). Activation of TRPV4 channels with GSK101 (30 nmol/L) increased DAF‐FM fluorescence in EC and SMC layers in small PAs (Figure 3A and 3B). Consistent with the NOS‐independent nature of TRPV4‐vasodilation in small mesenteric arteries4 (Figure 2C and 2D), TRPV4 channel activation failed to increase NO levels in ECs and SMCs from MAs (Figure 3C). In PAs, GSK101 produced a 1.8‐fold increase in DAF‐FM fluorescence in ECs and a 2‐fold increase in SMCs (Figure 3C). Pretreatment with L‐NNA inhibited the GSK101‐induced increases in DAF‐FM fluorescence in both ECs and SMCs (Figure 3A through 3C). Moreover, in PAs from eNOS−/− mice, GSK101 was unable to increase DAF‐FM fluorescence in ECs or SMCs (Figure 3B). These results support the concept that local TRPV4‐eNOS signaling regulates NO levels and endothelium‐dependent vasodilation in small PAs.
To test the possibility that TRPV4 sparklets regulate NO release under basal conditions (without TRPV4 agonist) in small PAs, we studied the effect of GSK219 on NO levels. The basal DAF‐FM fluorescence in both ECs and SMCs was reduced by ≈30% (Figure 3D, left) in the presence of GSK219 supporting TRPV4 regulation of basal eNOS activity in PAs. TRPV4 channels are Ca2+‐selective channels, but they can also conduct other ions including Na+ and K+.36 To determine whether Ca2+ influx through TRPV4 channels is solely responsible for the regulation of eNOS activity by TRPV4 channels, we studied the effect of TRPV4 activation on NO levels in the presence of 0 mmol/L extracellular Ca2+. TRPV4 agonist GSK101 was unable to increase NO levels under these conditions (Figure 3D, right), confirming that Ca2+ influx through TRPV4 channels activated eNOS in small PAs.
To determine whether TRPV4 sparklets potentiate Ca2+ release from the ER in PAs, Ca2+ signals were recorded in PAs from mice that express the Ca2+ biosensor GCaMP2 selectively in ECs.27, 28 The effect of GSK101 on the activity of Ca2+ release events and TRPV4 Ca2+ sparklets was studied by utilizing the differences in kinetics between Ca2+ release signals from the ER (Ca2+ pulsars,2, 4 spikes with duration <300 ms, Figure S5A and S5B)4 and TRPV4 Ca2+ sparklets (discrete, square amplitudes, duration >300 ms, Figure S5A and S5B). Ca2+ release signals were unaffected by 0 mmol/L extracellular Ca2+ and TRPV4 inhibitor, but were inhibited by CPA (Figure S5C). TRPV4 sparklet activity was unaffected by CPA, but was inhibited by 0 mmol/L extracellular Ca2+ and TRPV4 inhibitor. GSK101 stimulated Ca2+ sparklet activity, but had no effect on the frequency of Ca2+ release events (Figure S5B and S5C), suggesting that Ca2+ release from the ER did not contribute to eNOS activation by TRPV4 sparklets.
NO‐Dependent Negative Feedback Loop Limits Ca2+ Influx Through TRPV4 Channels in PAs
Studies in expression systems reveal an increase in the activity of TRPV4 channels by S‐nitrosylation18 and a decrease by GC‐PKG signaling,19 and NO can activate both these mechanisms. We hypothesized that NO released by TRPV4‐eNOS signaling serves as an immediate feedback regulator of TRPV4 channel activity in small PAs. To determine the effect of NO on baseline TRPV4 sparklet activity, PAs were treated with L‐NNA (100 μmol/L) in the absence of GSK101. L‐NNA produced a 3‐fold increase in TRPV4 sparklet activity (Figure 4A), revealing TRPV4 channel inhibition by NO. TRPV4 inhibitor GSK219 completely blocked the L‐NNA‐induced increase in sparklet activity, confirming the specific inhibition of TRPV4 sparklets by NO (Figure 4A). Consistent with the NOS‐independent nature of vasodilation in small MAs, L‐NNA did not increase baseline sparklet activity in small MAs (Figure 4A, right). For a more detailed analysis of the effect of NO on TRPV4 sparklets in PAs, we increased the open state probability of TRPV4 channels by using a small concentration of GSK101 (6 nmol/L). L‐NNA also increased sparklet activity in the presence of GSK101 (Figure 4B). To further assess the inhibitory effect of NO on TRPV4 channel function, we examined the effect of NONOate on TRPV4 sparklet activity. At 10 and 30 μmol/L, NONOate increased DAF‐FM fluorescence in ECs and SMCs to levels similar to GSK101 (30 nmol/L; Figure S4B). Moreover, PA dilation caused by 30 nmol/L GSK101 was similar to that caused by 10 and 30 μmol/L NONOate (Figure S4C). We therefore used both 10 and 30 μmol/L NONOate for determining the effect of NO on TRPV4 sparklet activity. In the presence of L‐NNA, the activity of TRPV4 sparklets was inhibited by ≈2‐fold with 10 μmol/L NONOate and by 3‐fold with 30 μmol/L NONOate (Figure 4B, right) confirming that NO limits Ca2+ influx through TRPV4 channels in the endothelium from PAs.
We recently demonstrated that Ca2+‐dependent cooperative openings of TRPV4 channels amplify Ca2+ influx through the channels by 2‐ to 3‐fold in MAs.4, 5 The F/F0 traces for TRPV4 sparklets in PAs in the presence of L‐NNA displayed simultaneous openings of multiple channels at a site under control conditions, but a lesser number of channels opened simultaneously in the presence of NONOate (Figure 4B), implying that NO may interfere with cooperative openings of TRPV4 channels at a site. Using a coupled Markov chain model in Matlab, we determined coupling coefficients (κ) as an indicator of coupling strength among TRPV4 channels, as we have done previously.5 The κ values range between 0, which indicates no coupling, and 1, which indicates maximum coupling. Addition of L‐NNA increased the coupling strength among TRPV4 channels at a site, and NONOate reduced the coupling strength (Figure 4C). Using an arbitrary cutoff of κ=0.1, ≈50% of the sparklet sites showed coupled openings under control conditions, a number that was increased to 95% in the presence of L‐NNA and reduced to 35% by NONOate. These results confirmed that NO disrupted the functional coupling among TRPV4 channels at a site, thereby reducing Ca2+ influx in ECs.
NO Limits Ca2+‐Dependent Cooperativity of TRPV4 Channels via GC‐PKG Signaling
The commonly used inhibitors of S‐nitrosylation (N‐acetyl cysteine, dithiothreitol, and ultraviolet light43) did not affect the NO‐inhibition of TRPV4 channels (Figure 5A), suggesting that S‐nitrosylation does not play a major role in the NO‐inhibition of TRPV4 channels in small PAs. GC‐PKG signaling has been shown to inhibit TRPV4 channels in expression systems.19 We therefore tested the hypothesis that NO activates endothelial GC‐PKG signaling to limit Ca2+ influx through TRPV4 channels. Both ECs and SMCs from PAs showed a strong expression of PKG (Figure 5B). Similar to L‐NNA, GC inhibitor ODQ (3 μmol/L)44 and PKG inhibitor Rp‐8‐Br‐PET‐CGMPS (PET; 30 μmol/L)45 increased the baseline sparklet activity by ≈2‐fold (Figure 5C), confirming that GC‐PKG signaling constitutively inhibits TRPV4 channels in PA endothelium. In the presence of GC or PKG inhibitor, L‐NNA was unable to further increase the activity of TRPV4 sparklets (Figure 5C, left). Moreover, NONOate did not attenuate TRPV4 sparklet activity in the presence of GC or PKG inhibitor (Figure 5C, right). GC or PKG inhibitor also enhanced the coupling strength among TRPV4 channels (Figure 5D) by ≈2‐fold. In the presence of GC or PKG inhibitors, neither L‐NNA nor NONOate (10 and 30 μmol/L) was able to alter the coupling strength among TRPV4 channels at a site (Figure 5D). These results supported the novel concept that in small PAs, NO limits TRPV4 channel activity through GC‐PKG signaling in the native endothelium.
Ca2+‐dependent cooperativity of TRPV4 channels is a key endogenous regulatory mechanism for TRPV4 channel activity.4, 5, 20, 21 Using EGTA to chelate local Ca2+, we previously demonstrated that coupled openings of channels in a cluster were dependent on local increases in Ca2+.5 To determine whether NO‐GC‐PKG signaling disrupts the functional coupling between the channels by interfering with Ca2+‐dependent cooperativity, we studied the effect of NONOate on sparklet activity in the presence of EGTA‐AM, a cell‐permeable form of EGTA. EGTA lowered the sparklet activity and coupling strength among TRPV4 channels in PAs in the presence of L‐NNA (Figure 6A and 6B). Moreover, NONOate was unable to further reduce the activity of sparklets and coupling strength among TRPV4 channels in the presence of EGTA (Figure 6A and 6B). Whereas 43% of the total sparklet sites showed cooperative openings (κ>0.1) in the presence of EGTA alone, 41% of the sparklet sites showed cooperative openings after the addition of NONOate in the presence of EGTA, suggesting that NO did not further reduce the cooperativity of TRPV4 sparklets in the presence of EGTA. GSK219, however, was able to cause a further decrease in sparklet activity in the presence of EGTA (n=4 PAs). These results suggest that NO interferes with Ca2+‐dependent activation of TRPV4 channels, thereby limiting cooperative channel openings.
Based on the NO‐GC‐PKG negative feedback mechanism for regulating TRPV4 channel activity, we postulated that removing this inhibition would augment TRPV4‐vasodilation in PAs. In the presence of GC inhibitor ODQ, the dilation to GSK101 was markedly increased at each concentration (3–30 nmol/L) when compared with vasodilation in the absence of ODQ (Figure 6C and 6D), further supporting the role of NO‐GC‐PKG negative feedback mechanism as a “limiter” of Ca2+ influx through TRPV4 channels and TRPV4‐vasodilation in PAs.
ATP Is an Endogenous Activator of TRPV4 Sparklets in Endothelium From Small PAs
In small mesenteric and cremaster arteries, physiological muscarinic receptor agonist acetylcholine caused vasodilation predominantly through activation of endothelial TRPV4 sparklets.1, 4, 5 In small PAs, however, neither muscarinic receptor agonist (carbachol or CCh) nor bradykinin receptor agonist (bradykinin) was able to activate TRPV4 sparklets (Figure S6). Carbachol dilated large PAs (>400 μm), but was unable to evoke dilation in small PAs (Figure 7A), underscoring functional differences in the endothelium from large and small PAs. We, therefore, postulated that TRPV4 channels in ECs from small PAs couple to novel physiological activators. Previous studies in large PAs revealed that endogenous purinergic receptor activator ATP increases endothelial Ca2+ and causes endothelium‐dependent vasorelaxation.22, 23, 24, 46, 47 We, therefore, hypothesized that ATP dilates small PAs via activation of TRPV4 sparklets. ATP (10 μmol/L) induced an 8‐fold increase in TRPV4 sparklet activity in PAs, an effect that was inhibited by a general P2 purinergic receptor inhibitor suramin and TRPV4 inhibitor GSK219, and was absent in PAs from TRPV4−/− mice (Figure 7B), suggesting P2 purinergic receptor‐dependent activation of TRPV4 channels by ATP. In cannulated, pressurized small PAs, ATP (1–10 μmol/L) induced a concentration‐dependent dilation, which was inhibited by GSK219 and L‐NNA, and was absent in endothelium‐denuded PAs and PAs from TRPV4−/− mice (Figure 7C). It is possible that catabolism of ATP to ADP and/or adenosine may activate TRPV4 channels through P2Y148 and adenosine receptors, respectively. ADP itself did not induce dilation in PAs (Figure 7D). Adenosine dilated PAs, but this effect was not inhibited by TRPV4 inhibitor, thus ruling out a role for adenosine in ATP‐induced vasodilation of PAs (Figure 7E). Taken together, these results reveal that ATP is a novel endogenous activator of local TRPV4‐eNOS signaling in small PAs.
Despite the functional differences between systemic and pulmonary circulations, the identity of local Ca2+ signals that regulate vascular function in small PAs remains entirely unknown. Our discoveries of spatially distinct TRPV4 sparklets in small PAs and local TRPV4‐eNOS signaling network not only support a novel paradigm that eNOS can be activated by spatially restricted Ca2+ signals, but also identifies TRPV4 channels as a key regulator of basal and induced eNOS activity in pulmonary microcirculation. Moreover, inhibition of TRPV4 channel cooperativity by NO through GC‐PKG signaling represents a novel endogenous mechanism for the regulation of TRPV4 channel function. Although endothelial TRPV4 channels have been studied for several years, their physiological roles remain unclear. Our findings of ATP as a novel endogenous activator of TRPV4 channels may prove crucial for deciphering the physiological roles of TRPV4 channels. Because small PAs regulate vascular resistance, TRPV4 channel‐induced vasodilation of small PAs may be important for the regulation of pulmonary vascular resistance under normal and disease conditions. Pulmonary vascular disorders, including pulmonary arterial hypertension, are associated with reduced NO bioavailability and loss of endothelial function.49, 50, 51 It is plausible that an impairment in the TRPV4‐eNOS signaling results in endothelial dysfunction in these disorders. Inhaled NO represents a major advancement in acute treatment of pulmonary arterial hypertension, but sustained improvements in the clinical outcomes have not been achieved in adult patients.52 Inhibition of endothelial TRPV4 channels by inhaled NO may contribute to the negative outcome in pulmonary arterial hypertension. Inhibition of TRPV4 channel activity by NO‐GC‐PKG signaling will also have implications in the diseases characterized by excessive activation of TRPV4 channels, such as pulmonary edema and lung injury.53, 54, 55
Previous studies of endothelial Ca2+ signals have demonstrated that localized Ca2+ signals predominantly activate IK and SK channels to cause vasodilation in small systemic arteries including mesenteric, cremaster, and cerebral arteries.1, 2, 3, 4, 56 In small PAs, TRPV4 sparklets preferentially activated eNOS to cause vasodilation. Activation of eNOS by TRPV4 channels was absent in PAs from eNOS−/− mice (Figure 3B), was inhibited by TRPV4 channel inhibitor (Figure 3D, left), and required influx of extracellular Ca2+ (Figure 3, right). TRPV4 sparklets did not activate Ca2+ release from intracellular stores (Figure S5C). Additionally, all the Ca2+ influx signals in response to TRPV4 channel agonist were inhibited by TRPV4 channel inhibitor and were absent in PAs from TRPV4−/− mice (Figure 1A). These results, in combination with the local nature of TRPV4 sparklets, support a direct activation of eNOS by TRPV4 sparklets.
Selective coupling of TRPV4 sparklets with eNOS for vasodilation in small PAs supports differential Ca2+ signaling networks in small PAs and mesenteric/cremaster/cerebral arteries. Our data suggest that the presence of U46619 alone (Figure 2C and 2D) does not alter the IK/SK channel‐dependent nature of TRPV4‐vasodilation in MAs. The differences in signaling pathways may arise from drastic physiological differences in intravascular pressures in pulmonary and systemic circulations. The pressures used in this study mimicked the physiological intravascular pressures of the 2 vascular beds. Localization of Ca2+ signals and IK/SK channels at MEPs has been proposed as a mechanism for preferential activation of IK/SK channels by TRPV4 sparklets in small MAs and cremaster arterioles.1, 2 The non‐MEP localization of sparklets in small PAs is consistent with the IK/SK channel‐independent nature of TRPV4‐vasodilation in small PAs; however, the molecular mechanism underlying preferential TRPV4‐eNOS coupling in PAs is not clear. In MAs, the MEP‐localization of TRPV4 sparklet activity is attributed to A‐kinase anchoring protein 150 (AKAP150)‐mediated cooperative opening of TRPV4 channels at the MEPs.5 Surprisingly, AKAP150 staining was not observed at the MEPs in PAs (Figure 1E), and only a small fraction of TRPV4 sparklets occurred at the MEPs (Figure 1D). Caveolin, a major structural protein of caveolae, directly interacts with and inhibits eNOS activity.57 Endothelial hemoglobin‐α and cytochrome b5 reductase 3 (CytB5R3) have also been shown to regulate the effects of NO on vascular reactivity.58 It is, therefore, conceivable that differential expression of caveolin, hemoglobin‐α, and CytB5R3, along with the absence of AKAP150, is responsible for selective activation of eNOS over IK/SK channels in small PAs. Additionally, differences in the aforementioned mechanisms could be responsible for species‐, vessel size‐, and vascular bed‐specific coupling of TRPV4 sparklets with their signaling targets.
Although TRPV4 channels have been proposed as a key Ca2+ influx pathway in vascular endothelium, their endogenous activators remain elusive. The discovery of ATP as an endogenous activator of TRPV4‐eNOS signaling may be a crucial step towards deciphering the physiological and pathological roles of TRPV4 channels in the pulmonary circulation. Purinergic signaling is an essential component to the pulmonary vasculature,59, 60, 61, 62, 63 and ATP activation of TRPV4 channels represents a mechanism that may link physiological stimuli to the regulation of pulmonary vascular function. ATP can be released from sympathetic nerves during synaptic transmission, or can be released into the circulation by ECs, SMCs, or erythrocytes.25, 26, 64, 65 Shear stress, which is a well‐known activator of endothelial TRPV4 channels,66, 67, 68 also induces the release of ATP,65 suggesting a possible involvement of P2 purinergic receptor‐TRPV4 signaling in flow‐induced vasodilation. Endothelial Gq‐protein coupled receptor agonists carbachol and bradykinin activated TRPV4 channels through a phospholipase C‐diacylglycerol‐protein kinase C mechanism in MAs.5 ATP may also activate endothelial TRPV4 channels through Gq‐coupled P2 purinergic receptors, a possibility that will be explored in future studies.
While TRPV4 channels play a physiologically important role at a low level of activation, excessive TRPV4 channel activity can cause Ca2+ overload and EC death.4 In this context, a dual role for NO—as a mediator of TRPV4‐vasodilation and as a limiter of TRPV4 channel activity—provides a more precise control over channel activation and vasodilation. Prior studies in the expression systems reveal 2 possibilities for the modulation of TRPV4 channel function by NO: an increase in channel activity by S‐nitrosylation,18 and a decrease in channel activity by GC‐PKG signaling.19 Although S‐nitrosylation and activation of TRPV4 channels by NO cannot be ruled out, our results suggest that NO‐induced TRPV4 channel inhibition via GC‐PKG pathway predominates in intact PAs. Unlike PKG, protein kinase A and protein kinase C activation potentiates TRPV4 channel function via channel phosphorylation.69 These findings point to an interesting possibility that PKG phosphorylates the channel at a site that is different from protein kinase C or protein kinase A phosphorylation, and results in channel inhibition. Previous studies by Yin et al17 using Fura‐2 indicated that NO‐cGMP signaling lowers TRPV4‐induced increases in global Ca2+ levels in lung venular capillary endothelium. Whether this effect of NO is because of a direct effect on single channel function of TRPV4 channels remains unknown. Moreover, a functional effect of NO on TRPV4 channel‐induced vasodilation of small PAs is not known. The effect of NO‐GC‐PKG signaling on Ca2+‐dependent cooperative openings of TRPV4 channels represents a novel mechanism to control the diameter of small PAs in response to TRPV4 channel activation. How PKG phosphorylation of TRPV4 channel inhibits the coupling among TRPV4 channels is not known, but 1 possible mechanism could be inhibition of Ca2+ potentiation of the channel, thereby limiting the effect of Ca2+ on channel cooperativity. In support of this claim, our results demonstrate that in the presence of Ca2+ chelator EGTA, which limits TRPV4 channel cooperativity,5 NO had no effect on channel activity (Figure 6).
Our results also point to distinct functional roles of GC‐cGMP‐PKG signaling pathways from ECs and SMCs (Figure 8). Activation of the canonical GC‐PKG pathway in SMCs leads to vasodilation.70 Indeed, SMC GC has been targeted to dilate PAs and reduce pulmonary vascular resistance in pulmonary arterial hypertension.71 Interestingly, the finding that the endothelial GC‐PKG pathway has an inhibitory effect on vasodilation suggests that EC and SMC pathways work in opposite directions, and excessive activation of endothelial GC‐PKG signaling could lead to increased vasoconstriction and vascular resistance under pathological conditions. Moreover, the finding that GC inhibitor did not inhibit the vasodilation caused by EC‐derived NO (Figure 6C and 6D) suggests that in small PAs there is a vasodilatory component that is activated by NO and that acts independently of the SMC GC‐PKG signaling.
In conclusion, our results represent the first direct evidence that localized Ca2+ signals regulate NO release in small PAs. Reduced NO bioavailability is a major contributor to endothelial dysfunction in many pulmonary vascular disorders including pulmonary arterial hypertension. The perturbations of TRPV4‐eNOS pathway may provide a mechanistic explanation for reduced NO levels and endothelial dysfunction in these disorders. Our results also reveal a potent inhibitory role of NO on TRPV4 channel function, defining it as a novel physiological regulator (limiter) of TRPV4 channels (Figure 8). TRPV4 channels are Ca2+‐selective cation channels with a large single channel conductance; the amount of Ca2+ entering through 1 TRPV4 channel is ≈100 times higher than an L‐type Ca2+ channel.72 A slight overactivation of TRPV4 channels could lead to Ca2+ overload. It is, therefore, critical to tightly control the activity of these channels without inhibiting their physiological effect. The TRPV4‐eNOS signaling achieves this feat by mediating the functional effect of TRPV4 channels, and by limiting the channel function through endothelial GC‐PKG signaling. While some pulmonary vascular disorders show reduced NO levels,10, 50, 51, 73 others show excessive TRPV4 channel activity.53, 54, 55, 74, 75 Thus, we propose that abnormalities in the NO‐PKG‐TRPV4 feedback mechanism may be involved in both types of pulmonary dysfunctions.
Sources of Funding
This work was funded by grants from NIH to Sonkusare (HL121484 and HL138496), Marziano (4T32HL007284), Isakson (HL088554), UVA School of Medicine, and Robert M. Berne Cardiovascular Research Center startup funds to Sonkusare, and American Heart Association predoctoral Fellowship (17PRE33660762) to Marziano.
Figure S1. Specific TRPV4 channel agonists increase the number of sparklet sites per field of view in native ECs from fourth‐order PAs. EC indicates endothelial cells; TRPV4, transient receptor potential vanilloid 4.
Figure S2. TRPV4 sparklets increase local, but not whole‐cell Ca2+ levels in the ECs. EC indicates endothelial cells; TRPV4, transient receptor potential vanilloid 4.
Figure S3. TRPV4 sparklets represent unitary Ca2+ influx events through TRPV4 channels in the native endothelium from small PAs. PAs, pulmonary arteries; TRPV4, transient receptor potential vanilloid 4.
Figure S4. Spermine NONOate, a NO donor, increases DAF‐FM fluorescence in ECs and SMCs from small PAs and causes vasodilation in a concentration‐dependent manner. NO, nitric oxide; NONOate, ; PAs, pulmonary arteries; SMCs, smooth muscle cells; TRPV4, transient receptor potential vanilloid 4.
Figure S5. Ca2+ influx via endothelial TRPV4 channels does not increase Ca2+ release from the endoplasmic reticulum (ER) in PAs. PAs indicates pulmonary arteries; TRPV4, transient receptor potential vanilloid 4.
Figure S6. Classical activators of endothelial Gq protein‐coupled receptors, carbachol (CCh) and bradykinin, do not increase EC TRPV4 Ca2+ sparklet activity in small PAs. EC indicates endothelial cells; PAs, pulmonary arteries; TRPV4, transient receptor potential vanilloid 4.
Video S1. TRPV4 sparklets in fourth‐order pulmonary arteries (PAs) in an en face preparation. PAs were loaded with fluo‐4AM (10 μmol/L). Cyclopiazonic acid (CPA, 20 μmol/L) was used in order to eliminate the interference from ER Ca2+ release. The activity of TRPV4 sparklets was increased using TRPV4 channel agonist GSK101 (3 nmol/L). ER indicates endoplasmic reticulum; TRPV4, transient receptor potential vanilloid 4.
We would like to acknowledge GlaxoSmithKline for providing TRPV4−/− breeder mice, Dr Adrian Bonev (University of Vermont) for SparkAn, Dr Luis F. Santana (University of California, Davis) for providing the software program for determining coupling coefficients, and Dr Avril Somlyo for her comments on the manuscript.
↵† Ms Marziano and Dr Hong are co‐first authors.
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