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cANF causes endothelial cell hyperpolarization by activation of chloride channels
Peptides 30 (2009) 2337–2342
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cANF causes endothelial cell hyperpolarization by activation of chloride channels Aaron Simon a, Gong Xin Liu b,c, Gideon Koren b,c, Gaurav Choudhary a,c,* a
Vascular Research Laboratory, Providence VA Medical Center, USA Cardiovascular Research Center, Rhode Island Hospital, USA c Division of Cardiology, Warren Alpert Medical School of Brown University, USA b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2009 Received in revised form 6 July 2009 Accepted 5 August 2009 Available online 12 August 2009
Objectives: Natriuretic peptides bind with natriuretic peptide receptor (NPR)-C, which can alter cellular function through its interaction with the Gi protein complex. NPR-C has been found to mediate the activation of K+ channels and non-selective cation channels in vascular smooth muscle and cardiac fibroblast cells, respectively. However, the electrophysiological effect of NPR-C activation on endothelial cells (EC) has not been previously examined. In this study we sought to elucidate the effect of cANF(423), a selective NPR-C ligand, on EC membrane potential (Em). Methods/results: Changes in EC Em was measured through non-invasive fluorescence imaging. EC were preincubated in the potentiometric dye, DiBAC4(3) and subsequently exposed to cANF(4-23), in the presence of selective inhibitors of ionchannels or second messengers. NPR-C expression in rat lung microvascular endothelial cells was assessed by RT-PCR. cANF(4-23) induced a sustained decrease in EC cellular fluorescence, indicating endothelial cell hyperpolarization. The cANF-induced hyperpolarization could not be attenuated by TEA, barium, ouabain or by the reduction of extracellular Ca2+. Further, the cANF-induced hyperpolarization was insensitive to inhibition of Gi and protein kinase G (PKG), downstream messengers of NPRs. However, the Cl channel inhibitors, 4,40 -diisothiocyanatostilbene-2,20 -disulfonic acid, niflumic acid, and hypertonic saline attenuated the cANF-induced hyperpolarization. Perforated patch clamp recordings confirmed the cANF-induced current was carried by Cl and could be inhibited by niflumic acid. RT-PCR confirmed expression of NPR-C in vascular smooth muscle cells but not in EC. Conclusions: cANF causes hyperpolarization that is most likely mediated via activation of Cl channels by a PKG and Gi independent mechanism. Published by Elsevier Inc.
Keywords: Natriuretic peptide Natriuretic peptide receptor-C Endothelial cell Ion channel Membrane potential
1. Introduction The natriuretic peptide system consists of atrial natriuretic peptide, B-type natriuretic peptide and C-type natriuretic peptide (ANP, BNP and CNP, respectively) and three natriuretic peptide receptors (NPRs). NPR-A binds to ANP and BNP, NPR-B binds to CNP and NPR-C binds to all three natriuretic peptides. cANF(4-23) is a ring deleted ANP fragment that selectively binds to NPR-C [13]. NPR-A and -B are particulate guanylate cyclase receptors that increase intracellular cGMP while NPR-C was thought to be a clearance receptor that recently has been demonstrated to activate a pertussis (PTX) sensitive Gi protein resulting in anti-proliferative effects in vascular smooth muscle cells and modulation of vascular
* Corresponding author at: Providence VA Medical Center, 830 Chalkstone Ave., Providence, RI 02908, USA. E-mail address: [email protected] (G. Choudhary). 0196-9781/$ – see front matter . Published by Elsevier Inc. doi:10.1016/j.peptides.2009.08.006
tone [2,3,5,6,12,20]. Moreover, experiments with cANF in mesenteric circulation led to the hypothesis that CNP might act as an endothelium dependent hyperpolarizing factor (EDHF), and that this effect is mediated via NPR-C receptor [7]. There is a controversy as to whether CNP is EDHF in these vascular beds because of the lack of evidence of CNP-mediated effect on endothelial cell membrane potential and intracellular calcium [21]. In addition to the paracrine effects of EDHF and endotheliumderived CNP on vascular smooth muscle cells, the autocrine effects on the endothelium are also important. One of the mechanisms proposed for the effects of EDHF is hyperpolarization of endothelial cells with a tonic hyperpolarizing effect on vascular smooth muscle cells mediated through gap junctions. Hyperpolarization of endothelial cells would also increase the driving gradient for extracellular calcium to enter the cells via non-specific action and/ or trp channels. Hence, we sought to elucidate if cANF, a NPR-C receptor agonist, is involved in the hyperpolarization of lung microvascular endothelial cells (LMVECs) and evaluated the down stream mechanism of this effect.
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2. Methods 2.1. Cell culture Rat LMVEC used for all experiments were commercially isolated from rat lungs, characterized as endothelial cells by VEC technologies (Rochester, NY), and confirmed as such by demonstrating uptake of acetylated LDL and expression of von-Willebrand factor. Also, LMVEC stained with the lectins, Helix pomatia and Griffonia (Bandeiraea) simplicifolia, in a pattern consistent with that previously reported, with a greater level of Griffonia (Bandeiraea) simplicifolia staining, relative to Helix pomatia [22]. The cells were used at passages 3 through 8 for all experiments. The cells were cultured on 0.2% gelatin coated glass cover slips in complete molecular, cellular, and developmental biology (MCDB) media (VEC Technologies, NY) for 24–48 h prior to study. Rat pulmonary artery vascular smooth muscle cells (RPASMC) were purchased from Cellprogen (Cellprogen Inc., San Pedro, CA) and cultured in media and flasks with extracellular matrix supplied by Cellprogen (Cellprogen Inc.). 2.2. Measurement of membrane potential Fluorescent measurement of Em was performed using cells loaded with the Em-sensitive dye, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) (493 nM Abs/516 nM Em) [22]. LMVEC were grown on glass coverslips, washed with phosphate buffered saline (PBS), and incubated with 50–100 nM DiBAC4(3) in physiological salt solution (PSS) containing 141 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES buffer, and 10 mM glucose for 30 min. Subsequently, the cover slips were transferred to a closed bath-imaging chamber mounted to an inverted fluorescence microscope interfaced with a digital camera, which was contained within a Solent incubator maintaining a temperature of 37 8C. The chamber was superfused with PSS containing 50–100 nM DiBAC4(3) at a constant rate of 1 ml/ min, and the cells were allowed to reach steady baseline fluorescence prior to treatment. At time of treatment, samples were superfused with either a control media consisting of PSS and 50–100 nM DiBAC4(3) or the control media containing, in addition, a chemical agonist. Images were acquired using automated acquisition sequence with Phylum Software every 10 s. At each time point, the image was focused under DIC immediately prior to acquisition of fluorescent images. Fluorescent images were obtained with lEx(405 nm)/lEm(516 nM) under 30 magnification using constant exposure times. For each image, the cellular fluorescence was isolated through background subtraction and normalized to baseline fluorescence at the time of treatment. 2.3. Calibration of bis-oxanol dye The fluorescent dye DiBAC4(3) permeates the cell in a Emdependant manner. Increasing cell Em corresponds with increased intracellular DiBAC4(3) concentration and cellular fluorescence while decreasing Em corresponds with decreased fluorescence. A 1% change in fluorescence intensity corresponds roughly to a 0.6 mV change in Em. Membrane potentials were determined based on the Nernst equation.
the inhibitors along with the agonist in the superfusate. The following inhibitors were used in the study: 10 mM tetraethylammonium (TEA), 100 mM BaCl2, 1 mM KT5823, 250 mM ouabain, 100 mM niflumic acid, 1 mM DIDS and 4 mg/ml PTX. Ca2+-free PSS was used for selected experiments. High Potassium PSS (95.7 mM NaCl, 50 mM KCl, 1.2 mM MgCl2, 10 mM HEPES buffer and 10 mM glucose) was also used in selected experiments. Hypertonic solution was prepared by adding 90 mM sucrose to PSS. 2.5. Electrophysiology Perforated patch clamp recordings were performed on freshly dissociated cells plated on 35 mm culture dishes. Recordings were made with an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA). Briefly, the pipette resistances were 2–4 MV when filled with 200 mg/ml Nystatin, 30 mM KCl, 110 mM Kglutamate, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The extracellular bath solution contained (in mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 7.5 glucose, and 5 HEPES (pH 7.2). In selected experiments high chloride pipette solution was used: 200 mg/ml Nystatin, 140 KCl mM, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The currents were recorded at a temperature of 21–23 8C. The holding potential was 0 mV. The cells were superfused with the bath solution for 10 min prior to the start of recording. The patch pipettes were briefly dipped in nystatin free pipette solution prior to backfilling with the nystatin containing pipette solution. Pulse train of 5 mV steps was use to monitor for perforation of the patch that occurred in 10–15 min. Once the access resistance was <20 MV and the cell capacitance was constant, the acquisition protocols were started. Two protocols were used with voltageclamp configuration: a step protocol with the test potential ranging from 100 to +100 mV and lasting 400 ms and a ramp protocol with ramps from 100 to +100 mV for 600 ms. All data was acquired using Clampex software and analyzed with Clampfit software (Molecular Devices). Data are expressed as mean SE. Junction potential was calculated for all solution combinations using Clampex and the holding potentials were corrected for junction potential for analysis. 2.6. Confirmation of NPR-C mRNA expression Cultured adherent cells were disaggregated by application of 2 trypsin in PBS and suspended in media. Cells were pelleted by centrifugation at 93 (gravity) for 5 min and RNA was extracted following the Trizol (Invitrogen Inc., Carlsbad, CA) protocol. cDNA was created from extracted RNA following the recommended protocol for use with iScript cDNA Synthesis Kit (BioRad Laboratories, Hercules, CA). PCR was subsequently performed following protocol for iTaq Polymerase Kit (BioRad Laboratories) using primers for GAPD (www.realtimeprimers.com) (forward: 50 AGA CAG CCG CAT CTT CTT GT-30 ; reverse: 50 -CTT GCC GTG GGT AGA GTC AT-30 ) and NPR-C (Integrated DNA Technologies Inc.). Two primers were used for NPR-C: NPR-C1 (forward: 50 -ATC CAG CAG ACT TGG AAC AGG ACA-30 ; reverse: 50 -AGC TGT TGG TGT GCT CCA CAA TTC-30 ) and NPR-C2 (forward: 50 -TCC AGC AGA CTT GGA ACA GGA CAT-30 ; reverse: 50 -AGC TGT TGG TGT GCT CCA CAA TTC30 ). Electrophoresis of PCR product was run a on a 1.5% agarose gel. 2.7. Statistics
2.4. Agonists and inhibitors The ion channel inhibitors used were purchased from Sigma (Sigma–Aldrich Inc., St. Louis, MO) In the experiments with inhibitors, the cells were preincubated with the inhibitors for 30 min during the loading step with DiBAC4(3). The concentration of inhibitors was kept constant during the experiment by adding
The values of fluorescent intensity in the individual cells (5– 10 cells/field) on each coverslip were calculated, averaged, and represented as n. Experimental treatments were repeated in separate cover slips 3–14 times for each treatment. Statistical differences were determined by one-way ANOVA and Student’s ttest and were considered significant at p < 0.05.
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3. Results 3.1. cANF causes endothelial cell hyperpolarization In order to non-invasively evaluate Em changes in the LMVEC, we utilized the fluorescent and potentiometric dye DiBAC4(3). In order to correlate observed changes in cellular fluorescence with changes in Em, we exposed samples to 10 mM gramicidin and variable concentrations of external Na+ [8]. Based on the Nernst equation, assuming equal permeability for K+ and Na+, we calculated that a 1% increase in background-subtracted fluorescence corresponded to a 0.6 mV increase in membrane potential in our experimental setup [22]. Adding cANF to the PSS/DiBAC4(3) perfusate caused a time dependent decrease in mean cellular fluorescence, indicating the hyperpolarization of the LMVEC. Fluorescence began to decrease immediately upon exposure to 1 mM cANF and reached, on average, a level of 79 3% below baseline after 6 min (n = 7), representing an average Em decrease of 13 2 mV (Fig. 1A). There
Fig. 2. K+ channels do not mediate cANF-induced hyperpolarization in rat lung microvascular endothelial cells. Comparison of cellular fluorescence relative to baseline after 6 min of treating cells with vehicle or 1 mM cANF in the absence or presence of K+ channel blockers. *p < 0.05 vs. control. Mean SEM.
was a dose dependent effect of cANF on endothelial cell hyperpolarization. At doses of 0.1, 0.03, and 0.01 mM; cANF reduced the Em by 15 4, 11 3, and 4 4 mV, respectively (Fig. 1B). The maximum dose of cANF (1 mM) was chosen for subsequent experiments to be comparable with prior publications studying the effect of cANF on endothelial cells [10,11]. 3.2. Ion-channels mediating cANF-induced hyperpolarization
Fig. 1. cANF hyperpolarizes pulmonary microvascular endothelial cells. (A) Effects of 1 mM cANF (open circles, n = 10) or vehicle (closed circles, n = 5) on cellular fluorescence over time. Background-subtracted fluorescence is normalized to baseline at each time point. Membrane potential changes were measured by incubating cells in DiBAC4(3) and monitoring treatment-dependent changes in cellular fluorescence over time. With DiBAC4(3), a decrease in cellular fluorescence indicates a decrease in Em. Arrow points to time = 0, when cANF/vehicle was added. (B) Changes in cellular fluorescence after 6 min of treating cells with variable cANF doses and vehicle.
Next, we sought to determine the ion-channels responsible for the cANF-induced hyperpolarization using chemical inhibitors. Pretreatment with a relatively high concentration of TEA (10 mM), a non-specific K+ channel blocker did not affect the cANF-induced hyperpolarization (Fig. 2), suggesting K+ channels do not play an important role in cANF-induced EC hyperpolarization. Pretreatment with 250 mM ouabain, a Na+/K+ ATPase blocker, 100 mM Ba2+, a selective blocker of the inwardly rectifying potassium channel (Kir), or both did not inhibit cANF-induced hyperpolarization either (Fig. 2). In contrast, pretreatment of cells with 100 mM niflumic acid or 1 mM DIDS significantly attenuated the cANF-induced hyperpolarization suggesting that Cl channels play a role in this process (Fig. 3). Depletion of extracellular calcium for 30 min using Ca2+ free PSS had no effect on cANF-induced hyperpolarization, suggesting that this is a Ca2+ independent effect (Fig. 3) while pretreatment with hypertonic PSS abolished the cANF-induced hyperpolarization (Figs. 3 and 4). Interestingly, preincubation with niflumic acid and hypertonic saline attenuated CNP-mediated hyperpolarization too (decrease in DiBAC4(3) fluorescence at 6 min: hypertonic saline + 1 mM CNP: 13 5%; 100 mM niflumic acid + 1 mM CNP: 3 1%; 1 mM CNP alone: 33 4% [22]). In order to confirm that cANF-induced current is mediated by a chloride channel; we performed perforated patch clamp recordings on LMVEC. The cells were clamped at 0 mV and voltage steps ranging from +100 to 100 mV were delivered at baseline (Fig. 5A, panel a), after exposure to cANF in absence (Fig. 5A, panel b) or presence of niflumic acid (Fig. 5A, panel c). 1 mM cANF induced an outwardly rectifying current with a reversal potential of 29 mV (predicted Erev for Cl: 39 mV) in 5/7 cells (Fig. 5B). Niflumic acid blocked this current (Fig. 5C). To confirm that this was chloride current we changed the pipette solution to high chloride solution (Clin = 142 mM, predicted Erev for Cl: 1.3 mV). Under these conditions, the outwardly rectifying current had a reversal potential of 8 mV (n = 4).
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Fig. 4. Effects of 1 mM cANF in presence (n = 6) and absence of hypertonic PSS on cellular fluorescence over time. Background-subtracted fluorescence is normalized to baseline at each time point. Reference line indicates time = 0, when cANF was added. Mean SEM. Fig. 3. Cl channels may mediate cANF-induced hyperpolarization in LMVEC. Comparison of average cellular fluorescence relative to baseline after 6 min of treating cells with either vehicle or 1 mM cANF in the absence or presence of Ca2+ free PSS (n = 5), 1 mM DIDS (n = 11), 100 mM niflumic acid (n = 14), and hypertonic PSS (n = 6). * Indicates p < 0.05 vs. control, # indicated p < 0.05 vs. cANF. Mean SEM.
Next, we sought to evaluate the role of downstream signaling mechanisms associated with NPRs. Pretreatment with 4 mg/ml PTX did not attenuate cANF-induced hyperpolarization (Fig. 6). Also, KT5823, a PKG inhibitor, did not inhibit the effect of cANF on endothelial cell Em (Fig. 6). 3.3. Microvascular cells do not express NPR-C The lack of sensitivity of PTX on the effects of cANF prompted us to evaluate the NPR-C receptor expression in LMVEC. We performed RT-PCR of mRNA from rat lung and heart MVEC as well as rat pulmonary VSMC. Using two different primer sets, we were unable to detect any NPR-C mRNA in microvascular lung endothelial cells (Fig. 7). These results were consistent across passage 4–6. Conversely, the VSMC exhibited the NPR-C mRNA.
4. Discussion Endothelial cells are non-excitable and depend on modulation of their Em to facilitate calcium entry in order to fill intracellular calcium stores. In addition, extracellular calcium entry may play an important role in release of vasoactive mediators, such as nitric oxide and EDHF, in modulation of proliferation, monolayer permeability and inflammation. It is believed that the three classes of ion-channels that play an important role in modulating membrane potential are K+ channels, Cl channels and nonspecific cation channels [16]. We found that cANF hyperpolarizes endothelial cells via Cl channel dependent mechanism by using pharmacological blockers of Cl channels and fluorescent measurement of Em. The perforated patch clamp recordings confirmed that the cANF-activated current was carried by Cl. The shift in reversal potential of the cANF-induced current was in the same direction as the shift in calculated reversal potential of Cl. Furthermore, the cANF-inducible current and the current blocked by niflumic acid are similar confirming that the current was indeed carried by Cl channels. These results were very different than our
Fig. 5. (A) cANF induces outward current that is inhibited by niflumic acid. Representative family of current tracings using voltage-clamp step protocol (100 to +100 mV steps of 25 mV) at baseline (a), after 10 min of exposure to 1 mM cANF (b), and subsequent exposure to 1 mM cANF + 100 mM niflumic acid (c). (B) I–V curve showing mean subtracted currents observed in presence of 1 mM cANF from baseline (b–a) (n = 5). (C) I–V curve showing mean subtracted currents observed in presence of cANF and niflumic acid from cANF alone (c–b) (n = 5). Mean SEM.
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Fig. 6. cANF-induced hyperpolarization is not mediated by traditional natriuretic peptide receptor second messengers. Comparison of cellular fluorescence relative to baseline after 6 min of treating cells with either vehicle or 1 mM cANF in the absence or presence of 1 mM KT5823 (n = 4) or 4 mg/ml Pertussis toxin (Ptx) (n = 3). * Indicates p < 0.05 vs. control. Mean SEM.
Fig. 7. Rat LMVEC do not express NPR-C receptor. Representative agarose gel electrophoresis result of PCR products obtained from RNA isolated from Rat PASMC (top panel) and rat LMVEC (bottom panel) using 2 different NPR-C specific DNA primers and GAPD as positive control.
recently reported affect of CNP on membrane potential [22]. While TEA, Ca2+ free PSS, and KT5823 completely blocked the CNPinduced hyperpolarization, they did not attenuate the cANFinduced hyperpolarization. Interestingly, we did observe that pretreatment with niflumic acid and hypertonic saline inhibited the CNP-induced hyperpolarization. These effects could be related to the effect of cell volume changes on K+ channel activity that has been observed by others [9]. Also, it is possible that availability of Cl channels is important in maintaining the hyperpolarization in response to BK channel activation. Further studies are required to evaluate this interaction between BK channels, cell volume, and Cl channels. We observed that DIDS and niflumic acid inhibited the cANFmediated hyperpolarization. Both these compounds block Ca2+ activated Cl (CaCl) current as well as volume regulated Cl current at these doses [15]. The lack of response to Ca2+ free solution suggests that extracellular entry is not required to mediate the change in Em. The prolonged Ca2+ free duration (30 min) in the presence of flow likely led to depletion of intracellular stores, making CaCl an unlikely candidate. In fact, we saw that in presence of Ca2+ free solution, the hyperpolarization was significantly greater. This observation suggests that extracellular calcium entry causes attenuation of cANF-induced hyperpolarization. Volume regulated Cl channels (VRC) are typically activated by cell swelling and exposure to hypotonic solution and inhibited by hypertonic solution. In our experiments, the osmolality of the vehicle and cANF solutions was similar. Pretreatment with hypertonic solution inhibited the cANF-induced hyperpolarization, suggesting that most likely cANF-activated VRC. Also, the ratio of
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outward to inward current induced by cANF during voltage steps was 1.77 0.2, closer to the reported rectification ratio of VRC (1.36 0.07) than that of CaCl (5.37 0.49) [17]. It has been noted that depending on membrane potential and the reversal potential for Cl (Ecl), activation of these channels can cause either hyper- or depolarization [15]. The membrane potential of endothelial cells has been reported to vary from 0 to 70 [18,23]. The Ecl of LMVEC is not known. Others have suggested that the [Cl]i in endothelial cells is 35 mM leading to a reversal potential of 39.2 mV. This would suggest that the endothelial cells Em was >39.2. In addition to osmotic regulation, VRC have been implicated in mechanosensitive repose of the endothelium, cell proliferation, angiogenesis, differentiation, and apoptosis (reviewed in [15]). Also, thrombin has been shown to activate the VRC in endothelial cells in a calcium independent manner suggesting a possible link of this channel in regulating endothelial permeability [14]. Further studies are needed to elucidate if cANF has any effect on these important endothelial functions mediated via its effect on Cl channels. We did not find any effect of TEA or Ba2+ on EC hyperpolarization. TEA and Ba2+ would block most calcium activated potassium channels, inwardly rectifying potassium channels and shear stress activated potassium channels [1] making these unlikely candidates underlying cANF-induced hyperpolarization. In presence of ouabain, we noted that the cANF-induced hyperpolarization was significantly greater. This finding could be explained by the voltage dependence of the Cl current. By inhibiting Na+/K+ ATPase, ouabain depolarizes the cell. Since, the Cl current is greater in depolarized cells it would likely lead to more Cl entry leading to more hyperpolarization. There are three natriuretic peptide receptors. NPR-A and NPR-B contain intrinsic guanylate cyclase domains and alter cellular physiology by increasing cellular cGMP. NPR-C is thought to function via coupling to the PTX-sensitive Gi protein complex. However, the electrophysiological effect of cANF was insensitive to preincubation in inhibitors of both of these signaling pathways. A number of studies have characterized the important role that NPRC plays in mediating vasodilatory effects in mesenteric arteries [7], anti-proliferative effects, ischemia-reperfusion in coronary circulation, activation of trp channels in cardiac fibroblasts, etc. [2,3,5,6,12,19]. By using cANF as a selective NPR-C agonist, it has been suggested that activation of NPR-C delays the barrier restoration in LMVEC after exposure to thrombin [11]. Since thrombin has been shown to activate VRC [14], and we show that cANF activates Cl channels; it is conceivable that the effect of cANF causing delayed restoration of barrier function after exposure to thrombin [11] is related to cANF actions on the Cl channels. Whether, any of the other effects of cANF could be mediated by its effect on Cl channels remains to be determined. NPR-C has been reported to be the most prevalent NPR in tissues [2,4]. However, the cellular distribution of the receptor has been less well studied. Unfortunately, due to lack of a specific inhibitor, cANF has been used extensively to isolate the role NPR-C plays in mediating these actions. A thorough evaluation of receptor expression and use of molecular suppression techniques such as siRNA have not been utilized in many of the studies. We report that cANF can have actions in the absence of NPR-C expression. The underlying receptor/ligand that mediates this action is yet to be determined. This is a novel finding that has significant implications in the interpretation of the effects that have been attributed to NPR-C. We found that the effect of cANF on endothelial membrane potential was insensitive to pertussis toxin, further confirming that the underlying mechanism is independent of NPR-C/Gi pathway. Also, these results would be consistent with the lack of a NPR-B independent effect of C-type natriuretic peptide, which binds to both NPR-B and NPR-C, observed with LMVEC [22]. Though, there have been no splice variants reported for NPR-C in rats, it is
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possible that rat endothelial cells have a distinct isoform of NPR-C that was not detected by PCR. The focus of our study was on endothelial cells. It remains to be determined if cANF activates Cl channels in other cell types, such as VSMC that do have the NPR-C receptor and respond to cANF via NPR-C/Gi pathway. In summary, we report that cANF causes microvascular lung endothelial cell hyperpolarization mediated via Cl channel, an effect that is not mediated via NPR-C receptors. Funding Department of Veteran Affairs CDA-2 Award to GC. Conflict of interest None declared. Acknowledgements This material is based upon work supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. The contents of this manuscript do not represent the views of the Department of Veterans Affairs or the United States Government. References [1] Adams DJ, Hill MA. Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells. J Cardiovasc Electrophysiol 2004;15:598–610. [2] Anand-Srivastava MB. Natriuretic peptide receptor-C signaling and regulation. Peptides 2005;26:1044–59. [3] Anand-Srivastava MB, Srivastava AK, Cantin M. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. J Biol Chem 1987;262: 4931–4. [4] Anand-Srivastava MB, Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev 1993;45:455–97. [5] Cahill PA, Hassid A. Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells. Biochem Biophys Res Commun 1991;179:1606–13.
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Peptides journal homepage: www.elsevier.com/locate/peptides
cANF causes endothelial cell hyperpolarization by activation of chloride channels Aaron Simon a, Gong Xin Liu b,c, Gideon Koren b,c, Gaurav Choudhary a,c,* a
Vascular Research Laboratory, Providence VA Medical Center, USA Cardiovascular Research Center, Rhode Island Hospital, USA c Division of Cardiology, Warren Alpert Medical School of Brown University, USA b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2009 Received in revised form 6 July 2009 Accepted 5 August 2009 Available online 12 August 2009
Objectives: Natriuretic peptides bind with natriuretic peptide receptor (NPR)-C, which can alter cellular function through its interaction with the Gi protein complex. NPR-C has been found to mediate the activation of K+ channels and non-selective cation channels in vascular smooth muscle and cardiac fibroblast cells, respectively. However, the electrophysiological effect of NPR-C activation on endothelial cells (EC) has not been previously examined. In this study we sought to elucidate the effect of cANF(423), a selective NPR-C ligand, on EC membrane potential (Em). Methods/results: Changes in EC Em was measured through non-invasive fluorescence imaging. EC were preincubated in the potentiometric dye, DiBAC4(3) and subsequently exposed to cANF(4-23), in the presence of selective inhibitors of ionchannels or second messengers. NPR-C expression in rat lung microvascular endothelial cells was assessed by RT-PCR. cANF(4-23) induced a sustained decrease in EC cellular fluorescence, indicating endothelial cell hyperpolarization. The cANF-induced hyperpolarization could not be attenuated by TEA, barium, ouabain or by the reduction of extracellular Ca2+. Further, the cANF-induced hyperpolarization was insensitive to inhibition of Gi and protein kinase G (PKG), downstream messengers of NPRs. However, the Cl channel inhibitors, 4,40 -diisothiocyanatostilbene-2,20 -disulfonic acid, niflumic acid, and hypertonic saline attenuated the cANF-induced hyperpolarization. Perforated patch clamp recordings confirmed the cANF-induced current was carried by Cl and could be inhibited by niflumic acid. RT-PCR confirmed expression of NPR-C in vascular smooth muscle cells but not in EC. Conclusions: cANF causes hyperpolarization that is most likely mediated via activation of Cl channels by a PKG and Gi independent mechanism. Published by Elsevier Inc.
Keywords: Natriuretic peptide Natriuretic peptide receptor-C Endothelial cell Ion channel Membrane potential
1. Introduction The natriuretic peptide system consists of atrial natriuretic peptide, B-type natriuretic peptide and C-type natriuretic peptide (ANP, BNP and CNP, respectively) and three natriuretic peptide receptors (NPRs). NPR-A binds to ANP and BNP, NPR-B binds to CNP and NPR-C binds to all three natriuretic peptides. cANF(4-23) is a ring deleted ANP fragment that selectively binds to NPR-C [13]. NPR-A and -B are particulate guanylate cyclase receptors that increase intracellular cGMP while NPR-C was thought to be a clearance receptor that recently has been demonstrated to activate a pertussis (PTX) sensitive Gi protein resulting in anti-proliferative effects in vascular smooth muscle cells and modulation of vascular
* Corresponding author at: Providence VA Medical Center, 830 Chalkstone Ave., Providence, RI 02908, USA. E-mail address: [email protected] (G. Choudhary). 0196-9781/$ – see front matter . Published by Elsevier Inc. doi:10.1016/j.peptides.2009.08.006
tone [2,3,5,6,12,20]. Moreover, experiments with cANF in mesenteric circulation led to the hypothesis that CNP might act as an endothelium dependent hyperpolarizing factor (EDHF), and that this effect is mediated via NPR-C receptor [7]. There is a controversy as to whether CNP is EDHF in these vascular beds because of the lack of evidence of CNP-mediated effect on endothelial cell membrane potential and intracellular calcium [21]. In addition to the paracrine effects of EDHF and endotheliumderived CNP on vascular smooth muscle cells, the autocrine effects on the endothelium are also important. One of the mechanisms proposed for the effects of EDHF is hyperpolarization of endothelial cells with a tonic hyperpolarizing effect on vascular smooth muscle cells mediated through gap junctions. Hyperpolarization of endothelial cells would also increase the driving gradient for extracellular calcium to enter the cells via non-specific action and/ or trp channels. Hence, we sought to elucidate if cANF, a NPR-C receptor agonist, is involved in the hyperpolarization of lung microvascular endothelial cells (LMVECs) and evaluated the down stream mechanism of this effect.
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2. Methods 2.1. Cell culture Rat LMVEC used for all experiments were commercially isolated from rat lungs, characterized as endothelial cells by VEC technologies (Rochester, NY), and confirmed as such by demonstrating uptake of acetylated LDL and expression of von-Willebrand factor. Also, LMVEC stained with the lectins, Helix pomatia and Griffonia (Bandeiraea) simplicifolia, in a pattern consistent with that previously reported, with a greater level of Griffonia (Bandeiraea) simplicifolia staining, relative to Helix pomatia [22]. The cells were used at passages 3 through 8 for all experiments. The cells were cultured on 0.2% gelatin coated glass cover slips in complete molecular, cellular, and developmental biology (MCDB) media (VEC Technologies, NY) for 24–48 h prior to study. Rat pulmonary artery vascular smooth muscle cells (RPASMC) were purchased from Cellprogen (Cellprogen Inc., San Pedro, CA) and cultured in media and flasks with extracellular matrix supplied by Cellprogen (Cellprogen Inc.). 2.2. Measurement of membrane potential Fluorescent measurement of Em was performed using cells loaded with the Em-sensitive dye, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) (493 nM Abs/516 nM Em) [22]. LMVEC were grown on glass coverslips, washed with phosphate buffered saline (PBS), and incubated with 50–100 nM DiBAC4(3) in physiological salt solution (PSS) containing 141 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES buffer, and 10 mM glucose for 30 min. Subsequently, the cover slips were transferred to a closed bath-imaging chamber mounted to an inverted fluorescence microscope interfaced with a digital camera, which was contained within a Solent incubator maintaining a temperature of 37 8C. The chamber was superfused with PSS containing 50–100 nM DiBAC4(3) at a constant rate of 1 ml/ min, and the cells were allowed to reach steady baseline fluorescence prior to treatment. At time of treatment, samples were superfused with either a control media consisting of PSS and 50–100 nM DiBAC4(3) or the control media containing, in addition, a chemical agonist. Images were acquired using automated acquisition sequence with Phylum Software every 10 s. At each time point, the image was focused under DIC immediately prior to acquisition of fluorescent images. Fluorescent images were obtained with lEx(405 nm)/lEm(516 nM) under 30 magnification using constant exposure times. For each image, the cellular fluorescence was isolated through background subtraction and normalized to baseline fluorescence at the time of treatment. 2.3. Calibration of bis-oxanol dye The fluorescent dye DiBAC4(3) permeates the cell in a Emdependant manner. Increasing cell Em corresponds with increased intracellular DiBAC4(3) concentration and cellular fluorescence while decreasing Em corresponds with decreased fluorescence. A 1% change in fluorescence intensity corresponds roughly to a 0.6 mV change in Em. Membrane potentials were determined based on the Nernst equation.
the inhibitors along with the agonist in the superfusate. The following inhibitors were used in the study: 10 mM tetraethylammonium (TEA), 100 mM BaCl2, 1 mM KT5823, 250 mM ouabain, 100 mM niflumic acid, 1 mM DIDS and 4 mg/ml PTX. Ca2+-free PSS was used for selected experiments. High Potassium PSS (95.7 mM NaCl, 50 mM KCl, 1.2 mM MgCl2, 10 mM HEPES buffer and 10 mM glucose) was also used in selected experiments. Hypertonic solution was prepared by adding 90 mM sucrose to PSS. 2.5. Electrophysiology Perforated patch clamp recordings were performed on freshly dissociated cells plated on 35 mm culture dishes. Recordings were made with an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA). Briefly, the pipette resistances were 2–4 MV when filled with 200 mg/ml Nystatin, 30 mM KCl, 110 mM Kglutamate, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The extracellular bath solution contained (in mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 7.5 glucose, and 5 HEPES (pH 7.2). In selected experiments high chloride pipette solution was used: 200 mg/ml Nystatin, 140 KCl mM, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The currents were recorded at a temperature of 21–23 8C. The holding potential was 0 mV. The cells were superfused with the bath solution for 10 min prior to the start of recording. The patch pipettes were briefly dipped in nystatin free pipette solution prior to backfilling with the nystatin containing pipette solution. Pulse train of 5 mV steps was use to monitor for perforation of the patch that occurred in 10–15 min. Once the access resistance was <20 MV and the cell capacitance was constant, the acquisition protocols were started. Two protocols were used with voltageclamp configuration: a step protocol with the test potential ranging from 100 to +100 mV and lasting 400 ms and a ramp protocol with ramps from 100 to +100 mV for 600 ms. All data was acquired using Clampex software and analyzed with Clampfit software (Molecular Devices). Data are expressed as mean SE. Junction potential was calculated for all solution combinations using Clampex and the holding potentials were corrected for junction potential for analysis. 2.6. Confirmation of NPR-C mRNA expression Cultured adherent cells were disaggregated by application of 2 trypsin in PBS and suspended in media. Cells were pelleted by centrifugation at 93 (gravity) for 5 min and RNA was extracted following the Trizol (Invitrogen Inc., Carlsbad, CA) protocol. cDNA was created from extracted RNA following the recommended protocol for use with iScript cDNA Synthesis Kit (BioRad Laboratories, Hercules, CA). PCR was subsequently performed following protocol for iTaq Polymerase Kit (BioRad Laboratories) using primers for GAPD (www.realtimeprimers.com) (forward: 50 AGA CAG CCG CAT CTT CTT GT-30 ; reverse: 50 -CTT GCC GTG GGT AGA GTC AT-30 ) and NPR-C (Integrated DNA Technologies Inc.). Two primers were used for NPR-C: NPR-C1 (forward: 50 -ATC CAG CAG ACT TGG AAC AGG ACA-30 ; reverse: 50 -AGC TGT TGG TGT GCT CCA CAA TTC-30 ) and NPR-C2 (forward: 50 -TCC AGC AGA CTT GGA ACA GGA CAT-30 ; reverse: 50 -AGC TGT TGG TGT GCT CCA CAA TTC30 ). Electrophoresis of PCR product was run a on a 1.5% agarose gel. 2.7. Statistics
2.4. Agonists and inhibitors The ion channel inhibitors used were purchased from Sigma (Sigma–Aldrich Inc., St. Louis, MO) In the experiments with inhibitors, the cells were preincubated with the inhibitors for 30 min during the loading step with DiBAC4(3). The concentration of inhibitors was kept constant during the experiment by adding
The values of fluorescent intensity in the individual cells (5– 10 cells/field) on each coverslip were calculated, averaged, and represented as n. Experimental treatments were repeated in separate cover slips 3–14 times for each treatment. Statistical differences were determined by one-way ANOVA and Student’s ttest and were considered significant at p < 0.05.
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3. Results 3.1. cANF causes endothelial cell hyperpolarization In order to non-invasively evaluate Em changes in the LMVEC, we utilized the fluorescent and potentiometric dye DiBAC4(3). In order to correlate observed changes in cellular fluorescence with changes in Em, we exposed samples to 10 mM gramicidin and variable concentrations of external Na+ [8]. Based on the Nernst equation, assuming equal permeability for K+ and Na+, we calculated that a 1% increase in background-subtracted fluorescence corresponded to a 0.6 mV increase in membrane potential in our experimental setup [22]. Adding cANF to the PSS/DiBAC4(3) perfusate caused a time dependent decrease in mean cellular fluorescence, indicating the hyperpolarization of the LMVEC. Fluorescence began to decrease immediately upon exposure to 1 mM cANF and reached, on average, a level of 79 3% below baseline after 6 min (n = 7), representing an average Em decrease of 13 2 mV (Fig. 1A). There
Fig. 2. K+ channels do not mediate cANF-induced hyperpolarization in rat lung microvascular endothelial cells. Comparison of cellular fluorescence relative to baseline after 6 min of treating cells with vehicle or 1 mM cANF in the absence or presence of K+ channel blockers. *p < 0.05 vs. control. Mean SEM.
was a dose dependent effect of cANF on endothelial cell hyperpolarization. At doses of 0.1, 0.03, and 0.01 mM; cANF reduced the Em by 15 4, 11 3, and 4 4 mV, respectively (Fig. 1B). The maximum dose of cANF (1 mM) was chosen for subsequent experiments to be comparable with prior publications studying the effect of cANF on endothelial cells [10,11]. 3.2. Ion-channels mediating cANF-induced hyperpolarization
Fig. 1. cANF hyperpolarizes pulmonary microvascular endothelial cells. (A) Effects of 1 mM cANF (open circles, n = 10) or vehicle (closed circles, n = 5) on cellular fluorescence over time. Background-subtracted fluorescence is normalized to baseline at each time point. Membrane potential changes were measured by incubating cells in DiBAC4(3) and monitoring treatment-dependent changes in cellular fluorescence over time. With DiBAC4(3), a decrease in cellular fluorescence indicates a decrease in Em. Arrow points to time = 0, when cANF/vehicle was added. (B) Changes in cellular fluorescence after 6 min of treating cells with variable cANF doses and vehicle.
Next, we sought to determine the ion-channels responsible for the cANF-induced hyperpolarization using chemical inhibitors. Pretreatment with a relatively high concentration of TEA (10 mM), a non-specific K+ channel blocker did not affect the cANF-induced hyperpolarization (Fig. 2), suggesting K+ channels do not play an important role in cANF-induced EC hyperpolarization. Pretreatment with 250 mM ouabain, a Na+/K+ ATPase blocker, 100 mM Ba2+, a selective blocker of the inwardly rectifying potassium channel (Kir), or both did not inhibit cANF-induced hyperpolarization either (Fig. 2). In contrast, pretreatment of cells with 100 mM niflumic acid or 1 mM DIDS significantly attenuated the cANF-induced hyperpolarization suggesting that Cl channels play a role in this process (Fig. 3). Depletion of extracellular calcium for 30 min using Ca2+ free PSS had no effect on cANF-induced hyperpolarization, suggesting that this is a Ca2+ independent effect (Fig. 3) while pretreatment with hypertonic PSS abolished the cANF-induced hyperpolarization (Figs. 3 and 4). Interestingly, preincubation with niflumic acid and hypertonic saline attenuated CNP-mediated hyperpolarization too (decrease in DiBAC4(3) fluorescence at 6 min: hypertonic saline + 1 mM CNP: 13 5%; 100 mM niflumic acid + 1 mM CNP: 3 1%; 1 mM CNP alone: 33 4% [22]). In order to confirm that cANF-induced current is mediated by a chloride channel; we performed perforated patch clamp recordings on LMVEC. The cells were clamped at 0 mV and voltage steps ranging from +100 to 100 mV were delivered at baseline (Fig. 5A, panel a), after exposure to cANF in absence (Fig. 5A, panel b) or presence of niflumic acid (Fig. 5A, panel c). 1 mM cANF induced an outwardly rectifying current with a reversal potential of 29 mV (predicted Erev for Cl: 39 mV) in 5/7 cells (Fig. 5B). Niflumic acid blocked this current (Fig. 5C). To confirm that this was chloride current we changed the pipette solution to high chloride solution (Clin = 142 mM, predicted Erev for Cl: 1.3 mV). Under these conditions, the outwardly rectifying current had a reversal potential of 8 mV (n = 4).
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Fig. 4. Effects of 1 mM cANF in presence (n = 6) and absence of hypertonic PSS on cellular fluorescence over time. Background-subtracted fluorescence is normalized to baseline at each time point. Reference line indicates time = 0, when cANF was added. Mean SEM. Fig. 3. Cl channels may mediate cANF-induced hyperpolarization in LMVEC. Comparison of average cellular fluorescence relative to baseline after 6 min of treating cells with either vehicle or 1 mM cANF in the absence or presence of Ca2+ free PSS (n = 5), 1 mM DIDS (n = 11), 100 mM niflumic acid (n = 14), and hypertonic PSS (n = 6). * Indicates p < 0.05 vs. control, # indicated p < 0.05 vs. cANF. Mean SEM.
Next, we sought to evaluate the role of downstream signaling mechanisms associated with NPRs. Pretreatment with 4 mg/ml PTX did not attenuate cANF-induced hyperpolarization (Fig. 6). Also, KT5823, a PKG inhibitor, did not inhibit the effect of cANF on endothelial cell Em (Fig. 6). 3.3. Microvascular cells do not express NPR-C The lack of sensitivity of PTX on the effects of cANF prompted us to evaluate the NPR-C receptor expression in LMVEC. We performed RT-PCR of mRNA from rat lung and heart MVEC as well as rat pulmonary VSMC. Using two different primer sets, we were unable to detect any NPR-C mRNA in microvascular lung endothelial cells (Fig. 7). These results were consistent across passage 4–6. Conversely, the VSMC exhibited the NPR-C mRNA.
4. Discussion Endothelial cells are non-excitable and depend on modulation of their Em to facilitate calcium entry in order to fill intracellular calcium stores. In addition, extracellular calcium entry may play an important role in release of vasoactive mediators, such as nitric oxide and EDHF, in modulation of proliferation, monolayer permeability and inflammation. It is believed that the three classes of ion-channels that play an important role in modulating membrane potential are K+ channels, Cl channels and nonspecific cation channels [16]. We found that cANF hyperpolarizes endothelial cells via Cl channel dependent mechanism by using pharmacological blockers of Cl channels and fluorescent measurement of Em. The perforated patch clamp recordings confirmed that the cANF-activated current was carried by Cl. The shift in reversal potential of the cANF-induced current was in the same direction as the shift in calculated reversal potential of Cl. Furthermore, the cANF-inducible current and the current blocked by niflumic acid are similar confirming that the current was indeed carried by Cl channels. These results were very different than our
Fig. 5. (A) cANF induces outward current that is inhibited by niflumic acid. Representative family of current tracings using voltage-clamp step protocol (100 to +100 mV steps of 25 mV) at baseline (a), after 10 min of exposure to 1 mM cANF (b), and subsequent exposure to 1 mM cANF + 100 mM niflumic acid (c). (B) I–V curve showing mean subtracted currents observed in presence of 1 mM cANF from baseline (b–a) (n = 5). (C) I–V curve showing mean subtracted currents observed in presence of cANF and niflumic acid from cANF alone (c–b) (n = 5). Mean SEM.
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Fig. 6. cANF-induced hyperpolarization is not mediated by traditional natriuretic peptide receptor second messengers. Comparison of cellular fluorescence relative to baseline after 6 min of treating cells with either vehicle or 1 mM cANF in the absence or presence of 1 mM KT5823 (n = 4) or 4 mg/ml Pertussis toxin (Ptx) (n = 3). * Indicates p < 0.05 vs. control. Mean SEM.
Fig. 7. Rat LMVEC do not express NPR-C receptor. Representative agarose gel electrophoresis result of PCR products obtained from RNA isolated from Rat PASMC (top panel) and rat LMVEC (bottom panel) using 2 different NPR-C specific DNA primers and GAPD as positive control.
recently reported affect of CNP on membrane potential [22]. While TEA, Ca2+ free PSS, and KT5823 completely blocked the CNPinduced hyperpolarization, they did not attenuate the cANFinduced hyperpolarization. Interestingly, we did observe that pretreatment with niflumic acid and hypertonic saline inhibited the CNP-induced hyperpolarization. These effects could be related to the effect of cell volume changes on K+ channel activity that has been observed by others [9]. Also, it is possible that availability of Cl channels is important in maintaining the hyperpolarization in response to BK channel activation. Further studies are required to evaluate this interaction between BK channels, cell volume, and Cl channels. We observed that DIDS and niflumic acid inhibited the cANFmediated hyperpolarization. Both these compounds block Ca2+ activated Cl (CaCl) current as well as volume regulated Cl current at these doses [15]. The lack of response to Ca2+ free solution suggests that extracellular entry is not required to mediate the change in Em. The prolonged Ca2+ free duration (30 min) in the presence of flow likely led to depletion of intracellular stores, making CaCl an unlikely candidate. In fact, we saw that in presence of Ca2+ free solution, the hyperpolarization was significantly greater. This observation suggests that extracellular calcium entry causes attenuation of cANF-induced hyperpolarization. Volume regulated Cl channels (VRC) are typically activated by cell swelling and exposure to hypotonic solution and inhibited by hypertonic solution. In our experiments, the osmolality of the vehicle and cANF solutions was similar. Pretreatment with hypertonic solution inhibited the cANF-induced hyperpolarization, suggesting that most likely cANF-activated VRC. Also, the ratio of
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outward to inward current induced by cANF during voltage steps was 1.77 0.2, closer to the reported rectification ratio of VRC (1.36 0.07) than that of CaCl (5.37 0.49) [17]. It has been noted that depending on membrane potential and the reversal potential for Cl (Ecl), activation of these channels can cause either hyper- or depolarization [15]. The membrane potential of endothelial cells has been reported to vary from 0 to 70 [18,23]. The Ecl of LMVEC is not known. Others have suggested that the [Cl]i in endothelial cells is 35 mM leading to a reversal potential of 39.2 mV. This would suggest that the endothelial cells Em was >39.2. In addition to osmotic regulation, VRC have been implicated in mechanosensitive repose of the endothelium, cell proliferation, angiogenesis, differentiation, and apoptosis (reviewed in [15]). Also, thrombin has been shown to activate the VRC in endothelial cells in a calcium independent manner suggesting a possible link of this channel in regulating endothelial permeability [14]. Further studies are needed to elucidate if cANF has any effect on these important endothelial functions mediated via its effect on Cl channels. We did not find any effect of TEA or Ba2+ on EC hyperpolarization. TEA and Ba2+ would block most calcium activated potassium channels, inwardly rectifying potassium channels and shear stress activated potassium channels [1] making these unlikely candidates underlying cANF-induced hyperpolarization. In presence of ouabain, we noted that the cANF-induced hyperpolarization was significantly greater. This finding could be explained by the voltage dependence of the Cl current. By inhibiting Na+/K+ ATPase, ouabain depolarizes the cell. Since, the Cl current is greater in depolarized cells it would likely lead to more Cl entry leading to more hyperpolarization. There are three natriuretic peptide receptors. NPR-A and NPR-B contain intrinsic guanylate cyclase domains and alter cellular physiology by increasing cellular cGMP. NPR-C is thought to function via coupling to the PTX-sensitive Gi protein complex. However, the electrophysiological effect of cANF was insensitive to preincubation in inhibitors of both of these signaling pathways. A number of studies have characterized the important role that NPRC plays in mediating vasodilatory effects in mesenteric arteries [7], anti-proliferative effects, ischemia-reperfusion in coronary circulation, activation of trp channels in cardiac fibroblasts, etc. [2,3,5,6,12,19]. By using cANF as a selective NPR-C agonist, it has been suggested that activation of NPR-C delays the barrier restoration in LMVEC after exposure to thrombin [11]. Since thrombin has been shown to activate VRC [14], and we show that cANF activates Cl channels; it is conceivable that the effect of cANF causing delayed restoration of barrier function after exposure to thrombin [11] is related to cANF actions on the Cl channels. Whether, any of the other effects of cANF could be mediated by its effect on Cl channels remains to be determined. NPR-C has been reported to be the most prevalent NPR in tissues [2,4]. However, the cellular distribution of the receptor has been less well studied. Unfortunately, due to lack of a specific inhibitor, cANF has been used extensively to isolate the role NPR-C plays in mediating these actions. A thorough evaluation of receptor expression and use of molecular suppression techniques such as siRNA have not been utilized in many of the studies. We report that cANF can have actions in the absence of NPR-C expression. The underlying receptor/ligand that mediates this action is yet to be determined. This is a novel finding that has significant implications in the interpretation of the effects that have been attributed to NPR-C. We found that the effect of cANF on endothelial membrane potential was insensitive to pertussis toxin, further confirming that the underlying mechanism is independent of NPR-C/Gi pathway. Also, these results would be consistent with the lack of a NPR-B independent effect of C-type natriuretic peptide, which binds to both NPR-B and NPR-C, observed with LMVEC [22]. Though, there have been no splice variants reported for NPR-C in rats, it is
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possible that rat endothelial cells have a distinct isoform of NPR-C that was not detected by PCR. The focus of our study was on endothelial cells. It remains to be determined if cANF activates Cl channels in other cell types, such as VSMC that do have the NPR-C receptor and respond to cANF via NPR-C/Gi pathway. In summary, we report that cANF causes microvascular lung endothelial cell hyperpolarization mediated via Cl channel, an effect that is not mediated via NPR-C receptors. Funding Department of Veteran Affairs CDA-2 Award to GC. Conflict of interest None declared. Acknowledgements This material is based upon work supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. The contents of this manuscript do not represent the views of the Department of Veterans Affairs or the United States Government. References [1] Adams DJ, Hill MA. Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells. J Cardiovasc Electrophysiol 2004;15:598–610. [2] Anand-Srivastava MB. Natriuretic peptide receptor-C signaling and regulation. Peptides 2005;26:1044–59. [3] Anand-Srivastava MB, Srivastava AK, Cantin M. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. J Biol Chem 1987;262: 4931–4. [4] Anand-Srivastava MB, Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev 1993;45:455–97. [5] Cahill PA, Hassid A. Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells. Biochem Biophys Res Commun 1991;179:1606–13.
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