Donate Help Contact The AHA Sign In Home
American Heart Association
Hypertension
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Hypertension. 2006;48:157-164
Published online before print May 8, 2006, doi: 10.1161/01.HYP.0000223652.29338.77
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
48/1/157    most recent
01.HYP.0000223652.29338.77v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herrera, M.
Right arrow Articles by Garvin, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herrera, M.
Right arrow Articles by Garvin, J. L.
Related Collections
Right arrow Ion channels/membrane transport
Right arrow Endothelium/vascular type/nitric oxide
Right arrowRelated Article

(Hypertension. 2006;48:157.)
© 2006 American Heart Association, Inc.

Original Articles

Aquaporin-1 Transports NO Across Cell Membranes

Marcela Herrera; Nancy J. Hong; Jeffrey L. Garvin

From the Hypertension and Vascular Research Division (M.H., N.J.H., J.L.G.), Henry Ford Hospital, Detroit, Mich, and Department of Physiology (J.L.G.), Wayne State University, Detroit, Mich.

Correspondence to Jeffrey L. Garvin, PhD, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202. E-mail jgarvin1{at}hfhs.org


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
NO plays a role in the regulation of blood pressure through its effects on renal, cardiovascular, and central nervous system function. It is generally thought to freely diffuse through cell membranes without need for a specific transporter. The water channel aquaporin-1 transports low molecular weight gases in addition to water and is expressed in cells that produce or are the targets of NO. Consequently, we tested the hypothesis that aquaporin-1 transports NO. In cells expressing aquaporin-1, NO permeability correlated with water permeability. NO transport was reduced by 71% by HgCl2, an inhibitor of aquaporin-1. Transport of NO by aquaporin-1 saturated at 3 µmol/L NO and displayed a K1/2 (the concentration of NO that produces half of the maximum transport rate) of 0.54 µmol/L. Reconstitution of purified aquaporin-1 into lipid vesicles increased NO influx by 316%. In endothelial cells, lowering aquaporin-1 expression with a small interfering RNA (siRNA) blunted aquaporin-1 expression by 54% and NO release by 44%. We conclude that NO transport by aquaporin-1 may allow cells to control intracellular NO levels and effects. NO transport by aquaporin-1 may play a role in central nervous system, vascular and renal function, and consequently blood pressure. Disruption of NO transport by aquaporin-1 offers an alternate cause for diseases currently explained by inadequate NO bioavailability.


Key Words: nitric oxide


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Nitric oxide is a hydrophobic gas that plays an important role in the regulation of blood pressure1–6 through effects on the kidney,1,7–9 vasculature,10 and central nervous system.11 In the kidney it may promote natriuresis and diuresis by effects on glomerular filtration,3,12,13 salt and water absorption along the nephron,7 and tubuloglomerular feedback.14–17 In the vasculature NO can reduce blood pressure by dilating vessels.10,18 In the brain NO acts as a neurotransmitter.19,20 NO is thought to exit cells where it is produced and enter cells where it acts by freely diffusing through the lipid bilayer of the cell membrane without need for a specific transporter. Thus the cell membrane reportedly does not represent a significant barrier to diffusion of NO.

Aquaporins (AQPs) are intrinsic membrane proteins that form water-permeable protein complexes and are ubiquitously expressed in animals.21 Although the role of AQPs in water transport is clear in some cells, such as the renal epithelium,21 they are often highly expressed in cells where one might think high water permeability is not important. This raises the question of whether AQPs have a function other than water transport. The water channel aquaporin-1 (AQP-1) has been shown to transport small gas molecules such as carbon dioxide and ammonia in oocytes.22,23 Because AQP-1 is highly expressed in cells that produce NO,24–27 we investigated whether AQP-1 transports NO across cell membranes.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Cell lines were purchased from the American Type Culture Collection (Manassas, VA). CHO-K1 cells were cultured in low-glucose DMEM (Gibco, Carlsbad, Calif) supplemented with 10% FBS (Hyclone, Logan, Utah). The pancreatic endothelial cell line, MS1, was cultured in high-glucose DMEM supplemented with 5% FBS. Trypan blue solution, HgCl2, dimethyl sulfoxide (DMSO), L-arginine, acetylcholine (Ach), phenylephrine, Triton-X 100, PMSF, and N-laurosylsarcosine were obtained from Sigma (St. Louis, Mo). The polyclonal AQP-1 and the monoclonal NOS 3 antibodies were purchased from BD Biosciences (San Jose, Calif). The monoclonal antibody for GAPDH was obtained from Chemicon International (Temecula, Calif) and the anti-rabbit and anti-mouse IgG peroxidase–linked antibodies were from Amersham Pharmacia Biotech (Arlington Heights, Ill). The NO donor spermine NONOate was obtained from Cayman Chemicals (Ann Arbor, Mich) and the fluorescent dyes Cell Trace calcein red-orange, DAF-2 DA, and DAF-2 were from Molecular Probes (Eugene, Ore) and EMD Biosciences (San Diego, Calif), respectively. Octylglucoside (octyl-ß-D-glucopyranoside) was obtained from EMD. Purified Escherichia coli phospholipids were from Avanti Polar Lipids (Alabaster, Ala). Anticoagulated outdated normal human blood was obtained from the blood bank at Henry Ford Hospital.

Protein Content Determination
Total protein content was determined using Coomassie Plus reagent (Pierce, Rockford, Ill), based on Bradford’s colorimetric method.

Western Blot
Western blot was performed as described previously.28,29 Briefly, cells were scraped using 100 µL of lysis buffer. Equal amounts of total protein were loaded into each lane of a 12% or 8% sodium dodecyl sulfate (SDS)–polyacrylamide gel, separated by electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass). Membranes were incubated with a 1:2000 dilution of an AQP-1–specific antibody for 60 minutes and then with a 1:1000 dilution of secondary antibody against the appropriate IgG conjugated to horseradish peroxidase. For NOS 3 and GAPDH detection, 1:1000 and 1:100 000 dilutions of primary antibodies were used, respectively.

Plasmid Construction
The cDNA-encoding human bone marrow water channel AQP-1 in a Xenopus expression vector (ATCC)30 was subcloned in the mammalian expression vector pcDNA3.1/zeo(+) (Invitrogen, Carlsbad, Calif) at the HindIII and PstI restriction sites.

Transient Transfection
Lipid-mediated transfection of CHO-K1 cells was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were grown on 60-mm plates containing glass coverslips and transfected with 8-µg DNA [either AQP-1 subcloned in pcDNA3.1/zeo(+) or empty vector] per plate. AQP-1 expression was confirmed by Western blot.

Stable Transfection
Lipid-mediated transfection of CHO-K1 cells was carried out using Lipofectamine 2000. Briefly, cells were grown in 12-well plates and transfected with 1.6-µg DNA [either AQP-1 subcloned in pcDNA3.1/zeo(+) or empty vector] per well. Cells were transferred to 100-mm plates in fresh growth medium 24 hours after transfection. The selective antibiotic zeocin (Invitrogen, 250 µg/mL) was added the following day. After selection for 12 to 14 days, resistant colonies were isolated using cloning disks and transferred to separate culture dishes for expansion and analysis. AQP-1 expression was confirmed by Western blot.

NO Influx and Water Permeabilities
CHO-K1 cells transfected with either AQP-1 or empty vector were grown on glass coverslips until confluent and placed in a temperature-regulated chamber at 37°C. The composition of the bath (HEPES-buffered physiological saline) was (in mmol/L) NaCl 130, NaH2PO4 2.5, KCl 4, MgSO4 1.2, alanine 6, Na3 citrate 1, glucose 5.5, Ca (lactate)2 2, HEPES 10 at pH 7.4. The flow rate of the bath was 1 mL/min. Cells were first loaded by adding 2 µmol/L of the NO-specific fluorescent dye DAF-2 DA for 60 minutes. Then they were washed with physiological saline for 15 minutes. The dye was excited at 488 nm with an argon/krypton laser, and fluorescence emitted by NO-bound dye was measured with a scanning laser confocal microscope (Noran Instruments). After stable baseline fluorescence was established, 500 µmol/L spermine NONOate (SPM) (5 µmol/L NO) or a solution gassed with NO (from 0.1 to 100 µmol/L NO) was added to the bath. Then the increase in fluorescence, representing the increase in intracellular NO, was measured once every 5 or 10 s for 80 s. When inhibitors of AQP-1 were used, HgCl2 and DMSO were added to the cells after loading them with DAF-2 DA. The initial NO concentration was quantified using a precalibrated amperometric NO sensor (amiNO-2000, Harvard Apparatus). Because the NO-sensitive dye cannot be calibrated, NO permeability was expressed in fluorescence units/s adjusted for surface area and NO concentration gradient. Varying concentrations of NO gas were obtained as follows: HEPES-buffered physiological saline, pH 8.3 was gassed for 1 minute. This solution was then diluted 1:1 with HEPES-buffered physiological saline, pH 7.5, and NO concentration was measured with an NO-sensitive electrode (amiNO-2000). The half-life measured for NO decay under these conditions was used to determine when the desired NO concentration would be reached.

In some experiments, after completion of NO influx measurements, cells were loaded with 2.5 µmol/L Cell Trace calcein red-orange for 5 minutes and then washed for 5 minutes with physiological saline to measure water permeability (Pf). The dye was excited at 568 nm with an argon/krypton laser and stable baseline fluorescence established. The osmolality of the bath was decreased by 200 mOsm/kg (to 90 mOsm/kg) and the decrease in fluorescence, representing increase in cell volume, was measured once every 5 s for 50 s. Absolute Pf values were calculated as reported previously.31 Surface area of the cells was assessed by confocal microscopy.

AQP-1 and Scrambled AQP-1 Small Interfering RNA Synthesis
Small interfering RNAs (siRNAs) were constructed following the method of Splinter et al.32 Briefly, AQP-1 siRNA, which targets nucleotides 673 to 693 of the AQP-1 mRNA sequence, and a nonspecific siRNA containing the same nucleotides but in random sequence (scrambled AQP-1 siRNA), were prepared by a transcription-based method using the Silencer siRNA construction kit (Ambion) according to the manufacturer’s instructions. The 29-mer sense and antisense DNA oligonucleotide templates (21 oligonucleotides specific to AQP-1 and 8 nucleotides specific to T7 promoter primer sequence 5'-CCTGTCTC-3') were synthesized by TIB Molbiol (Adelphia).

Transfection with siRNAs
Sixty-millimeter plates of confluent MS1 cells were treated for 12 hours with 30 nmol/L AQP-1 siRNA, scrAQP-1 siRNA, or GAPDH siRNA (provided by the Silencer siRNA construction kit). The siRNAs were delivered with the lipid carrier Lipofectamine 2000. AQP-1 expression was assessed by Western blot of total cell homogenates.

NO Release
Confluent MS1 endothelial cells were incubated in fresh DMEM high-glucose medium supplemented with 5% FBS and 500 µmol/L L-arginine (the substrate for NO synthase; at this concentration L-arginine does not cause NO release) in the presence or absence of either HgCl2 or DMSO. Basal NO levels were recorded using an amperometric electrode selective for NO. Ach (1 µmol/L) was added to the bath to increase NO synthase activity. NO release was recorded for 2 minutes. Measurements were performed at 37°C.

AQP-1 Purification
AQP-1 from human red blood cells was purified following the method used by Bennet et al33 and Zeidel et al.34 Briefly, 1 U of outdated human blood was centrifuged and the buffy coat completely removed. Red cells were then washed and erythrocyte "ghosts" obtained by hypotonic treatment in the presence of protease inhibitors. Membrane vesicles were obtained after extraction with 1 mol/L potassium iodide and were further extracted with 1% (wt/vol) N-lauroylsarcosine. The pellet was collected by centrifugation at 4°C. It was washed once in 7.5 mmol/L sodium phosphate and solubilized by shaking for 1 hour at room temperature in 800 mL of a buffer containing 4% Triton-X 100. After centrifugation at 30 000g for 6 h, the sample was filtered through a 0.22-µm Millex GV membrane (Millipore) and loaded onto a POROS HQ anion exchanger column (Applied Biosystems) equilibrated with a buffer containing 1% Triton-X 100 running at 3 mL/min while attached to a Perkin-Elmer fluorometer. Eluted peaks were detected by their emission at 345 nm on excitation at 289 nm. The column was eluted with a 120-mL gradient of 0.10 to 1.20 mol/L NaCl in the same buffer while monitoring fluorescence. The peak eluted between 0.25 to 0.30 mol/L NaCl was collected, diluted in a buffer containing 20 mmol/L Tris-HCl, pH 7.5; 1 mmol/L sodium azide; 1 mmol/L dithiothreitol (DTT); and 1.20% (wt/vol) octylglucoside and reloaded onto the same column. Peaks were collected, protein content determined by BCA protein method (Pierce), and purity evaluated by SDS-polyacrylamide electrophoresis gel stained with silver and Western blotting from AQP-1.

Reconstitution of AQP-1 Into Lipid Vesicles
Five milligrams of E. coli phospholipids was sonicated in 0.45 mL of a buffer containing 20 mmol/L Tris-HCl, pH 7.5; 1 mmol/L sodium azide; 1 mmol/L DTT; and 1.2% (wt/vol) octylglucoside. Then, 35 µg (50 µL) of purified AQP-1 or 50 µL of buffer alone was added, and the mixture briefly blended and incubated for 20 minutes on ice. Proteoliposomes (and liposomes prepared without protein) were formed at room temperature by rapidly injecting the mixture into 12.5 mL of a buffer containing 50 mmol/L 3-(N-Morpholino) propanesulfonic acid (MOPS), pH 7.5; 15 mmol/L n-methyl-D-glucamine chloride, pH 7.0; 0.1 mmol/L DAF-2; 1 mmol/L DTT; and 0.5 mmol/L PMSF. The suspension was incubated for 20 minutes at room temperature and proteoliposomes or liposomes collected by centrifugation for 1 hour at 123 000g at 4°C. Proteoliposomes and liposomes were washed twice in a buffer without DAF-2 and finally resuspended in 400 µL of the same buffer.34

NO Influx Into Proteoliposomes
Proteoliposomes or liposomes were placed in a glass cuvette at room temperature. The dye was excited at 495 nm and fluorescence emitted by NO-bound dye was measured with a fluorometer (Perkin-Elmer). After stable baseline fluorescence was established, 1 µmol/L NO was applied by adding the NO donor SPM-NONOate to the bath (75 µmol/L). The increase in fluorescence emitted at 515 nm, representing the increase in intravesicular NO, was continuously measured for 1 minute. Initial rates (typically 0 to 20 s after addition of SPM) were used to calculate NO influx.

Statistics
Data were analyzed using ANOVA and/or paired or unpaired t tests as appropriate. All statistical analyses were performed by the Biostatistics Department at Henry Ford Hospital.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
To test our hypothesis, we first investigated whether NO permeability would correlate with water permeability in cells manipulated to express AQP-1. For this, we stably transfected CHO-K1 cells with control or AQP-1–expressing vectors and studied AQP-1–dependent NO permeability (PNO) and water permeability (Pf) (Figure 1). NO and water transport were measured in the same cells and permeabilities calculated from the initial rate of NO and water fluxes. In CHO-K1 cells stably transfected with or without AQP-1, PNO correlated significantly with Pf ([y=0.64x+20.23]; r=0.70; n=71). These data suggest that NO may be transported by AQP-1.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Relationship between NO permeability and water permeability of CHO-K1 cells stably transfected with a control or AQP-1–expressing vector. The line plotted through the data is the result of linear regression [y=0.65x +20.23, r=0.70], n=71 (7 to 8 cells from each of 5 different transfections were used). {circ}: control-transfected cells (n=34). •: AQP-1–transfected cells (n=37).

To investigate whether the increase in NO permeability was in fact attributable to AQP-1 transport, we measured NO transport in CHO-K1 cells transiently transfected with a vector designed to express AQP-1 or a control vector. In control transfected cells, NO influx caused by the 5-µmol/L NO concentration gradient was 10.2±3.4 fluorescence units/s (n=5). In contrast, in cells transfected to express AQP-1, NO influx was 32.5±6.9 fluorescence units/s (n=5), a 219% increase (P<0.03). These data indicate that expression of AQP-1 augments NO transport into CHO-K1 cells and that the cell membrane is a significant barrier to NO diffusion.

The increase in NO transport induced by AQP-1 transfection could be caused by alterations such as a change in lipid composition, increased expression of a native gas channel, or direct mediation of NO transport by AQP-1. To show that AQP-1 transports NO, we measured NO influx in transiently transfected CHO-K1 cells in the presence and absence of the AQP-1 inhibitors DMSO and HgCl2. In the absence of inhibitors (control conditions), AQP-1 expression increased NO influx from 11.5±4.3 to 41.5±11.3 fluorescence units/s (P<0.005; n=9). Addition of 500 mmol/L DMSO reduced NO influx to 14.8±4.7 fluorescence units/s (P<0.001 versus AQP-1 transfected cells; n=9), a 64% inhibition (Figure 2B). In another set of experiments, AQP-1 expression increased NO influx from 4.7±1.4 to 21.0±5.0 fluorescence units/s (P<0.005; n=5). Addition of 20 µmol/L HgCl2 decreased NO influx to 5.9±0.7 fluorescence units/s (P<0.001 versus AQP-1 transfected cells; n=5), a 71% inhibition (Figure 2C). Neither DMSO nor HgCl2 affected NO transport in control cells.


Figure 2
View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Effect of transiently transfecting CHO-K1 cells with an AQP-1–expressing vector on NO influx in the presence and absence of AQP-1 inhibitors. (A) Representative traces of NO influx in CHO-K1 cells transfected to express AQP-1 in the presence and absence of DMSO. The initial rate of increase in fluorescence after addition of NO (typically the first 50 s) was used to calculate NO influx. The lines plotted through the data represent the initial rates. (B) NO influx after addition of 5 µmol/L NO using SPM NONOate (SPM) in the presence and absence of 500 mmol/L DMSO. (C) NO influx after addition of 5 µmol/L NO using SPM NONOate in the presence and absence of 20 µmol/L HgCl2. (D) NO influx after addition of 5 µmol/L native NO gas in the presence and absence of 500 mmol/L DMSO. *P<0.03 vs control (C); {ddagger}P<0.005 vs control (C); {dagger} P<0.001 vs AQP-1; ¥P<0.03 vs AQP-1.

To make sure the NO transport we measured using the NO donor was caused by NO, we repeated these experiments using native NO gas. NO influx was initiated by generating a 5-µmol/L concentration gradient (Figure 2D). In control transfected CHO-K1 cells, NO influx was 13.7±0.9 fluorescence units/s (n=4). Transfecting CHO-K1 cells so that they expressed AQP-1 raised NO influx to 37.8±3.3 fluorescence units/s (P<0.03; n=4), a 175% increase. Addition of 500 mmol/L DMSO decreased NO influx to 19.4±4.2 fluorescence units/s (P<0.03 versus AQP-1 transfected cells; n=4), a 49% inhibition. DMSO had no effect on NO influx in control transfected cells. These results are similar to our findings with the NO donor and further support our hypothesis that the AQP-1 water channel facilitates NO transport across the cell membrane.

If NO is transported by AQP-1, then the transport process should be saturable as a function of NO. Consequently, we measured NO influx in response to different NO concentration gradients. Varying NO concentrations were prepared by gassing solutions with native NO and quantified using a precalibrated NO-selective sensor. In AQP-1–transfected cells, NO influx was 7.0±0.9, 13.8±0.8, 20.1±1.8, 33.7±4.4, and 37.8±2.6 fluorescence units/s in response to 0.1, 0.3, 1.0, 3.0. and 5.0 µmol/L NO, respectively (Figure 3). NO influx in response to 100 µmol/L NO was 40.4±4.9 fluorescence units/s. The apparent concentration of NO that produces half of the maximum transport rate (K1/2) for the process was 0.54 µmol/L. These data indicate that NO transport is saturable and thus further support our hypothesis that NO is transported by AQP-1. Furthermore, they indicate that AQP-1 transports NO at physiologically relevant concentrations.


Figure 3
View larger version (7K):
[in this window]
[in a new window]
  		Figure 3. NO transport as a function of NO concentration in CHO-K1 cells expressing AQP-1. The line plotted through the data is the best fit assuming Michaelis-Menten kinetics. The K1/2 for the process is 0.54 µmol/L.

Although our data show that AQP-1 transports NO across the cell membrane, we cannot rule out the possibility that NO permeability is influenced by other proteins expressed in the cells we used. To eliminate this possibility, we reconstituted purified AQP-1 into lipid vesicles, trapped the NO-selective impermeant dye DAF-2 inside, and measured the initial rate of NO influx (Figure 4). NO influx increased from 0.05±0.01 fluorescence units/s in control liposomes to 0.22±0.06 fluorescence units/s in proteoliposomes containing AQP-1, a 316% increase (P<0.024; n=6). Because AQP-1 is the only protein present in our lipid vesicles, these data indicate that AQP-1 transports NO across the lipid bilayer.


Figure 4
View larger version (10K):
[in this window]
[in a new window]
  		Figure 4. NO transport by purified AQP-1 in lipid vesicles. NO influx was measured in lipid vesicles containing either purified AQP-1 (AQP-1) or no protein (control) after a 1-µmol/L NO gradient was created with spermine NONOate. Upper panel: representative experiment. The initial rate of increase in fluorescence intensity after addition of NO (typically the first 20 s) was used to calculate NO influx. Bottom panel: mean data for NO influx. *P<0.024; n=6.

To study if AQP-1 transport of NO is biologically relevant, we investigated whether endogenously produced NO is transported by AQP-1 in endothelial cells, which normally express AQP-1 and produce NO in response to Ach. We first measured NO release by cultured endothelial cells using an NO-selective sensor in the presence and absence (control) of the AQP-1 inhibitor DMSO or HgCl2. Under control conditions, 1 µmol/L Ach induced an NO release of 2.7±0.3 pAmps/µg protein. In the presence of 100 mmol/L DMSO, NO release was 0.9±0.3 pAmps/µg protein (n=6), an inhibition of 67% (P<0.005 versus control) (Figure 5A). In another set of experiments, 1 µmol/L Ach increased NO release by 4.5±0.7 pAmps/µg protein. In the presence of 20 µmol/L HgCl2, Ach-induced NO release was only 0.5±0.7 pAmps/µg protein (n=6), an 89% inhibition (P<0.005 versus control) (Figure 5B). Neither DMSO nor HgCl2 showed cytotoxicity as assessed by Trypan Blue exclusion assay. These results are similar to those found using CHO-K1 cells and support the hypothesis that the AQP-1 water channel facilitates NO transport across the cell membrane.


Figure 5
View larger version (8K):
[in this window]
[in a new window]
  		Figure 5. Effect of AQP-1 inhibitors on acetylcholine-induced release of NO by endothelial cells. (A) Effect of 100 mmol/L DMSO. (B) Effect of 20 µmol/L HgCl2.{dagger}P<0.005 vs control.

Neither DMSO nor HgCl2 is a selective inhibitor of AQP-1. To be sure their effects on NO release were because of inhibition of AQP-1, we decreased AQP-1 expression by transfecting cultured endothelial cells with a siRNA directed against AQP-1 (AQP-1 siRNA). As controls, we used siRNAs directed against GAPDH (GAPDH siRNA) and scrambled AQP-1 (scrAQP-1 siRNA). In cells transfected with GAPDH siRNA, 1 µmol/L Ach increased NO release to 2.3±0.3 pAmps/µg protein. However, NO release was only 1.3±0.3 pAmps/µg protein in cells transfected with AQP-1 siRNA, 44% less (P<0.05; n=7) (Figure 6A). Moreover, NO release was decreased by 55% in cells transfected with AQP-1 siRNA compared with scrAQP-1 siRNA (P<0.002; n=5). The NO release data correlated with AQP-1 expression. AQP-1 siRNA decreased expression by 54±7% (P<0.001; n=6) compared with GAPDH siRNA (Figure 6B), and by 41±8% (P<0.01; n=3) when compared with scrambled AQP-1 siRNA. To show that the AQP-1 siRNA is specific and does not downregulate other genes, we transfected endothelial cells with either scrAQP-1 siRNA, AQP-1 siRNA, or GAPDH siRNA and blotted for NOS 3 and GAPDH. No changes in NOS 3 or GAPDH expression were found (Figure 6C). These data indicate that the AQP-1 siRNA is specific and that it does not knock down other genes, such as NOS 3, that could confound interpretation of the results. These data show that the AQP-1 water channel facilitates efflux of NO across the membrane of cells that normally express AQP-1 and produce NO.


Figure 6
View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Effect of AQP-1 siRNA on AQP-1 expression and acetylcholine-induced release of NO by endothelial cells. (A) Effect of AQP-1 or GAPDH siRNA. *P<0.05 vs GAPDH siRNA. (B) Representative Western blots and mean densitometry data showing effects of AQP-1 or GAPDH siRNA on AQP-1 expression (P<0.001; n=6). (C) Representative Western blots showing the effect of scrAQP-1 siRNA, AQP-1 siRNA, and GAPDH siRNA on NOS 3 and GAPDH expression.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Our data show that AQP-1 transports NO across cell membranes. We found that NO permeability of CHO-K1 cells increases with water permeability, which is a marker of AQP-1 expression. In transiently transfected CHO-K1 cells, the increased AQP-1–dependent NO transport is blocked by DMSO as well as HgCl2. In addition, we found that 3 µmol/L NO saturates the AQP-1–dependent NO transport. The fact that influx can be saturated indicates that NO transport is facilitated by a specific carrier such as AQP-1. Furthermore, AQP-1 transports NO at physiologically relevant concentrations. DMSO and HgCl2 have been shown to reduce water permeability of AQP-1 in several studies35–38; however, neither of these compounds exclusively inhibits AQP-1. Therefore, we studied the effects of reconstituting pure AQP-1 into lipid vesicles. We found that AQP-1 significantly enhances NO influx when included in the lipid bilayer of the vesicles. Since AQP-1 is the only protein present in our lipid vesicles, these data rule out the possibility of NO transport being influenced by proteins other than AQP-1.

To begin to study whether transport of NO by AQP-1 actually has physiological significance, we examined the effects of AQP-1 inhibitors, as well as reducing AQP-1 expression by siRNA, on NO efflux out of endothelial cells (which endogenously express AQP-1). We found that DMSO and HgCl2 both inhibited NO release, as did decreasing AQP-1 expression with siRNA. Both AQP-1 expression and NO release were reduced to the same extent by the AQP-1 siRNA, whereas similar levels of NOS 3 and GAPDH were found. These data indicate that the effects of DMSO and HgCl2 were attributable to inhibition of aquaporin activity. From our data we conclude that: (1) NO is transported across the cell membrane by AQP-1; (2) the cell membrane represents a significant barrier to diffusion of NO; and (3) transport of NO by AQP-1 may be physiologically important.

Non-water transport functions of AQPs are increasingly being recognized as a property that may have physiological significance. The ability of AQPs to transport NO may permit tight control of intracellular NO concentrations in target cells and directional release from cells where it is produced. Free diffusion of NO through the cell membrane does not allow control of intracellular NO concentrations or directional release. Given that the intracellular environment is tightly controlled with regard to ions and signaling molecules, it seems unlikely that there would be no means of regulating entry and exit of NO. Transport of NO by AQPs would permit such a controlled diffusion mechanism, which would be required for cell homeostasis.

NO regulates numerous physiological functions involved in control of blood pressure where AQP-1 is expressed. In vessels, NO regulates blood flow.10,18 In the central nervous system, NO functions as a neurotransmitter.19,20,39 In the kidney, NO regulates sodium and water excretion,21 blood flow,40 and pressure natriuresis.5,40,41 Additionally, NO is produced and AQPs are expressed in alveolar pneumocytes,42 where NO protects the airways from excessive vasoconstriction, and the liver, where NO serves to maximize blood perfusion, prevent platelet aggregation, and neutralize toxic oxygen radicals.43 In all of these tissues, transport of NO by AQPs may play a role in regulating NO fluxes and local concentrations. Thus, the potential influence of NO transport by AQP-1 is great.

Although AQP-1 null individuals lack apparent clinical alterations and AQP-1 knockout mice are not hypertensive, this may be a result of several factors. First, the rapid reaction of NO with hemoglobin would be expected to be reduced in AQP-1 knockout mice because of the lack of AQP-1 in the membrane of red blood cells. This would increase NO concentrations, which in turn would enhance diffusion. Second, AQP-1 knockout mice have reduced ability to concentrate urine.44 The antihypertensive effect of polyuria would be expected to overwhelm the increased vascular tone resulting from the reduced effects of NO in the vasculature. Finally, other members of the AQP family may compensate for the lack of AQP-1.

Perspectives
We found that NO permeability of cells transfected to express AQP-1 correlated with osmotic water permeability. Transiently transfecting CHO-K1 cells with AQP-1 increased NO influx. AQP-1–dependent NO influx was inhibited by agents that decrease AQP-1 activity. Transport of NO by AQP-1 became saturated at 3 µmol/L NO, with a K1/2 of 0.54 µmol/L. NO transport was significantly increased when AQP-1 was reconstituted into lipid vesicles. NO release by endothelial cells was diminished by AQP-1 inhibitors and by reducing AQP-1 expression with siRNAs. We conclude that AQP-1 transports NO across cell membranes. Our results also indicate that the cell membrane is a significant barrier to NO diffusion. In addition, transport of NO by AQP-1 occurs at physiological concentration ranges. We believe this is the first direct evidence that an integral membrane transport protein facilitates diffusion of NO. Alterations in AQP-mediated NO transport may be an alternate explanation for many diseases, especially hypertension, currently thought to be due to reduced NO production or availability.


*    Acknowledgments
 
Sources of Funding

This work was supported in part by National Heart, Lung, and Blood Institute grants HL-28982 and HL-70985 (to J.L.G.) and a predoctoral fellowship to M.H. from the American Heart Association (0415404Z).

Disclosures

None.

Received January 27, 2006; first decision February 15, 2006; accepted March 24, 2006.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Pollock DM. Chronic studies on the interaction between nitric oxide and endothelin in cardiovascular and renal function. Clin Exp Pharmacol Physiol. 1999; 26: 258–261.[Medline] [Order article via Infotrieve]
  2. Manning RD Jr, Hu L, Tan DY, Meng S. Role of abnormal nitric oxide systems in salt-sensitive hypertension. Am J Hypertens. 2001; 14: 68S–73S.[CrossRef][Medline] [Order article via Infotrieve]
  3. Lahera V, Salom MG, Miranda-Guardiola F, Moncada S, Romero JC. Effects of NG-nitro-L-arginine methyl ester on renal function and blood pressure. Am J Physiol. 1991; 261: F1033–F1037.[Medline] [Order article via Infotrieve]
  4. Kone BC. Nitric oxide in renal health and disease. Am J Kidney Dis. 1997; 30: 311–333.[Medline] [Order article via Infotrieve]
  5. Granger JP, Alexander BT. Abnormal pressure-natriuresis in hypertension: role of nitric oxide. Acta Physiol Scand. 2000; 168: 161–168.[CrossRef][Medline] [Order article via Infotrieve]
  6. Alexander BT, Llinas MT, Kruckeberg WC, Granger JP. L-arginine attenuates hypertension in pregnant rats with reduced uterine perfusion pressure. Hypertension. 2004; 43: 832–836.[Abstract/Free Full Text]
  7. Ortiz PA, Garvin JL. Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol. 2002; 282: F777–F784.[Abstract/Free Full Text]
  8. Brands MW, Bell TD, Gibson B. Nitric oxide may prevent hypertension early in diabetes by counteracting renal actions of superoxide. Hypertension. 2004; 43: 57–63.[Abstract/Free Full Text]
  9. Majid DS, Omoro SA, Chin SY, Navar LG. Intrarenal nitric oxide activity and pressure natriuresis in anesthetized dogs. Hypertension. 1998; 32: 266–272.[Abstract/Free Full Text]
  10. Sasser JM, Sullivan JC, Elmarakby AA, Kemp BE, Pollock DM, Pollock JS. Reduced NOS3 phosphorylation mediates reduced NO/cGMP signaling in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats. Hypertension. 2004; 43: 1080–1085.[Abstract/Free Full Text]
  11. Kenney MJ, Weiss ML, Haywood JR. The paraventricular nucleus: an important component of the central neurocircuitry regulating sympathetic nerve outflow. Acta Physiol Scand. 2003; 177: 7–15.[CrossRef][Medline] [Order article via Infotrieve]
  12. Granger JP, Alberola AM, Salazar FJ, Nakamura T. Control of renal hemodynamics during intrarenal and systemic blockade of nitric oxide synthesis in conscious dogs. J Cardiovasc Pharmacol. 1992; 20 Suppl 12: S160–S162.
  13. Manning RD Jr, Hu L. Nitric oxide regulates renal hemodynamics and urinary sodium excretion in dogs. Hypertension. 1994; 23: 619–625.[Abstract/Free Full Text]
  14. Wilcox CS, Welch WJ. Macula densa nitric oxide synthase: expression, regulation, and function. Kidney Int Suppl. 1998; 67: S53–S57.[Medline] [Order article via Infotrieve]
  15. Navar LG. Integrating multiple paracrine regulators of renal microvascular dynamics. Am J Physiol. 1998; 274: F433–F444.[Medline] [Order article via Infotrieve]
  16. Welch WJ, Tojo A, Lee JU, Kang DG, Schnackenberg CG, Wilcox CS. Nitric oxide synthase in the JGA of the SHR: expression and role in tubuloglomerular feedback. Am J Physiol. 1999; 277: F130–F138.[Medline] [Order article via Infotrieve]
  17. Wilcox CS, Welch WJ, Murad F, Gross SS, Taylor G, Levi R, Schmidt HH. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci U S A. 1992; 89: 11993–11997.[Abstract/Free Full Text]
  18. Sullivan JC, Giulumian AD, Pollock DM, Fuchs LC, Pollock JS. Functional NOS 1 in the rat mesenteric arterial bed. Am J Physiol Heart Circ Physiol. 2002; 283: H658–H663.[Abstract/Free Full Text]
  19. Ott SR, Delago A, Elphick MR. An evolutionarily conserved mechanism for sensitization of soluble guanylyl cyclase reveals extensive nitric oxide-mediated upregulation of cyclic GMP in insect brain. Eur J Neurosci. 2004; 20: 1231–1244.[Medline] [Order article via Infotrieve]
  20. Snyder SH, Bredt DS. Nitric oxide as a neuronal messenger. Trends Pharmacol Sci. 1991; 12: 125–128.[CrossRef][Medline] [Order article via Infotrieve]
  21. Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper MA. Aquaporins in the kidney: from molecules to medicine. Physiol Rev. 2002; 82: 205–244.[Abstract/Free Full Text]
  22. Nakhoul NL, Davis BA, Romero MF, Boron WF. Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. Am J Physiol. 1998; 274: C543–C548.[Medline] [Order article via Infotrieve]
  23. Nakhoul NL, Hering-Smith KS, Abdulnour-Nakhoul SM, Hamm LL. Transport of NH(3)/NH in oocytes expressing aquaporin-1. Am J Physiol -Renal Fluid Electrol Physiol. 2001; 281: F255–F263.[Abstract/Free Full Text]
  24. Holbrook NM, Zwieniecki MA. Plant biology: water gate. Nature. 2003; 425: 361.[Medline] [Order article via Infotrieve]
  25. Au CG, Cooper ST, Lo HP, Compton AG, Yang N, Wintour EM, North KN, Winlaw DS. Expression of aquaporin 1 in human cardiac and skeletal muscle. J Mol Cell Cardiol. 2004; 36: 655–662.[CrossRef][Medline] [Order article via Infotrieve]
  26. Dolman D, Drndarski S, Abbott NJ, Rattray M. Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture. J Neurochem. 2005; 93: 825–833.[CrossRef][Medline] [Order article via Infotrieve]
  27. Devuyst O, Nielsen S, Cosyns JP, Smith BL, Agre P, Squifflet JP, Pouthier D, Goffin E. Aquaporin-1 and endothelial nitric oxide synthase expression in capillary endothelia of human peritoneum. Am J Physiol. 1998; 275: H234–H242.[Medline] [Order article via Infotrieve]
  28. Herrera M, Garvin JL. A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1. Am J Physiol Renal Physiol. 2005; 288: F58–F64.[Abstract/Free Full Text]
  29. Herrera M, Silva G, Garvin JL. A high-salt diet dissociates NOS3 expression and NO production by the THAL. Hypertension. 2005; 47: 95–101.[Medline] [Order article via Infotrieve]
  30. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science. 1992; 256: 385–387.[Abstract/Free Full Text]
  31. Agre P, Mathai JC, Smith BL, Preston GM. Functional analyses of aquaporin water channel proteins. Methods Enzymol. 1999; 294: 550–572.[Medline] [Order article via Infotrieve]
  32. Splinter PL, Masyuk AI, LaRusso NF. Specific inhibition of AQP1 water channels in isolated rat intrahepatic bile duct units by small interfering RNAs. J Biol Chem. 2003; 278: 6268–6274.[Abstract/Free Full Text]
  33. Bennett V. Proteins involved in membrane–cytoskeleton association in human erythrocytes: spectrin, ankyrin, and band 3. Methods Enzymol. 1983; 96: 313–324.[Medline] [Order article via Infotrieve]
  34. Zeidel ML, Ambudkar SV, Smith BL, Agre P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry (Mosc). 1992; 31: 7436–7440.
  35. Preston GM, Jung JS, Guggino WB, Agre P. The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem. 1993; 268: 17–20.[Abstract/Free Full Text]
  36. Van Hoek AN, de Jong MD, Van Os CH. Effects of dimethylsulfoxide and mercurial sulfhydryl reagents on water and solute permeability of rat kidney brush border membranes. Biochim Biophys Acta. 1990; 1030: 203–210.[Medline] [Order article via Infotrieve]
  37. Huebert RC, Splinter PL, Garcia F, Marinelli RA, LaRusso NF. Expression and localization of aquaporin water channels in rat hepatocytes. Evidence for a role in canalicular bile secretion. J Biol Chem. 2002; 277: 22710–22717.[Abstract/Free Full Text]
  38. Ma T, Frigeri A, Tsai ST, Verbavatz JM, Verkman AS. Localization and functional analysis of CHIP28k water channels in stably transfected Chinese hamster ovary cells. J Biol Chem. 1993; 268: 22756–22764.[Abstract/Free Full Text]
  39. Venero JL, Vizuete ML, Machado A, Cano J. Aquaporins in the central nervous system. Prog Neurobiol. 2001; 63: 321–336.[CrossRef][Medline] [Order article via Infotrieve]
  40. Romero JC, Lahera V, Salom MG, Biondi ML. Role of the endothelium-dependent relaxing factor nitric oxide on renal function [editorial]. [Review] [114 refs]. J Am Soc Nephrol. 1992; 2: 1371–1387.[Abstract]
  41. Patel AR, Granger JP, Kirchner KA. L-arginine improves transmission of perfusion pressure to the renal interstitium in Dahl salt-sensitive rats. Am J Physiol. 1994; 266: R1730–R1735.[Medline] [Order article via Infotrieve]
  42. Effros RM, Darin C, Jacobs ER, Rogers RA, Krenz G, Schneeberger EE. Water transport and the distribution of aquaporin-1 in pulmonary air spaces. J Appl Physiol. 1997; 83: 1002–1016.[Abstract/Free Full Text]
  43. Garcia F, Kierbel A, Larocca MC, Gradilone SA, Splinter P, LaRusso NF, Marinelli RA. The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP. J Biol Chem. 2001; 276: 12147–12152.[Abstract/Free Full Text]
  44. Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem. 1998; 273: 4296–4299.[Abstract/Free Full Text]

Related Article:

NO Break-Ins at Water Gate
Bruce C. Kone
Hypertension 2006 48: 29-30. [Full Text] [PDF]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
J. S. Hub and B. L. de Groot
Mechanism of selectivity in aquaporins and aquaglyceroporins
PNAS, January 29, 2008; 105(4): 1198 - 1203.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Echevarria, A. M. Munoz-Cabello, R. Sanchez-Silva, J. J. Toledo-Aral, and J. Lopez-Barneo
Development of Cytosolic Hypoxia and Hypoxia-inducible Factor Stabilization Are Facilitated by Aquaporin-1 Expression
J. Biol. Chem., October 12, 2007; 282(41): 30207 - 30215.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. Herrera and J. L. Garvin
Novel role of AQP-1 in NO-dependent vasorelaxation
Am J Physiol Renal Physiol, May 1, 2007; 292(5): F1443 - F1451.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
B. C. Kone
NO Break-Ins at Water Gate
Hypertension, July 1, 2006; 48(1): 29 - 30.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
48/1/157    most recent
01.HYP.0000223652.29338.77v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herrera, M.
Right arrow Articles by Garvin, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herrera, M.
Right arrow Articles by Garvin, J. L.
Related Collections
Right arrow Ion channels/membrane transport
Right arrow Endothelium/vascular type/nitric oxide
Right arrowRelated Article

Hypertension Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2006 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.