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(Hypertension. 2002;39:860.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Serine Protease Activity in M-1 Cortical Collecting Duct Cells

Lian Liu; Kathleen S. Hering-Smith; Faith R. Schiro; L. Lee Hamm

From the Department of Medicine and Physiology, Tulane University Health Science Center and VA Medical Center, New Orleans, La.

Correspondence to L. Lee Hamm, MD, Tulane University Health Science Center, Nephrology, SL 45, 1430 Tulane Ave, New Orleans, LA 70112. E-mail lhamm{at}tulane.edu

	Abstract

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An apical serine protease, channel-activating protease 1 (CAP1), augments sodium transport in A6 cells. Prostasin, a novel serine protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1. We have recently found functional evidence for a similar protease activity in the M-1 cortical collecting duct cell line. The purposes of the present studies were to determine whether prostasin (or CAP1) is present in collecting duct cells by use of mouse M-1 cells, to sequence mouse prostasin, and to further characterize the identity of the serine protease activity and additional functional features in M-1 cells. Using mouse expressed sequence tag sequences that are highly homologous to the published human prostasin sequence as templates, reverse transcription–polymerase chain reaction and RACE (rapid amplification of cDNA ends) were used to sequence mouse prostasin mRNA, which shows 99% identical to published mouse CAP1 sequence. A single 1800-bp transcript was found by Northern analysis, and this was not altered by aldosterone. Equivalent short-circuit current (I_eq), which represents sodium transport in these cells, dropped to 59±3% of control value within 1 hour of incubation with aprotinin, a serine protease inhibitor. Trypsin increased the I_eq in aprotinin-treated cells to the value of the control group within 5 minutes. Application of aprotinin not only inhibited amiloride sensitive I_eq but also reduced transepithelial resistance (R_te) to 43±2%, an effect not expected with simple inhibition of sodium channels. Trypsin partially reversed the effect of aprotinin on R_te. Another serine protease inhibitor, soybean trypsin inhibitor (STI), decreased I_eq in M-1 cells. STI inhibited I_eq gradually over 6 hours, and the inhibition of I_eq by 2 inhibitors was additive. STI decreased transepithelial resistance much less than did aprotinin. Neither aldosterone nor dexamethasone significantly augmented protease activity or prostasin mRNA levels, and in fact, dexamethasone decreased prostasin mRNA expression. In conclusion, although prostasin is present in M-1 cells and probably augments sodium transport in these cells, serine proteases probably have other effects (eg, resistance) in the collecting duct in addition to effects on sodium channels. Steroids do not alter these effects in M-1 cells. Additional proteases are likely also present in mouse collecting duct cells.

Key Words: epithelium • sodium channels • cortical collecting duct • serine protease • M-1 cells

	Introduction

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The amiloride-sensitive epithelial sodium channel (ENaC), located at the apical membrane of the collecting duct, plays a major role in the control of the blood pressure, as indicated by the hypertensive or hypotensive genetic diseases that have mutations in ENaC genes. A variety of regulating factors for ENaC have been described, but recent studies have suggested a potential novel regulation by an extracellular protease. An early study¹ reported that a serine-protease inhibitor, aprotinin, induced a decrease of the amiloride-sensitive short-circuit current in the toad bladder, a model for collecting duct sodium transport. In 1997, Vallet et al² described a novel serine protease called CAP1 (for channel-activating protease 1) that regulates the epithelial sodium channel. Their study indicated that the activity of the amiloride-sensitive sodium channel in A6 cells (a Xenopus kidney cell line expressing ENaC) is modulated by an endogenous protease that is active at the extracellular side of the apical membrane. The coexpression of xCAP1 (Xenopus CAP1) cDNA with ENaC induced a 3-fold increase in the sodium current in Xenopus oocytes. Northern blot analysis shows that CAP1 mRNA is highly expressed in kidney, intestine, stomach, skin, and lung, all epithelial tissues in Xenopus laevis tissue that express ENaC mRNA.² Recent studies from our laboratory³ and from Vuagniaux et al⁴ have shown similar effects of aprotinin and trypsin on mammalian cortical collecting duct cell lines, M-1 cells, and mpkCCD_c14 cells. Vuagniaux et al⁴ further cloned a mouse ortholog (mCAP1) that is 80% homologous to prostasin. Prostasin is a novel serine protease that was identified and purified from human seminal fluid⁵ and was subsequently cloned.⁶

Because we had previously identified functional changes in sodium transport in M-1 cells that were consistent with an extracellular protease, ³ the present study addressed the identity of this activity in M-1 cells and additional functional features. We were particularly interested in potential regulation by corticosteroids because of a recent report that showed that elevated circulating aldosterone (Aldo) induced a shift in the molecular weight of -ENaC from 85 to 70 kDa.⁷ Masilamani et al⁷ suggested that this shift is caused by physiological proteolytic cleavage.

	Methods

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Cell Culture
The M-1 cells used in this study were obtained and cultured as previously described.³ In brief, the cultures were initially maintained in a defined medium consisting of equal amounts of Ham’s F-12 and low-glucose Dulbecco’s modified Eagle’s medium (Sigma), supplemented with 2 mmol/L L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, 5% fetal bovine serum, growth promoting factors (6.25 mg/mL of transferrin, insulin, and sodium selenite), and 100 nmol/L dexamethasone (Dex). After cells reached confluence, they were passaged to semipermeable membranes (Transwells, Costar). The medium was changed to serum-free media 3 days later and was changed to media containing Aldo (1 µmol/L), Dex (100 nmol/L), Aldo+Dex (1 µmol/L, 100 nmol/L, respectively), or vehicle (ethanol) 1 day later. Cells were studied after 48 hours.

Electrophysiological Transepithelial Measurements
Transepithelial voltage (V_te) and resistance (R_te) were measured with an ohm/volt meter (EVOM, WPI). V_te was measured by means of a set of 2 Ag:AgCl electrodes and determined with the apical solution as reference. R_te was measured by passing DC current through the cell monolayer and measuring the resulting voltage gradient across the cells. The equivalent current (I_eq) was calculated as the ratio of V_te to R_te and was normalized by dividing I_eq by the surface area (1.13 cm²) of active membrane.

Reverse Transcription–Polymerase Chain Reaction and Sequencing of Mouse Prostasin
Before the recent cloning of mCAP1,⁴ we used reverse transcription–polymerase chain reaction (RT-PCR) to determine the presence of a mouse prostasin ortholog of xCAP1 in M-1 cells. Total RNA was extracted from confluent M-1 cells using TRIzol Reagent (GIBCO BRL). mRNA was isolated from purified total RNA using MessageMaker Reagent Assembly (GIBCO BRL). First-strand cDNA was generated from mRNA using T-Primed First-Strand Kit (Pharmacia Biotech).

Primers were initially designed (GENERUNR program) based on a mouse kidney expressed sequence tag (EST; G_i 6077679) from GenBank that is 80% homologous to human prostasin mRNA, because at the initiation of these studies, mCAP1 had not been reported. Recombinant Tag DNA polymerase was used for PCR (Platinum PCR SuperMIX, GIBCO BRL). Initial melting was at 95°C for 3 minutes, and then 35 cycles of the following were run: (1) melting at 93°C for 1 minute, (2) annealing at 55°C for 30 seconds, and (3) extension at 72°C for 1 minute. Final extension was 72°C for 5 minutes. RT-PCR products were visualized by ultraviolet light using ethidium bromide staining after 1% agarose gel electrophoresis.

Northern Blot Analysis
mRNA (2 to 3 µg) extracted from M-1 cells was run on a 1% agarose-formaldehyde gel and blotted onto nylon membranes. Membranes were hybridized with random-primed ³²P-labeled probes for mouse prostasin, ß-actin, and G3PDH. The mouse prostasin probe was the sequenced PCR product above. Bands were quantitated using AlphaEase program.

Statistical Analysis
Statistical analysis for Northern blot analysis was conducted by use of the Student’s paired t test (2-tailed analysis) comparing steroids groups with control group. All values were normalized to the ß-actin or G3PDH mRNA. P<0.05 was considered significant. Values are expressed as mean±SEM.

	Results

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To determine the time course of the effect of protease inhibitor, we measured the I_eq from 10 minutes to 24 hours after addition of aprotinin to the apical solution. Figure 1 shows the time course of aprotinin on I_eq. In aprotinin-treated cells, I_eq dropped to 59±3% of control value within 1 hour and reached the plateau of 70±2% of control value at 4 hours. After 24 hours, I_eq in both the aprotinin group and control group dropped significantly. Therefore, cells were treated by aprotinin for 4 hours in most subsequent studies.

View larger version (14K): [in this window] [in a new window]   Figure 1. Time course of aprotinin on equivalent current (Ieq) (n=11). In aprotinin-treated (20 µg/mL) cells, Ieq dropped to 60% of control value within 1 hour and reached the plateau of 71% of control value at 4 hours. After 24 hours, Ieq in both aprotinin-treated () and control () groups had dropped significantly.

Figure 2 shows the effect of trypsin on I_eq after 4-hour incubation with aprotinin. Trypsin only increased the I_eq in aprotinin-treated cells and reached the maximum effect within 5 minutes. No effect was seen in cells not exposed to aprotinin. After addition of trypsin, the I_eq in the aprotinin group was close to that in the control group. Amiloride abolished the currents in both aprotinin-treated and vehicle control groups.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of trypsin (a serine protease) on Ieq after 4-hour incubation with aprotinin (n=25). Trypsin (200 µg/mL) increased Ieq in aprotinin-treated cells (), reaching a maximum effect similar to the current in control group () within 5 minutes. Trypsin did not stimulate Ieq in the control group. Amiloride (10-5 M) abolished the currents in both aprotinin-treated and control groups.

Unexpectedly, aprotinin and trypsin not only had effects on the transepithelial sodium current but also significantly changed transepithelial resistance. As shown in Figure 3, R_te dropped to 43±2% after treatment with aprotinin; trypsin reversed this effect partially.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of trypsin on transepithelial resistance (Rte) after 4-hour incubation with aprotinin (n=25). Rte dropped to about 40% after treatment with aprotinin; trypsin reversed this effect partially.

We also tried another serine protease inhibitor, soybean trypsin inhibitor (STI), on M-1 cells. As shown in Figure 4, both inhibitors, aprotinin (20 µg/mL) and STI (200 µg/mL), decreased sodium transport, which is measured as I_eq. However, there were some differences between the effects of aprotinin and STI: the effect of aprotinin began rapidly and reached a maximum within 1 hour, whereas STI inhibited I_eq gradually over 6 hours. The inhibition of I_eq by the 2 inhibitors was additive. Also, STI decreased transepithelial resistance much less than did aprotinin.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of 2 serine protease inhibitors, aprotinin and STI, on equivalent current (n=11). The effect of aprotinin (20 µg/mL, ) started rapidly, whereas STI (200 µg/mL, inhibited Ieq more gradually. The inhibition of Ieq by 2 inhibitors was additive ().

PCR using the primers designed based on the mouse EST (G_i 6077679) yielded the predicted 422 bp products. The sequence of this PCR product was identical to the EST. Additional overlapping ESTs (G_i 5336751, 1701704, and 3681612) were identified and used to sequence PCR products of the entire coding region of mouse prostasin. The sequence was >99% identical to that recently reported for mCAP1 (data not shown).⁴ Northern analysis demonstrated a single band at 1800 bp Figure 5, consistent with the size of human prostasin and mCAP1. The changes in mRNA expression were studied next.

View larger version (47K): [in this window] [in a new window]   Figure 5. A representative Northern blot, hybridized with prostasin probe. The size of 1.8 kb is appropriate for mouse prostasin or mCAP1.

To test the effects of adrenal steroids on the abundance of prostasin mRNA, M-1 cells were divided into 4 groups: vehicle control, Aldo (10^-6 mol/L), Dex (10^-7 mol/L), and the combination of Aldo+Dex. Cells were studied after 48 hours. These doses and the time were based on our prior studies that demonstrated that sodium transport is stimulated by Aldo, Dex, and Aldo+Dex in M-1 cells.³ Figure 6 shows the relative abundance of prostasin mRNA in M-1 cells in steroids groups compared with the control group. The density of prostasin bands was normalized by either ß-actin or G3PDH mRNA. Neither Aldo nor Aldo+Dex increased prostasin mRNA. Prostasin mRNA was decreased to 79±5% and 68±11% of control value in Dex group, normalized by ß-actin and G3PDH, respectively (P<0.05).

View larger version (35K): [in this window] [in a new window]   Figure 6. Relative abundance of prostasin mRNA in M-1 cells in steroid groups compared with control group (n=5). Cells were divided into 4 groups: control (Con), Aldo, Dex, and Aldo+Dex (A+D). The density of prostasin bands was either normalized by ß-actin or G3PDH bands. *P<0.05.

To study the activity of prostasin in steroids treated cells, the cells were divided into 4 groups: vehicle control, Aldo, Dex, and Aldo+Dex . Aprotinin was added to the apical solution in each group, and trypsin was added 4 hours later. Figure 7A shows the I_eq before and after the treatments, with aprotinin and trypsin in the 4 groups of cells. Figure 7B shows the decreases of I_eq in different groups after 4 hours incubation with aprotinin, which indicates the endogenous activity of prostasin. There was a trend that steroids increase the reduction of I_eq by aprotinin, but this did not reach statistical significance.

View larger version (36K): [in this window] [in a new window]   Figure 7. A, The change of equivalent current after aprotinin and trypsin in the 4 different groups (n=6). Although Aldo (), Dex () and the combination (A+D, stimulated baseline current, all groups responded similarly to aprotinin and trypsin. B, The decrease of equivalent current after aprotinin in the 4 different groups (n=6). The decrease of Ieq after aprotinin indicates the function of endogenous serine protease. There is no significant difference among the 4 groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Collecting duct sodium transport is regulated by a variety of hormones, the most important of which are Aldo and, to a lesser extent, vasopressin. The mechanisms of regulation of sodium transport by these and other hormones are still being actively investigated. Regulation by an extracellular protease, CAP1, first clearly demonstrated by Vallet et al,² represents a unique mechanism of regulation that has been little explored. The primary sequence of CAP1 or prostasin indicates that it is likely bound to the apical membrane or secreted into the apical luminal fluid.^2,6 Findings by other groups indicate that CAP1 likely activates preexisting sodium channels rather than causing the insertion of new sodium channels into the apical membrane.^2,4,8 The findings of Masilamani et al⁷ that Aldo induces a shift in the molecular weight of the -subunit of sodium channels from 85 to 70 kDa, have suggested that this might be the mechanism of action of CAP1; however, proof of this mechanism is lacking, and some contrary data have been reported.² One group has presented preliminary data that demonstrate cleavage of ENaC subunits by trypsin.⁹

Our previous studies demonstrated that the M-1 cortical collecting duct cell line exhibits functional changes with aprotinin and trypsin, consistent with an endogenous protease activity activating sodium transport.³ The present study demonstrates several important characteristics of this activation. Importantly, several findings point to more complex and multiple actions of endogenous protease(s) in collecting duct cells rather than simple sodium channel activation. Two different serum protease inhibitors (STI and aprotinin) both decrease sodium transport, measured as I_eq. However, some significant differences are obvious between STI and aprotinin: the response to aprotinin is quicker and plateaus earlier than the effect of STI; the effect of aprotinin is reversed by trypsin; and aprotinin, but not STI, dramatically alters resistance. The effect of both inhibitors begins rapidly but does not have a full effect at least for an hour. With both, there is a more prolonged effect; even with aprotinin, there is a further decrease in I_eq after 24 hours. This rapid time course may provide some future insight into possible mechanisms of the protease activity activating sodium transport. The finding that both inhibitors inhibit sodium transport but that there are differences implies that there may be several actions of endogenous proteases (and multiple proteases, as discussed below) present in M-1 cells and the native cortical collecting duct. The alteration of transepithelial resistance with aprotinin, which is reversed by trypsin, implies that endogenous protease activity may also regulate transport properties in addition to those involving sodium channels. The functional data, furthermore, demonstrate this protease activity does not appear to be regulated dramatically by glucocorticoids or mineralocorticoids in these cells. The studies also demonstrate the presence of the mouse ortholog of human prostasin in M-1 cells. This protein has been called either prostasin or mCAP1. Similar to the functional studies, there is no increase in the mRNA levels of prostasin by adrenal steroids.

The rapid time course with trypsin found in the present studies is entirely consistent with activation of preexisting channels rather than induction of the synthesis of new channels or other processes that might take a more prolonged period of action. The rapidity of the early effects of aprotinin are also much faster than any of the reported rates of turnover of the sodium channel subunits, with a range of 1 hour¹⁰ to most recent estimates of >24 hours for the apical surface subunits.¹¹ Reversal of the effect of aprotinin with trypsin indicates that the effect is not nonspecific, but rather a reversible phenomenon.

Distinct characteristics for the inhibition of sodium transport by aprotinin versus that by STI possibly indicate that >1 protease activity, with different inhibitory sensitivities, is present in M-1 cells. In fact, preliminary reports indicate that there might be homologs of CAP1 present in mouse cortical collecting duct cells. Interestingly, aprotinin but not STI reversibly dramatically alters transepithelial resistance. Because a decrease of resistance is opposite to the finding expected, with simply an inactivation of sodium channels, some protease activity is likely altering paracellular resistance in these cells. A nonspecific effect of aprotinin on paracellular resistance is unlikely based on the finding that trypsin increases resistance in these cells. Notably, aprotinin and STI differ in their effect of transepithelial resistance. An alternative possibility to multiple proteases is for differing mechanisms of action of aprotinin and STI.

Consistent with our prior functional data, we now find the presence of an mRNA that is essentially identical to the recently reported mCAP1. Because this is highly homologous to human prostasin, this protein might alternately be called mouse prostasin. By Northern analysis, only a single mRNA band, of the expected molecular size, was found. This does not exclude the possible presence of other closely related proteins.

Regulation of the protease activity in M-1 cells was examined using the glucocorticoid Dex and Aldo. Although regulation of prostasin by Aldo seemed possible based on the findings of Masilamani et al,⁷ we could find no regulation of prostasin mRNA or of functional activity in M-1 cells. Dex decreased prostasin mRNA but resulted in no functional change in the response to aprotinin and trypsin. These findings are in spite of the fact that both Dex and Also do regulate sodium transport in these cells.³ Therefore, our experiments do suggest that the induction of Na transport by Aldo (and Dex) does not require an increase in CAP1 (or prostasin) message or activity. Aldo likely increases Na transport by several actions, only some of which may be dependent on the action of CAP1. Another group has preliminary data indicating regulation of prostasin by Aldo,¹² but there are also contrary data.¹³ The reasons for the differences in these studies are not apparent at the present time. Although the present studies expand our understanding of protease regulation of sodium transport in cortical collecting duct cells, many aspects remain to be explored regarding this unique mechanism of regulation of sodium transport. Whether altered protease regulation of sodium transport in the collecting duct contributes to some forms of hypertension needs to be investigated.

Note Added in Proof

Reference 12 has now been published in full (J Clin Invest. 2002;109:401–408) and demonstrates increased prostasin and its apparent effects on Na⁺ transport. Although the discrepant results are unexplained, the main differences in methods were their use of cells on solid support and the use of ²²Na⁺ uptake, in contrast to our use of cells on permeable support and Na⁺ transport measured by current.

	Acknowledgments

 
The present study was supported by research grants from Veterans Administration, National Institutes of Health, and Dialysis Clinics, Inc.

Received May 17, 2001; first decision October 19, 2001; accepted January 23, 2002.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liu, L. Articles by Hamm, L. L. Search for Related Content PubMed PubMed Citation Articles by Liu, L. Articles by Hamm, L. L. Related Collections Hypertension - basic studies Ion channels/membrane transport