Nitric Oxide-Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

Abstract

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.

Keywords: calcium channel; calcium signaling; endothelial nitric oxide synthase; endothelium; microcirculation; pulmonary artery; signaling pathways; transient receptor potential vanilloid 4 channel; vascular endothelial function.

Figure 1

Native endothelium from small pulmonary arteries (PAs) displays transient receptor potential vanilloid 4 (TRPV4) sparklets with distinct spatial localization. Local Ca²⁺ influx events through TRPV4 channels (TRPV4 sparklets) were recorded in en face fourth‐order PAs and third‐order mesenteric arteries (MAs) loaded with fluo‐4AM (10 μmol/L). Cyclopiazonic acid (CPA, 20 μmol/L) was used in order to eliminate interference from Ca²⁺ release from the endoplasmic reticulum (ER). A, Top, the diagram indicates a fourth‐order PA from left lung that was used in this study (left). A grayscale image of a field of view with ≈15 endothelial cells (ECs; right). The dotted line indicates the outline of a single EC. Square boxes represent the regions of interest (ROIs) placed at the sparklet sites detected within a recording duration of 1 min. Arrows point to the holes in internal elastic lamina (IEL) that represent myoendothelial projections (MEPs). Middle, fractional fluorescence (F/F₀) traces were obtained from the ROIs shown in the top panel. The traces indicate sparklet activity under basal conditions (CPA), with the TRPV4 agonist GSK1016790A (GSK101) alone and in the presence of TRPV4 inhibitor GSK2193784 (GSK219). Dotted lines represent the single‐channel levels derived from all‐points histogram in (B). Bottom, averaged TRPV4 sparklet activity under basal conditions (CPA), in the presence of TRPV4 agonist GSK101, GSK101 in the absence or presence of 2 different TRPV4 channel inhibitors (GSK219, 100 nmol/L and HC067047 or HC067, 1 μmol/L) or 0 mmol/L extracellular Ca²⁺, another TRPV4 channel agonist RN1747 in the absence or presence of TRPV4 inhibitors GSK219 (100 nmol/L) and HC067 (1 μmol/L), and GSK101 (10 nmol/L) and RN1747 (1 μmol/L) in the PAs from TRPV4^−/− mice. Data are mean±SEM; TRPV4 sparklet activity (NP_O per site) was calculated using the quantal amplitude derived from (B); N represents the number of channels at a site and P_O is the open state probability of the channels (n=5 fields; P<0.0001 using 1‐way ANOVA and post hoc Tukey test; * and ^# indicate statistical significance [P<0.05] vs 10 nmol/L GSK101 and 1 μmol/L RN1747, respectively). B, All‐points histogram was constructed from F/F₀ traces pooled from 3 PAs and was fit with a multi‐Gaussian curve. The quantal levels (single‐channel amplitudes) were derived from the peaks of the multi‐Gaussian curve. C, Experiments were performed in arteries loaded with fluo‐4AM and Alexa Fluor 633 hydrazide. Representative images show black holes in the IEL that represent MEPs. Sparklet ROIs were superimposed with IEL staining. Arrows indicate MEP sites in the IEL (white) and non‐MEP sites (green) that overlapped with sparklet sites. D, (Left) Averaged data for localization of sparklet sites at MEPs in PAs and MAs. Data are mean±SEM (n=10 fields; P<0.05 using 2‐way ANOVA and post hoc Tukey test; *P<0.05 vs MEP). (Right) Average number of IEL holes per field in PAs and MAs. Data are mean±SEM (n=10 fields; P<0.05 using 2‐sample t test). E, AKAP150 staining was performed in en face third‐order MAs and fourth‐order PAs as described in the Methods section. (Left) Representative AKAP150 staining images from PAs and MAs, where green color indicates the autofluorescence of the internal elastic lamina (IEL), black holes in the IEL indicate MEPs, and red color indicates AKAP150‐staining. (Middle) Plot profiles of AKAP150 immunostaining for representative horizontal transects. Dotted lines indicate the positions of MEPs located at the holes in IEL. Images were acquired along the z‐axis (0.05‐μm optical slice). (Right) Averaged AKAP150 localization from MAs and PAs; AKAP150 immunostaining within 5 μm from the center of the holes in IEL was considered to be localized at the IEL (n= 5 arteries; *P<0.0001 using independent t test).

Figure 2

TRPV4 channel activation dilates cannulated, pressurized small PAs through endothelial nitric oxide synthase (eNOS) activation, and small MAs through IK/SK channel activation. Fourth‐order PAs from left lung were cannulated and pressurized to 15 mm Hg and third‐order MAs were pressurized to 80 mm Hg to record the changes in internal diameter. Both PAs and MAs were preconstricted with thromboxane analog U46619 (100 nmol/L). Dilation to NS309 (1 μmol/L), an activator of endothelial IK and SK channels, was used as a criterion to confirm functional viability of the endothelium. A, Representative traces for GSK101‐induced vasodilation under control conditions, in the endothelium‐denuded PAs, in the PAs from TRPV4^−/− mice, in the presence of IK and SK channel inhibitors (Tram‐34 and apamin, respectively), NOS inhibitor L‐NNA, and in PAs from eNOS ^−/− mice. Experiments in eNOS ^−/− mice were performed in the presence of iNOS inhibitor 1400W to account for a possible compensation by iNOS.42 B, (left to right) Averaged diameter responses to NS309 in control (n=37 arteries) and endothelium‐denuded (n=5 arteries) PAs, GSK101 in control PAs (n=8 arteries), in endothelium‐denuded PAs (n=5 arteries), in PAs from TRPV4^−/− mice (n=9 arteries), in the presence of GSK219 (n=4 arteries), Tram‐34+apamin (n=9 arteries), L‐NNA (n=11 arteries), 1400W (n=5 arteries), NPLA (nNOS inhibitor, n=8 arteries), and in PAs from eNOS ^−/− mice (n=5 arteries). Data are mean±SEM; P<0.05 using 2‐way ANOVA and post hoc Tukey test; *P<0.05 vs corresponding concentration under control conditions. C, Representative diameter traces for the effect of GSK101 on MA diameter in the absence (left) or presence (right) of the IK and SK channel inhibitors Tram‐34 and apamin, respectively. D, Averaged diameter data for GSK101‐induced dilations in the absence or presence of Tram‐34 and apamin (n=4 arteries; *P<0.05 using 2‐way ANOVA and post hoc Tukey test). EC indicates endothelial cell; L‐NNA, l‐N ^G‐nitroarginine; MAs, mesenteric arteries; NPLA, N^ω‐Propyl‐L‐arginine hydrochloride ; PAs, pulmonary arteries; TRPV4, transient receptor potential vanilloid 4.

Figure 3

TRPV4‐eNOS Ca²⁺ signaling mechanism regulates nitric oxide (NO) levels in small PAs. NO levels were recorded in en face fourth‐order PAs and third‐order MAs loaded with DAF‐FM (fluorescent NO indicator; 5 μmol/L). Images were acquired along the z‐axis using a spinning‐disk confocal microscope. A, Representative images for DAF‐FM fluorescence in ECs (top) and SMCs (bottom) of small PAs under basal conditions (left), and in the presence of GSK101 (middle) alone or with L‐NNA (200 μmol/L, right). B, Averaged raw fluorescence in ECs and SMCs from PAs of control (left) and eNOS ^−/− mice (right); data are mean±SEM; individual data points represent averaged fluorescence of all ECs or SMCs in a field of view (n=6 fields; P<0.0001 using 1‐way ANOVA and post hoc Dunnett test; *P<0.05 vs the baseline). C, Averaged DAF‐FM fluorescence in ECs and SMCs from PAs and MAs relative to the baseline fluorescence (n=6 fields; *P<0.0001 vs GSK101 for PAs using 1‐way ANOVA and post hoc Tukey test). D, Left, the effect of TRPV4 channel inhibition (GSK219) on baseline DAF‐FM fluorescence in both ECs and SMCs from small PAs (n=6 fields; *P<0.0001 vs baseline with independent t test); right, the increase in DAF‐FM fluorescence induced by GSK101 was abolished in the presence of 0 mmol/L extracellular Ca²⁺ (n=6 fields, P=0.9793 for ECs and 0.8087 for SMCs using independent t test). DAF‐FM indicates 4‐amino‐5 methylamino‐2′,7′‐difluorofluorescenin diacetate; ECs, endothelial cells; eNOS, endothelial nitric oxide synthase; L‐NNA, l‐N ^G‐nitroarginine; MAs, mesenteric arteries; PAs, pulmonary arteries; SMCs, smooth muscle cells; TRPV4, transient receptor potential vanilloid 4.

Figure 4

Nitric oxide disrupts cooperative openings of TRPV4 channels and lowers channel activity in small PAs. TRPV4 sparklets were recorded in en face fourth‐order PAs or third‐order MAs loaded with fluo‐4AM (10 μmol/L). Cyclopiazonic acid (CPA, 20 μmol/L) was used in order to eliminate the interference from Ca²⁺ release from intracellular stores. L‐NNA (100 μmol/L) was used as an inhibitor of NOS and spermine NONOate (NONOate, 10–30 μmol/L) was used as a NO donor. A, Representative F/F₀ traces of 3 distinct sparklet sites in a field of view under basal conditions (CPA) and in the presence of L‐NNA (left) in small PAs. Averaged TRPV4 sparklet activity expressed as NP_O per field, which is a summation of NP_O per site for all the sparklet sites in a field of view. N is the number of channels at a site and P_O is the probability of finding the channel in an open state (n=6 fields; P<0.05 using 2‐way ANOVA and post hoc Tukey test; *P<0.05 vs CPA) (right). B, Representative F/F₀ traces from 3 distinct sparklet sites in a field of view in the presence of GSK101, after the addition of L‐NNA, and NONOate in the presence of L‐NNA (left). Averaged TRPV4 sparklet activity (NP_O) per site; each data point represents the averaged sparklet activity per site for a field (n=7 fields; P<0.0001 using 1‐way ANOVA and post hoc Tukey test; *P<0.05 vs GSK101; ^# P<0.05 vs L‐NNA) (right). C, To estimate the coupling strength of TRPV4 channels at a sparklet site, we determined coupling coefficients or κ values using coupled Markov Chain model in Matlab. Representative F/F₀ traces with corresponding κ values for 4 distinct sparklet sites under control conditions (GSK101), in the presence of L‐NNA and in the presence of L‐NNA and 30 μmol/L NONOate (left). Dotted lines represent the quantal levels derived from all‐points histograms. Averaged κ values, data are mean±SEM (n=47 sites; P<0.0001 using 1‐way ANOVA and post hoc Tukey test; *P<0.05 vs GSK101) (right). L‐NNA indicates l‐N ^G‐nitroarginine; MAs, mesenteric arteries; NONOate, (Z)‐1‐[N‐[3‐aminopropyl]‐N‐ [4‐(3‐aminopropylammonio) butyl]‐amino]diazen‐1‐ium‐ 1,2‐diolate; NOS, nitric oxide synthase; PAs, pulmonary arteries.

Figure 5

Endothelial NO‐guanylyl cyclase (GC)‐protein kinase G (PKG) signaling disrupts cooperative openings of TRPV4 channels in small PAs. NONOate (30 μmol/L)‐induced suppression of TRPV4 sparklet activity was assessed after pharmacological inhibition of S‐nitrosylation with dithiothreitol (DTT, 1 mmol/L) or N‐acetyl cysteine (NAC, 5 mmol/L). In addition, ultraviolet (UV) light was applied to physically disrupt serine (S)‐NO covalent bonds formed by NO on EC TRPV4 channels. PKG expression and Ca²⁺ signals were recorded in en face fourth‐order PAs. TRPV4 sparklets were recorded in small PAs loaded with fluo‐4AM (10 μmol/L). The experiments were performed in the presence of CPA (20 μmol/L) to eliminate the interference from Ca²⁺ release from intracellular stores. L‐NNA (100 μmol/L) was used as an inhibitor of NOS and spermine NONOate (NONOate, 10–30 μmol/L) was used as a NO donor. To estimate the coupling strength among TRPV4 channels at a sparklet site, we determined coupling coefficients or κ values using a coupled Markov Chain model in Matlab. Cyclopiazonic acid (CPA, 20 μmol/L) was used throughout the experiments to eliminate intracellular Ca²⁺ signaling and exclusively assess Ca²⁺ influx through TRPV4 channels. A, Averaged TRPV4 sparklet activity (NP_O per site) and the effect of NONOate (30 μmol/L) in the presence of DTT (left; n=3 fields; *P=0.0083 using paired t test), NAC (middle; n=3 fields; *P=0.0190 using paired t test), and after UV exposure (right; n=3 fields; P=0.8576 using paired t test). Data are mean±SEM. B, Representative images for PKG immunostaining (left), nuclear staining with DAPI (middle) and a merged image (right) in the ECs (top) and SMCs (bottom) from a small PA. The experiments were repeated in 4 small PAs. C, Averaged data indicating the effect of the PKG inhibitor (Rp‐8‐Br‐PET‐cGMPS, PET, 30 μmol/L) and the GC inhibitor (ODQ, 3 μmol/L) on TRPV4 sparklet activity, and on L‐NNA (100 μmol/L) activation of TRPV4 sparklets (n=6 fields; P<0.001 using 1‐way ANOVA and post hoc Dunnett test; *P<0.05 vs control) (left). Averaged TRPV4 sparklet activity indicating the effect of NONOate in the presence of PKG (PET) and GC (ODQ) inhibitors (n=10 fields; P=0.4708 vs PET or ODQ using 1‐way ANOVA and post hoc Dunnett test) (right). PET or ODQ were added in the presence of L‐NNA (right panel), and did not cause a further increase in sparklet activity in the presence of L‐NNA. D, Averaged κ values under control conditions (GSK101, 6 nmol/L) and with PET or ODQ before or after the addition of L‐NNA (100 μmol/L). Data are mean±SEM (n=47 sites; P<0.0001 using 1‐way ANOVA and post hoc Dunnett test; *P<0.05 vs control) (left). Averaged κ values in the presence of PET or ODQ before or after the addition of NONOate. Data are mean±SEM (n=47 sites; P=0.2315 vs PET or ODQ using 1‐way ANOVA and post hoc Dunnett test) (right). EC indicates endothelial cells; DAP I4',6‐Diamidino‐2‐Phenylindole, GC, guanylyl cyclase; L‐NNA, l‐N ^G‐nitroarginine; NAC, N‐acetyl cysteine; NO, nitric oxide; NONOate, (Z)‐1‐[N‐[3‐aminopropyl]‐N‐ [4‐(3‐aminopropylammonio) butyl]‐amino]diazen‐1‐ium‐ 1,2‐diolate; PAs, pulmonary arteries; PKG, protein kinase; SMCs, smooth muscle cells; TRPV4, transient receptor potential vanilloid 4; UV, ultraviolet.

Figure 6

NO‐GC‐PKG signaling impairs Ca²⁺‐dependent cooperative openings of TRPV4 channels in small PAs. TRPV4 sparklets were recorded in PAs loaded with fluo‐4AM (10 μmol/L). The experiments were performed in the presence of CPA (20 μmol/L) to eliminate the interference from Ca²⁺ release from intracellular stores. L‐NNA (100 μmol/L) was used as an inhibitor of NOS and spermine NONOate (NONOate, 10–30 μmol/L) was used as a NO donor. Coupling coefficients or κ values for coupling among TRPV4 channels were determined using a coupled Markov Chain model in Matlab. Changes in internal diameter were recorded in cannulated fourth‐order PAs pressurized to 15 mm Hg. A, Experiments were performed in the presence of GSK101 (6 nmol/L) and L‐NNA (100 μmol/L). Representative F/F₀ traces indicate the effect of membrane‐permeable Ca²⁺ chelator EGTA‐AM and NONOate in the presence of EGTA‐AM on TRPV4 sparklet activity. B, Averaged TRPV4 sparklet activity per site indicating a decrease in sparklet activity with EGTA‐AM and lack of effect of NONOate on TRPV4 sparklet activity in the presence of EGTA‐AM (n=24 sites; P<0.0001 using 1‐way ANOVA and post hoc Dunnett test; *P<0.05 vs GSK101 control) (left). Averaged κ values, data are mean±SEM (n=24 sites; P<0.0001 using 1‐way ANOVA; *P<0.05 vs GSK101 control) (right). C, A representative diameter trace for GSK101‐induced vasodilations in small PAs pretreated with GC inhibitor ODQ (3 μmol/L). D, Averaged percent dilation to GSK101 (3–30 nmol/L) in PAs treated with ODQ (3 μmol/L) compared with control PAs. Data are mean±SEM (n=8 and 4 arteries for control and ODQ, respectively; P<0.05 using 2‐way ANOVA and post hoc Tukey test; *P<0.05 vs control). CPA indicates cyclopiazonic acid; GC, guanyl cyclase; L‐NNA indicates l‐N ^G‐nitroarginine; NO, nitric oxide; NONOate, (Z)‐1‐[N‐[3‐aminopropyl]‐N‐ [4‐(3‐aminopropylammonio) butyl]‐amino]diazen‐1‐ium‐ 1,2‐diolate; NOS, nitric oxide synthase; PAs, pulmonary arteries; PKG, protein kinase; TRPV4, transient receptor potential vanilloid 4.

Figure 7

ATP is an endogenous activator of local TRPV4‐eNOS signaling in small PAs. Changes in internal diameter were recorded in cannulated large PAs and small fourth‐order PAs pressurized to 15 mm Hg. TRPV4 sparklets were recorded in en face fourth‐order PAs loaded with fluo‐4AM (10 μmol/L). CPA (20 μmol/L) was used in order to eliminate the interference from Ca²⁺ release from intracellular stores. A, Representative diameter traces for acetylcholine analog carbachol (CCh)–induced vasodilation in large second‐order PAs (≈400 μm, top left) and small fourth‐order PAs (100–200 μm, bottom left). Averaged diameter responses to CCh; data are mean±SEM; (n=5 large PAs, 9 small PAs; *P<0.01 using independent t test) (right). B, Representative F/F₀ traces of TRPV4 sparklets under baseline conditions (CPA) and with the addition of ATP (10 μmol/L) (left). Averaged TRPV4 sparklet activity (NP_O) per field of view under basal condition (CPA), in the presence of ATP, ATP in the presence of TRPV4 inhibitor GSK219 (100 nmol/L), P2 purinergic receptor inhibitor suramin (500 μmol/L), or in the PAs from TRPV4^−/− mice (n=10 fields; P<0.0004 using 1‐way ANOVA and post hoc Tukey test; *P<0.05 vs baseline; ^# P<0.05 vs ATP) (right). C, Representative diameter traces for ATP‐induced dilation in fourth‐order PAs before (top left) and after (top right) addition of TRPV4 inhibitor GSK219 (100 nmol/L). (Bottom) Averaged percent dilation to ATP (1–10 μmol/L, n=7 arteries) in control PAs, in the presence of GSK219 (n=5 arteries), in EC‐denuded PAs (n=5 arteries), in the presence of L‐NNA (100 μmol/L, n=6 arteries), and in the PAs from TRPV4^−/− mice (n=5 arteries); data are mean±SEM; (P<0.05 using 2‐way ANOVA and post hoc Tukey test; *P<0.05 vs control). D, Representative diameter trace (left) and averaged data (right) for the effect of adenosine diphosphate (ADP) on PA diameter in the absence or presence of TRPV4 inhibitor GSK219 (100 nmol/L; gray; n=5 PAs; P>0.05 using 2‐way ANOVA and post hoc Tukey test). E, Representative diameter trace (left) and averaged diameter data (right) for adenosine‐induced dilations in the absence or presence of GSK219 (100 nmol/L; gray; n=4 arteries; P>0.05 using 2‐way ANOVA and post hoc Tukey test). CPA indicates cyclopiazonic acid; eNOS, endothelial nitric oxide synthase; L‐NNA, l‐N ^G‐nitroarginine; PAs, pulmonary arteries; TRPV4, transient receptor potential vanilloid 4.

Figure 8

Local TRPV4 channel‐dependent Ca²⁺ signaling regulates endothelium‐dependent vasodilation in resistance‐sized PAs. The diagram depicts TRPV4 channel‐dependent endothelial Ca²⁺ signaling mechanisms in small PAs and small mesenteric arteries.1, 4, 7 In small PAs, TRPV4 sparklets promote eNOS activity and NO release. P2 purinergic receptor agonist ATP is an endogenous activator of the TRPV4‐eNOS signaling in PAs. Endothelium‐derived NO then causes vasodilation through the activation of SMC guanylyl cyclase (GC)‐protein kinase G (PKG) signaling and GC‐PKG‐independent mechanisms. Blue arrows indicate this pathway. NO also induces activation of endothelial GC‐PKG signaling, which lowers Ca²⁺‐dependent cooperative opening of TRPV4 channels and limits TRPV4‐mediated vasodilations. This pathway is indicated by red arrows. In small mesenteric arteries, TRPV4 Ca²⁺ sparklets selectively activate endothelial IK and SK channels, which hyperpolarize EC and SMC membranes. SMC membrane hyperpolarization deactivates voltage‐dependent Ca²⁺ channels (VDCCs), which results in vasodilation, as described earlier. EC indicates endothelial cells; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; PAs, pulmonary arteries; PKG, protein kinase G; SMC, smooth muscle cell; TRPV4, transient receptor potential vanilloid 4.4