Inhibition of Prostasin Secretion by Serine Protease Inhibitors in the Kidney
Kozo Iwashita,
Kenichiro Kitamura,
Takefumi Narikiyo,
Masataka Adachi,
Naoki Shiraishi,
Taku Miyoshi,
Junko Nagano,
Do Gia Tuyen,
Hiroshi Nonoguchi and
Kimio Tomita
Third Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto, Japan.
Correspondence to Dr. Kenichiro Kitamura, Assistant Professor, Third Department of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan. Phone: 81-96-373-5164; Fax: 81-96-366-8458;
ABSTRACT. A serine protease, prostasin, has been shown to stimulatethe activity of amiloride-sensitive sodium channels (ENaC).Prostasin is a glycosylphosphatidylinositol-anchored proteinthat is found free in physiologic fluids and tissue culturemedium, but the mechanism by which prostasin is secreted fromthe cells has not been elucidated. The current studies foundthat serine protease inhibitor aprotinin blocked the secretionof prostasin in a mouse cortical collecting duct (CCD) cellline (M-1 cells). A synthetic serine protease inhibitor, nafamostatmesilate (NM), which is commonly used for the treatment of pancreatitisand disseminated intravascular coagulation in Japan, also inhibitedthe secretion of prostasin in M-1 cells. Continuous infusionof NM into rats resulted in a substantial decrease in urinaryprostasin and urinary sodium excretion. p-guanidinobenzoic acidand 6-amidino-2-naphtol, catalytically inactive metabolitesof NM, had no effect on prostasin secretion both in M-1 cellsand in rats. These findings suggest that a serine protease-sensitivemechanism is involved in the secretion of prostasin in vitroas well as in vivo. Potassium secretion in the CCD is tightlylinked to sodium reabsorption through EnaC; therefore, NM-induceddecrease in prostasin secretion and subsequent inhibition ofENaC activity could account for the side effects of hyponatremiaand/or hyperkalemia that are found sometimes in patients treatedwith NM. The results indicate an important role for prostasinin sodium reabsorption in the kidney under pathophysiologicconditions. E-mail: ken@gpo.kumamoto-u.ac.jp
Proteases are involved in many biologic and physiologic processessuch as digestion of proteins in the gastrointestinal tract,clotting of blood, processing of precursors of peptide hormonesand growth factors, activation of proteases, and fertilization(1). Prostasin is a serine protease purified from human seminalfluid in 1994 (2). Purified prostasin displays a trypsin-likeenzymatic activity and cleaves synthetic substrates in vitro,preferentially at the carboxy-terminal side of Arg residue.Prostasin is found in the kidney, prostate gland, bronchi, colon,liver, lung, pancreas, and the salivary glands. At the timeprostasin was purified and the cDNA was cloned, the role ofprostasin in mammalian physiology was not known (2–4).Recently a couple of laboratories including our laboratory showedthat co-expression of prostasin and the epithelial sodium channel(ENaC) in Xenopus oocytes substantially increased the amiloride-sensitivesodium currents (5–9). In a mouse cortical collectingduct (CCD) cell line (M-1 cells), Nakhoul et al. and Liu etal. demonstrated that treatment of M-1 cells with serine proteaseinhibitors reduced sodium currents (10,11). In addition, wefound that the treatment of M-1 cells with aldosterone increasedthe secretion of prostasin into culture media, and that prostasinthen stimulated 22Na uptake (12). We also found that elevatedaldosterone levels in rats markedly increased the urinary secretionof prostasin (12). Furthermore, we demonstrated that the urinarysecretion of prostasin was substantially increased in patientswith primary aldosteronism, and that adrenalectomy significantlyreduced urinary prostasin secretion (12). These results indicatethat prostasin is an important physiologic regulator of sodiumreabsorption in the kidney through ENaC and suggest the possibilitythat prostasin is involved in the development of salt-sensitivehypertension in humans.
Molecular cloning of a full-length human prostasin cDNA revealedthat its predicted amino acid residue sequence contains a carboxy-terminalhydrophobic region that is a consensus sequence of glycosylphosphatidylinositol(GPI)-anchored membrane protein (13). Although prostasin issynthesized as a GPI-anchored membrane protein, a free-formprostasin is secreted into the medium of cultured prostate cells(13). In M-1 cells, we previously demonstrated that prostasinis found only in the culture medium, but not in the either cytosolicor membrane fraction (12). We were also unable to detect prostasinin either cytosolic or membrane fractions of rat kidneys, butwe easily found prostasin in the urine of rats and humans (12).Therefore, we believe that prostasin is expressed as a secretoryprotein both in M-1 cells and in the kidneys rather than a membrane-boundprotein. However, the precise mechanism by which prostasin isexpressed in the kidney remains unknown. Understanding thismechanism could provide the new insight into the treatment ofhypertension and other sodium-retaining states.
In the present studies, we found that treatment of M-1 cellswith serine protease inhibitor aprotinin blocked the secretionof prostasin into the medium of cultured cells. We demonstratedthat treatment with nafamostat mesilate (NM), a synthetic serineprotease inhibitor that is commonly used for the treatment ofpancreatitis and disseminated intravascular coagulation (DIC)in Japan, also inhibited prostasin secretion in M-1 cells aswell as in rat kidneys. Furthermore, we showed that urinarysodium excretion was substantially increased in NM-treated rats.NM has been shown to induce hyponatremia and/or hyperkalemiain some cases when used for the treatment of pancreatitis orDIC (14); therefore, our results suggest the possibility thatthe inhibition of prostasin secretion into urine and the subsequentinhibition of prostasin-induced ENaC activity in the kidneymay be responsible for the hyponatremia and/or hyperkalemiaassociated with NM treatment. Taken together, our results indicatethat the secretion of prostasin is mediated by a mechanism thatis sensitive to the serine protease inhibitors and suggest theinvolvement of prostasin action in pathophysiologic conditions.
Materials
Nafamostat mesilate, p-guanidinobenzoic acid (PGBA), and 6-amidino-2-naphtol(AN) are kind gifts from Torii Pharmaceutical Co., Ltd. (Nihonbashi,Tokyo, Japan). Other chemicals are obtained from Sigma (St.Louis, MI, USA).
Cell Culture
M-1 cell, an SV40-transformed mouse CCD cell line, was obtainedfrom the American Type Culture Collection (Rockville, MD). Cellswere maintained in Dulbecco modified Eagle medium/Ham’sF-12 (1:1) mixture (Life Technologies-BRL, Rockville, MD) supplementedwith 5% FBS and 100 nM dexamethasone in a humidified incubatorat 37°C and 5% CO2 as described previously (12). Experimentswere performed when cells were confluent. Serum and dexamethasonewere removed 48 h before experiments. All studies describedin this paper were performed on cells between the fifth andtwentieth passages.
Northern Blot Analyses
Total RNA from M-1 cells grown in 10-cm plastic dishes underexperimental conditions was isolated by using an RNeasy kit(QIAGEN, Hilden, Germany). Total RNA (20 µg) of each samplewas resolved on agarose-formaldehyde gels and transferred ontonylon membranes. A full-length cDNA of mouse prostasin and rat-actin was individually labeled with [-32P] dCTP, and probeswere hybridized with the membranes as described previously (5).
Preparation of Proteins and TCA Precipitation
After incubation under each experimental condition, culturemedium (10 ml/10 cm dish) was collected, and centrifuged at1200 x g to pellet cell debris. Total protein in the culturemedium was precipitated with TCA (final concentration, 15%).The samples were centrifuged at 12,000 x g, and the pelletswere washed three times with ice-cold 80% acetone. The precipitatedproteins were dried and solubilized at 100°C for 5 min in1 x TCA buffer (200 mM unbuffered Tris, 1% SDS, 10% Glycerol,1% -mercaptoethanol).
Immunoblotting
Samples were size fractionated using SDS-PAGE with 12% gelsand transferred electrophoretically onto nitrocellulose filters.After blocking with 5 g/dl nonfat dry milk overnight at 4°C,the blots were probed with a polyclonal antibody against prostasinpeptide in Tris-buffered saline with 0.05% Tween-20 for 1 hat room temperature, followed by a secondary antibody (goatanti-rabbit IgG-conjugated with horseradish peroxidase, dilutedto 1:10,000) for 1 h at room temperature. Bands were visualizedusing chemiluminescence substrate (ECL; Amersham Pharmacia Biotech,Buckinghamshire, England) before exposure to x-ray film. Theband densities were quantitated by densitometry (Densitograph4.0; ATTO, Tokyo, Japan).
Serine Protease Inhibitor Infusion Studies
Experiments were conducted in male Sprague-Dawley rats (180to 200 g) from Charles River Japan, Inc. (Yokohama, Japan).Rats were anesthetized with 2% isoflurane before the implantationof catheters. The external jugular vein of rats was exposedas it crossed the clavicle via a ventral skin incision on theneck. After phlebotomy, a fine silicone catheter (Silascon,Kaneka Medics, Osaka, Japan) was introduced into the vein untilthe intravascular tip was located at the junction of the cranialvena cava and right atrium. The catheter was tied onto the veinby two 4-0 silk ligatures just proximal and distal of the phlebotomy.A trocar was inserted subcutaneously from the dorsal aspectof the mid-cervical region exiting at the ventral skin incisionon the neck. The external end of the catheter was then threadedthrough the trocar to exit at the scapula, and the trocar waswithdrawn. Next, the external end of the catheter was tightlyattached to the swivel (Instech 375/22; Lomir Biomedical Inc.,Malone, NY) through a vinyl tube, and an infusion pump was connectedto the swivel. The swivel was kept over the middle of the cageby a metal hook. To keep daily sodium, potassium, and waterintake constant, total parenteral nutrition was administeredwith this swivel system. The rats were received 260 ml/kg ofbody weight per day of a formulated solution composed of 100ml of 50% glucose, 40 ml of essential amino acids (Amiparen;Ohtsuka Pharmaceutical Co., Ltd., Tokushima, Japan), 2.8 mlof 1 M NaCl, 1.5 ml of 1 M K2HPO4, 1.0 ml of 1 M MgSO4, 1.5ml of 1 M potassium aspartate, 2.5 ml of 0.2 M CaCl2, 110 mlof sterile H2O, and vitamins. The solutions were continuouslyinfused with the syringe pump. Continuous infusion of a serineprotease inhibitor or its stable metabolites was started 24h after the initiation of parenteral nutrition. The rats werecontinuously infused with 2 mg/kg per hour of NM, 1 mg/kg perhour of PGBA, 1 mg/kg per hour of AN, or vehicle. All rats werekept in metabolic cages, and urine samples were collected andstored at 4°C. The urine electrolytes and creatinine concentrationswere measured in the 24-h urine samples. Urine samples, correctedfor creatinine excretion, were concentrated by TCA precipitation,and the amount of prostasin was determined by immunoblottingas described above.
Effect of Protease Inhibitors on Prostasin Secretion in M-1 Cells
Since an intracellular proteolytic mechanism has been implicatedin the secretion of soluble forms of GPI-anchored proteins (15),we hypothesized that protease(s) is/are involved in the secretionof prostasin in M-1 cells. To test our hypothesis, we measuredprostasin protein abundance in M-1 cells after the proteaseinhibitor treatment. M-1 cells (serum-deprived for 48 h) weretreated with aprotinin (28 µg/ml), phosphoramidon (1.1µg/ml), E-64 (7.2 µg/ml), leupeptin (2.0 µg/ml),antipain (2.5 µg/ml), or pepstatin A (1.0 µg/ml)in serum-free media for 24 h, and the amount of prostasin proteinin the culture media was determined by the immunoblotting withanti-prostasin our peptide antibody. As shown in Figure 1A,treatment with serine protease inhibitor aprotinin almost completelyinhibited the secretion of prostasin, whereas other types ofprotease inhibitors (cysteine, aspartic, or metalloproteaseinhibitors) had no effect on the amount of prostasin proteinin the medium. The prostasin protein was not found in membraneor cytosol fraction of M-1 cells under basal condition; evenin the presence of aprotinin, we were unable to detect the prostasinprotein in both membrane and cytosol fraction (data not shown).None of these protease inhibitors used for the experiments hadsignificant effects on the mRNA levels of prostasin (Figure 1B).These findings suggest that a serine protease inhibitoraprotinin inhibited the translation of prostasin mRNA or theprosttranslational processing of prostasin protein in M-1 cells.
Figure 1. Effect of protease inhibitors on the expression of prostasin protein and mRNA in M-1 cells. (A) Effect of protease inhibitors on the protein expression of prostasin in M-1 cells. M-1 cells were serum-deprived for 48 h and then treated with various protease inhibitors (as indicated) for another 24 h. Proteins were precipitated from the culture medium with TCA, and the expression level of prostasin protein was determined by immunoblotting. (B) Effect of protease inhibitors on the mRNA expression of prostasin in M-1 cells. The cells from these samples were harvested, and the expression of prostasin mRNA as well as -actin mRNA was determined by northern blotting. These blots are representative of four separate experiments. Values are mean ± SEM (n = 4). * P < 0.05.
Effect of NM, PGBA, and AN on Prostasin Secretion in M-1 Cells
NM, a synthetic serine protease inhibitor, inhibits variousserine proteases, including trypsin, thrombin, activated factorX, kallikrein, activated complements, and neutrophil elastase.Although NM has been shown to be effective in the treatmentof pancreatitis and DIC (16), it sometimes causes side effectsof hyponatremia and/or hyperkalemia (14). Muto et al. (17–19)reported that perfusion of rabbit CCD with NM inhibited theamiloride-sensitive sodium current, resulting in the inhibitionof potassium secretion. They suggested that hyponatremia and/orhyperkalemia associated with NM treatment could be caused inpart by the inhibition of amiloride-sensitive sodium conductance.We demonstrated that a serine protease inhibitor aprotinin inhibitsthe secretion of prostasin into culture media, and we showedthat prostasin increases sodium reabsorption through the activationof EnaC; we therefore hypothesized that hyponatremia and/orhyperkalemia associated with NM treatment is caused by the inhibitionof prostasin secretion and the subsequent inhibition of prostasin-inducedactivation of ENaC. To test this hypothesis, we treated M-1cells with NM or its catalytically inactive metabolites, PGBAor AN, and measured the level of prostasin protein in the culturemedia. The stable serum concentration of NM is 10-8 to 10-7M in patients receiving continuous infusion of NM at a doseof 0.06 to 0.20 mg/kg per hour (commonly used dosage for theDIC treatment); therefore, M-1 cells were incubated with 10-8M of NM, PGBA, or AN for 24 h. As shown in Figure 2A, treatmentwith NM completely blocked the secretion of prostasin into M-1culture media, whereas PGBA and AN had no effect on prostasinsecretion. The expression of prostasin was not demonstratedin either membrane or cytosol fraction in M-1 cells treatedwith NM as observed in M-1 cells treated with aprotinin (datanot shown). None of these compounds had significant effect onmRNA expression of prostasin in M-1 cells (Figure 2B). Next,we examined the dose-response relationship of the inhibitionof prostasin secretion by NM. NM blocked prostasin secretion,even at a dose of 10-9 M, a dose that is ten times lower thanthe serum concentration of NM used for the treatment of DIC(Figure 3). To address the question whether aprotinin or NMbinds to prostasin and reduces the ability of the antibody torecognize the epitope sequence, we added aprotinin or NM ingraded concentrations to the culture medium of M-1 cells producedunder the condition of no aprotinin or NM and performed immunoblottings.As shown in Figure 4, aprotinin and NM had no effect on thedetection of prostasin protein by the prostasin antibody. Thesedata suggest that serine protease activity is indeed involvedin the prostasin secretion.
Figure 2. Effect of nafamostat mesilate (NM), p-guanidinobenzoic acid (PGBA), and 6-amidino-2-naphtol (AN) on the expression of prostasin protein and mRNA in M-1 cells. (A) Effect of NM, PGBA, and AN on the protein expression of prostasin in M-1 cells. M-1 cells were serum-deprived for 48 h, and then NM, PGBA, or AN was added to the culture media for another 24 h. Each culture medium was TCA precipitated and subjected to immunoblotting to evaluate the level of expression of prostasin protein. (B) Effect of NM, PGBA and AN on the mRNA expression of prostasin in M-1 cells. At the same time, total RNA was extracted from the cells, and northern blots were performed to determine the level of expression of prostasin as well as -actin mRNA. These blots are representative of five separate experiments. Values are mean ± SEM (n = 5). * P < 0.05.
Figure 3. Dose-dependent effect of NM on prostasin secretion in M-1 cells. M-1 cells were serum-deprived for 48 h and then treated with increasing doses of NM (0 to 10-7 M). The culture medium was TCA precipitated, and the level of prostasin protein expression was determined by immunoblotting. These blots are representative of three separate experiments. Values are mean ± SEM (n = 3). * P < 0.05.
Figure 4. Effect of aprotinin or NM on the detection of prostasin protein by the anti-peptide prostasin antibody in the culture medium of M-1 cells. M-1 cells were serum-deprived for 48 h in the absence of aprotinin or NM. Each culture medium was harvested and mixed with 28 µg/ml of aprotinin or with graded concentration (10-7 to 10-10 M) of NM. Then, each medium was TCA precipitated and subjected to immunoblotting to evaluate the expression level of prostasin protein. These blots are representative of three separate experiments. Values are mean ± SEM (n = 3).
NM Infusion Studies
To determine the effect of NM on prostasin secretion in vivo,we continuously infused NM, PGBA, or AN into rats receivingtotal parenteral nutrition as described in the Materials andMethods section. Urinary secretion of prostasin was measuredbefore and 7 d after the initiation of continuous infusion.As shown in Figure 5, continuous infusion of NM almost completelyinhibited the secretion of prostasin into urine, whereas PGBAand AN had no effect on prostasin secretion. Next, we examinedthe time course of urinary prostasin secretion and sodium excretionin rats receiving parenteral nutrition with or without NM. Interestingly,the inhibitory effect of NM on urinary prostasin secretion couldbe observed on day 1, and the effect lasted throughout the experimentalperiod (Figure 6A). In addition, urinary sodium excretion wassignificantly increased in rats receiving NM when compared withrats receiving vehicle. The NM-induced increase in urinary sodiumexcretion was observed during the whole experimental period(Figure 6B). We also measured urinary potassium excretion inthese rats. Urinary potassium excretion was as follows: at day0 (vehicle, 1.0 ± 0.05 mEq/d; NM, 0.9 ± 0.04 mEq/d),at day 1 (vehicle, 0.8 ± 0.06 mEq/d; NM, 0.6 ±0.04 mEq/d), at day 4 (vehicle, 1.6 ± 0.24 mEq/d; NM,1.2 ± 0.07 mEq/d), and at day 7 (vehicle, 0.9 ±0.12 mEq/d; NM, 0.9 ± 0.12 mEq/d). Although the differencein urinary potassium excretion between vehicle and NM was notstatistically significant at each time point, we found a tendencythat rats receiving NM had lower urinary potassium excretionlevel. These results demonstrate that a serine protease inhibitorNM almost completely blocks the secretion of prostasin in amouse CCD cell line as well as in whole rats and suggest thatan increase in urinary sodium excretion could be associatedwith the decrease in the urinary prostasin secretion. Furthermore,these findings indicate that inhibition of prostasin could beinvolved in NM-induced hyponatremia and/or hyperkalemia.
Figure 5. Effect of NM, PGBA, or AN on urinary prostasin secretion in rats. Experiments were conducted in male SD rats. Rats receiving parenteral nutrition were continuously infused with NM, PGBA or AN for 7 d. Urine samples that are equivalent to 80 µg of creatinine excretion were TCA precipitated, and urinary prostasin secretion was determined by immunoblotting. This blot is representative of five separate experiments.
Figure 6. Effect of NM on urinary prostasin and sodium excretion in rats. (A) Time course of NM-induced inhibition of urinary prostasin secretion. The inhibitory effect of NM on urinary prostasin secretion was observed the day after initiation of NM (day 1), and the effect of NM lasted throughout the experimental period. This blot is representative of six separate experiments. (B) Urinary sodium excretion in rats infused with NM or vehicle. Urinary sodium excretion was significantly increased in rats treated with NM when compared with vehicle-treated rats. The results are expressed as mean ± SEM (n = 6). NS, not significant; * P < 0.03.
In the present studies, we found that treatment of M-1 cellswith a serine protease inhibitor aprotinin almost completelyblocked the secretion of prostasin. We also demonstrated thattreatment with NM inhibited prostasin secretion in M-1 cellas well as urinary prostasin secretion in rats. Furthermore,we showed that urinary sodium excretion was substantially increasedin the NM-treated rats.
Serine Protease-Sensitive Mechanism of Prostasin Secretion
Prostasin is synthesized as a GPI-anchored membrane protein,whereas a free-form of prostasin is secreted into the culturemedium of the prostate cells (13). In the studies presentedhere, we demonstrated that serine proteases are involved inthe secretion of prostasin in M-1 cell as well as in rat kidneys.Prostasin is detected only in the culture media under basalconditions. When treated with serine protease inhibitors, prostasinwas not found in the culture media, membrane fraction, or cytosolfraction of M-1 cells, but mRNA expression of prostasin wasnot changed. These findings suggest that serine proteases areinvolved in the translation of prostasin mRNA or in the posttranslationalprocessing of prostasin protein. However, in the current studieswe have not addressed these possibilities. In the native tissuesuch as prostate gland, prostasin has been shown to be expressedas a GPI-anchored protein (13), but we were not able to demonstratethe expression of prostasin in either membrane fraction or cytosolfraction of the kidneys. We speculated that processing mechanismsof prostasin might be different between in the kidney and inthe prostate gland. Human folate receptors are demonstratedto be produced as either GPI-anchored proteins or secreted proteinsdepending on the cell or tissue type. An intracellular proteolyticmechanism that could result in the selective and efficient secretionof soluble forms of folate receptors has been suggested (15).A same kind of mechanisms might play a role in the secretionof prostasin in the kidneys. Although renal handling mechanismsof aprotinin is not completely elucidated, it is demonstratedthat aprotinin binds to the plasma membrane of renal tubulesand that radio-labeled aprotinin is found in the cytoplasm ofrenal tubules (20,21). Recently, NM has been shown to be transportedinside the renal tubular cells via organic cation transporter2 (OCT2) (22). Therefore we speculated that these serine proteaseinhibitors (at least NM) can gain access to the intracellularcompartments and then inhibit the intracellular proteolyticmechanism of prostasin processing. Further investigation isneeded to elucidate the precise mechanism by which a serineprotease regulates the secretion of prostasin.
Implications for NM-Induced Hyponatremia and/or Hyperkalemia
Previously Muto et al. (17–19) reported that perfusionof rabbit CCD with NM, PGBA, or AN inhibited the amiloride-sensitivesodium conductance in the apical membrane. They speculated thathyponatremia and/or hyperkalemia that sometimes occur in patientstreated with NM are induced by inhibition of the amiloride-sensitivesodium conductance and resultant inhibition of potassium secretion.In the current studies, we also demonstrated that continuousinfusion of NM into rats resulted in a substantial decreasein urinary sodium excretion and prostasin secretion. However,our data indicated that PGBA and AN had little effect on urinaryprostasin secretion as well as urinary sodium excretion. Thediscrepancy could be explained by the experimental systems usedfor the studies. Muto et al. used ex vivo renal tubule perfusionsystem and studied the acute effect of NM on sodium conductance,whereas we performed in vitro and in vivo experiments and observedrelatively long-term effects of NM.
Although Muto et al. speculated that NM, PGBA, and AN directlyinhibit the amiloride-sensitive sodium conductance in rabbitCCD, the precise mechanisms by which these protease inhibitorsblock the sodium conductance are not defined. In the presentstudies, we found that the secretion of prostasin is almostcompletely inhibited by NM treatment, but not by AN or PGBAtreatment. We previously demonstrated that prostasin is a potentENaC activator; therefore, it is reasonable to speculate thatNM blocks the secretion of prostasin into the lumen of the tubulesand consequently inhibits the prostasin-induced sodium uptakethrough ENaC. Thus, the NM-induced inhibition of sodium reabsorptionleads to a decrease in potassium secretion in the CCD. Herewe propose that hyponatremia and/or hyperkalemia associatedwith NM treatment in the clinical situation is probably causedby the inhibition of prostasin secretion in the kidney.
Our findings would be useful to develop a strategy for the preventionof side effects of NM in the future, and they also indicatethat an analogue of NM could be a candidate for the therapeuticdrug for disorders of sodium handling in the kidney such ashypertension and nephrogenic edema.
Acknowledgments
We thank Dr. R. Tyler Miller (Case Western Reserve University)for the critical reading of this manuscript. This work was supportedby the Grants-in-Aid for Scientific Research from the Ministryof Education, Culture, Sports, Science and Technology in Japan(11770599 and 13770602 to K. K., 10671000, 11470219, and 11877177to K. T.), The Salt Science Research Foundation (to K. T.).
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Received for publication April 19, 2002.
Accepted for publication October 4, 2002.
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683 - 688.
[Abstract][Full Text][PDF]
C. Planes, C. Leyvraz, T. Uchida, M. A. Angelova, G. Vuagniaux, E. Hummler, M. Matthay, C. Clerici, and B. Rossier In vitro and in vivo regulation of transepithelial lung alveolar sodium transport by serine proteases
Am J Physiol Lung Cell Mol Physiol,
June 1, 2005;
288(6):
L1099 - L1109.
[Abstract][Full Text][PDF]
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Abstract
Introduction
-actin was individually labeled with [
-32P] dCTP, and probes were hybridized with the membranes as described previously (5). 
