(Hypertension. 1995;26:891-898.)
© 1995 American Heart Association, Inc.
Articles |
Expression of Rat Kallikrein and Epithelial Polarity in Transfected Madin-Darby Canine Kidney Cells
From the Departments of Pharmacology (M.A., F.N., F.T., P.A.D., R.A.S., E.G.E.), Anesthesiology (F.N., F.T., R.A.S., E.G.E.), Anatomy and Cell Biology (P.A.D., R.P.B.), and Biochemistry (K.J.C.), University of Illinois College of Medicine at Chicago.
Correspondence to Ervin G. Erdös, MD, Laboratory of Peptide Research, Department of Pharmacology M/C 868, University of Illinois College of Medicine at Chicago, 835 S Wolcott St, Chicago, IL 60612. E-mail egerdos@uicvm.uic.edu.
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Abstract Many properties of urinary kallikrein are well characterized, but the intracellular processing of prokallikrein and release by kidney cells have yet to be clarified. We report here on the synthesis of prokallikrein in Madin-Darby canine kidney (MDCK) cells transfected with rat submaxillary gland kallikrein cDNA and on its activation by MDCK cells and by an enriched liver Golgi membrane preparation. Transfected MDCK cells secreted only prokallikrein at both the apical and basolateral sides in about a 4:1 ratio, but cells transfected with kallikrein cDNA in reverse orientation or untreated cells released only traces of the enzyme. Prokallikrein, in culture medium or in homogenized MDCK cells, was fully activated by trypsin but activated only to 44% by thermolysin. Prokallikrein was synthesized and released into the medium at a high rate: the enzyme secreted by 5x106 cells in 24 hours cleaved 46 nmol/min D-Val-Leu-Arg-7-amino-4-methylcoumarin and liberated 63 ng/min bradykinin after activation. Immunocytology indicated the association of prokallikrein with the Golgi apparatus in the transfected cells. Antiserum to rat urinary kallikrein detected a single band in a Western blot of conditioned medium and also immunoprecipitated the enzyme. Aprotinin inhibited activated prokallikrein. Although MDCK cells released prokallikrein, their homogenates activated prokallikrein at both pH 5.5 and 7.5. Prokallikrein was also activated by a highly enriched liver Golgi membrane fraction and by an endoplasmic reticulum preparation, but the Golgi preparation was 38-fold more active. The activation was blocked significantly by inhibitors of serine proteases and less by cysteine protease inhibitors. Thus, transfected MDCK cells synthesize and release prokallikrein without activating it from the apical (80%) and the basolateral (20%) sides.
Key Words: Golgi apparatus • DNA, complementary • recombinant proteins • prokallikrein • enzyme precursors
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Plasma and tissue kallikreins are serine proteases1 2 that belong to the kallikrein gene family, which can be large in some species (eg, up to 24 members in the mouse or 20 in the rat) but is much smaller in humans.3 4 5 6 7 The genes are tightly clustered on the same chromosome and encode the synthesis of a variety of closely related kallikrein-like enzymes. The term tissue kallikrein (or glandular kallikrein) refers to the best-studied member of the family, which is highly expressed in kidney, pancreas, and salivary gland.1 2 8 The sequences of most kallikreins are known,1 and naturally occurring and synthetic inhibitors and substrates of kallikreins have been characterized,8 9 10 but the exploration of their functions, which started in 1925,2 11 is still going on. Besides releasing bradykinin, plasma kallikrein has a complex but well-defined interrelationship with its substrate, kininogen, and with blood coagulation factors XII and XI.12 13 14 15 Tissue kallikrein was first thought to originate from glandular tissues only (eg, pancreas and submaxillary gland2 9 ), hence the name "glandular" kallikrein,2 8 9 16 17 but diverse organs and tissues such as brain,18 intestine,2 19 kidney,2 20 21 22 23 24 25 26 27 and blood vessels28 also contain the enzyme. Urinary kallikrein was discovered first,2 and because it is excreted less in hypertension26 29 it has been studied most by investigators. This enzyme is tissue kallikrein, which originates from the kidney and is excreted at the level of the connecting tubules.23 24 27 The enzyme liberates kinins from kininogen, which affect Na+ and water excretion and the autoregulation of the kidney by releasing prostaglandins and NO.1 30 The level of urinary kallikrein in the rat is very high,1 2 16 17 but in contrast to the presence of prokallikrein in human or rabbit urine,31 32 the rat enzyme is primarily in the active form, converted from prokallikrein.1 25 Prokallikrein can be activated in the renal tissue before entering the nephron, but kallikrein can also be released from the basolateral side of distal tubular cells and thereby be introduced into the renal circulation as well.22 33 34
To gain more information on the activation and release of renal kallikrein, we transfected MDCK cells with rat tissue kallikrein cDNA3 5 6 35 to study its potential activation and to establish whether its release is polarized to either the luminal or the basolateral side.
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D-Val-Leu-Arg-AMC and Z-Arg-Arg-AMC were purchased from Enzyme Systems Products; thermolysin and phosphoramidon were from Peninsula Laboratories Inc; Lipofectin reagent and G418 (Geneticin) were from Gibco BRL; and vector pHßAPr-3P-neo and pcDNAI were from Invitrogen. Trypsin, thermolysin, SBTI, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), and other chemicals were obtained from Sigma Chemical Co. Antiserum to rat tissue kallikrein (which reacts with both the pro and active forms of the enzyme) was a gift from Dr G. Scicli of Henry Ford Hospital, Detroit, Mich. Aprotinin (Trasylol) was donated by Bayer AG, Wuppertal, Germany, and human urinary trypsin inhibitor was donated by Prof C.-W. Chi of the Shanghai Institute of Biochemistry, Academia Sinica, PRC.
Construction of Kallikrein cDNA and Expression
Vectors
The full-length rat submaxillary gland kallikrein cDNA
(pPS-J) was a generous gift from Dr Raymond J. MacDonald of the
Southwestern Medical Center, University of Texas, Dallas. This cDNA
encodes the full preproenzyme of 265 amino acids with a calculated
molecular weight of 29 285.35 Two PCR primers with
Xba I cleavage sites were synthesized and used to amplify
the cDNA insert from the pPS-J plasmid. The cDNA thus obtained starts
from the translation initiation codon (-28) and ends after the
polyadenylation signal (864). This fragment, after being cloned into
the pGEM-7Z(-) vector, was completely sequenced to ensure that no
spurious mutations were introduced. The kallikrein cDNA was ligated
into the Xba I restriction site of the pcDNAI expression
vector.
Cell Culture
MDCK cells were purchased from the American Type Culture
Collection and maintained in Dulbecco's modified Eagle's medium
(DMEM) with L-glutamine and supplemented with 10% fetal
bovine serum and 50 µg/mL of gentamicin (complete DMEM) at 37°C in
an atmosphere of 95% air/5% CO2.36
Transfection
For the expression of rat kallikrein, pHBAPr-3P-neo and pcDNAI,
containing the kallikrein cDNA, were cotransfected into MDCK cells with
the use of Lipofectin reagent37 according to the
manufacturer's instructions. A 100-mm plate of cells (50% confluent)
was incubated in 2 mL DMEM containing 10 µg of plasmid DNA
pHßAPr-3P-neo, 20 µg pPS-J-pcDNAI plasmid DNA, and 30 µL
Lipofectin for 15 hours at 37°C in a 95% air/5% CO2
incubator. The pcDNAI plasmid DNA with pPS-J ligated in the inverted
direction was used as control with the same procedures. After the
DNA-containing medium was replaced with 5 mL complete DMEM, the cells
were incubated for 48 hours, then split and grown in complete DMEM
containing 600 µg/mL G418 (selective medium). Separate colonies were
isolated using cloning cylinders and were grown in 24-well culture
plates. After reaching 80% to 100% confluence, the colonies were
amplified and screened for expression of kallikrein in culture media
and/or cell homogenates by specific enzyme assays. Several
clones that expressed prokallikrein were isolated, as were several
control clones. One of the clones that expressed prokallikrein,
designated ExPKK, was chosen for further analysis. Another one,
transfected with inverted kallikrein cDNA, was designated ExPC. The
nontransfected MDCK cells also served as controls (cont-MDCK).
Protein Determination
The protein concentration was determined according to Bradford
with crystalline bovine serum albumin (BSA) as a
standard.38
Separation of Golgi and Endoplasmic Reticulum
Membranes
Golgi and ER vesicles were prepared from rat liver according to
Fleischer and Kervina,39 with a few modifications. Rat
livers were dissected and rinsed with ice-cold washing buffer (0.25
mol/L sucrose in 20 mmol/L phosphate buffer, pH 7.1) immediately after
the animals were killed. Rat livers were weighed, placed in two volumes
of homogenization buffer (0.25 mol/L sucrose, 10
mmol/L HEPES, pH 7.5, 100 mmol/L iodoacetamide, 10 µg/mL leupeptin,
and 10 µg/mL aprotinin), homogenized, and filtered
through two layers of cheesecloth. The filtrate was centrifuged
at 2000g for 10 minutes and filtered again. The filtrate was
centrifuged at 25 000g for 10 minutes and the
supernatant was centrifuged at 34 500g for 10
minutes. The supernatant was saved for isolation of the ER, the pellet
was dissolved gently in sucrose buffer (52% sucrose in 20 mmol/L
phosphate buffer, pH 7.1), and the final sucrose concentration was
adjusted to 43.7%. A discontinuous sucrose gradient was prepared by
placing the resuspension (7 mL per tube) at the bottom of an
ultracentrifuge tube and layering the different sucrose
solutions in 20 mmol/L phosphate buffer, pH 7.1, on top (from bottom to
top: 12 mL of 38.7%, 7 mL of 36%, 7 mL of 33%, and 8 mL of 29%).
This gradient was centrifuged at 100 000g for 1
hour. Golgi vesicles were collected at the interphase between 29% and
33% sucrose, diluted with an equal volume of cold distilled water, and
pelleted by centrifugation at 250 000g for
45 minutes. Golgi vesicles were stored in 20 mmol/L phosphate buffer,
pH 7.1, containing 20% glycerol, 10 µg/mL leupeptin, and 10 µg/mL
aprotinin. The enrichment of the Golgi membrane preparation was
monitored by measurement of ß-galactoside
I-2,6-sialyltransferase activity as described.40 The
specific activity of sialyltransferase in the Golgi preparations was
enriched approximately 300-fold over that in the crude
homogenate. ER vesicles were basically prepared according
to Fleischer and Kervina39 (and K.J.C., unpublished data,
1995). In control experiments, after dilution, it was ascertained that
the enzyme inhibitors added to the membrane fractions did
not affect prokallikrein activation or the assay of kallikrein
activity.
Cell Homogenization and
Fractionation
ExPKK cells, ExPC cells, and cont-MDCK cells were grown to
confluence on 100-mm plates36 in 10 mL of medium, yielding
5x106 cells per plate. Cells were washed and then
were incubated for 24 hours in 10 mL of serum-free DMEM. The medium
was removed, and the cells then were washed three times in PBS and
harvested by scraping, resuspended in PBS, centrifuged at
800g for 10 minutes, then resuspended in 20 mmol/L Tris, pH
7.4, containing 0.25 mol/L sucrose, and disrupted by sonication with a
Sonic Dismembranator (Artek System). The homogenates were
fractionated at 4°C by sequential centrifugation at
1000g for 15 minutes, 10 000g for 25 minutes,
and 100 000g for 60 minutes. The pellets gained in each
centrifugation step (P1, P2, and P3) were resuspended
in the same buffer. The pellets and the final supernatant (S3) were
either used immediately or stored at -70°C.
Enzyme Assay
Kallikrein activity was determined fluorometrically with the
substrate D-Val-Leu-Arg-AMC.41 Fifty
microliters of sample (culture medium or cell homogenate)
was added to 445 µL of 0.1 mol/L Tris-HCl buffer, pH 8.0, containing
10 mmol/L CaCl2 and 25 µg/mL SBTI. The reaction was
initiated by adding 5 µL of 10 mmol/L substrate dissolved in
dimethylsulfoxide, and the increase in fluorescence was
monitored directly by a recording spectrophotofluorometer at
380 nm excitation and 460 nm emission wavelengths. Kallikrein activity
was also assayed by measuring the release of bradykinin from heated dog
plasma (kininogen) by radioimmunoassay.42 Kallikrein
activity is expressed as units (1 U=1 pmol AMC produced per minute) per
milliliter of medium or per milligram of protein. Kininogenase activity
is expressed as nanograms of bradykinin equivalent released per minute
per milliliter of medium.
Cathepsin B
Cathepsin B activity was measured with Z-Arg-Arg-AMC
substrate according to Barrett.43
Activation of Prokallikrein
In routine assays, trypsin (and in additional studies,
thermolysin) was used as prokallikrein activator.
Serum-free media from cell culture or MDCK cell fractions were
treated with 2.5 µg/mL trypsin in 0.1 mol/L Tris, pH 7.4, for 30
minutes at 37°C.25 41 The reaction was stopped with 25
µg/mL SBTI, which completely inhibited trypsin in control
experiments. With thermolysin (5 µg/mL), the culture media or the
cell fractions were incubated for 30 minutes at 37°C in 0.1 mol/L
Tris, pH 8, containing 10 mmol/L CaCl2 and 25 µg/mL SBTI.
The reaction was stopped with 50 µmol/L
phosphoramidon.41 In control studies,
phosphoramidon completely inhibited the activation of
prokallikrein by thermolysin.
Activation by Tissues
To investigate whether the MDCK cell homogenate or
liver ER or Golgi fractions contain enzymes that activate
prokallikrein, we incubated the subcellular fractions with secreted
prokallikrein. A mixture of either 100 µL of 0.4 mol/L Tris, pH 7.4,
or 0.25 mol/L 2-(N-morpholino)ethanesulfonic
acid, pH 5.5, 50 µL distilled water, and either 50 µL liver ER (733
µg protein/mL), 50 µL Golgi extract (34 µg protein/mL), or ExPKK
MDCK cell homogenate (1.22 mg protein/mL) was incubated
with 200 µL culture medium at 37°C for 2 hours. When we
investigated the effects of protease inhibitors on
activation by Golgi membrane fractions, the 50 µL H2O was
replaced with 50 µL inhibitor in 0.1 mol/L Tris, pH 7.4,
to the final concentrations indicated. The tubes were incubated at
4°C for 15 minutes and then at 37°C for 2 hours.
Immunoprecipitation
Activated kallikrein from the culture medium or the cell
fractions was immunoprecipitated with rabbit antiserum to rat urinary
kallikrein and insoluble protein A.44 45 The extent of
immunoprecipitation was estimated from the amount of kallikrein
activity removed from the supernatant. Normal rabbit serum served as
control.
Immunocytochemistry
ExPKK cells, ExPC cells, and cont-MDCK cells were grown to
confluence on coverslips. The cells were fixed with 2%
paraformaldehyde in PBS at 4°C for 30 minutes. The
fixed cells were washed with PBS and dehydrated with 95% ethanol for 2
minutes. After being washed again with PBS, the cells were preincubated
with 5% BSA in PBS and then were incubated with antiserum to rat
kallikrein (diluted 1:300) for 1 hour at room temperature. Normal
rabbit serum was used as the control. The coverslips were washed three
times with PBS and incubated with donkey anti-rabbit IgG
F(ab')2 fragments conjugated to Texas red (Jackson
Immunoresearch) diluted 1:200 in 5% BSA in PBS for 1 hour at room
temperature. The cells were washed three times with PBS and covered
with Fluoromount G (Fisher Biotech) for viewing and photographing.
Apical and Basolateral Sorting
To study polarized delivery, the cells were plated at high
density in 24-mm tissue culture Transwell filter chambers (0.4-µm
pore size) and grown to confluence. For the assays of enzymatic
activity in apical and basolateral culture media, Transwell cells
were washed with DMEM three times and incubated with 2 mL DMEM in each
chamber. After incubation of varying durations (0 to 24 hours) in
serum-free medium, the apical and basolateral culture media were
collected and assayed for kallikrein activity.
Polyacrylamide Gel Electrophoresis and
Electroblotting
Aliquots of rat urine and cell culture media were
analyzed by SDS-PAGE for the presence of kallikrein. Kallikrein
bands were detected by Western blot analysis36
using antiserum to purified rat tissue kallikrein.
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Secretion of Prokallikrein
Transfected MDCK cells synthesized and released prokallikrein at a high level into the serum-free medium in a time-dependent manner. The enzyme secreted into the medium was almost entirely inactive proenzyme (Table 1), which could be activated by either trypsin or thermolysin, although trypsin was more efficient. The maximal specific activity of kallikrein released in 24 hours into the medium was 4.6 nmol of D-Val-Leu-Arg-AMC hydrolyzed in 1 minute per milliliter medium (n=3) in the first series of experiments (Table 1) and 3.7 nmol in the second series (n=3; Table 2). In other experiments measured by radioimmunoassay, 6.3 ng of kinin was released per minute from kininogen by activated kallikrein in 1 milliliter medium (Table 2). The total prokallikrein released by 5x106 cells in 24 hours after activation cleaved 46 or 37 nmol/min of the short peptide substrate and released 63 ng bradykinin per minute. In control experiments with medium from nontransfected (cont-MDCK) cells or with cells transfected with a plasmid ligated in reverse direction (ExPC), only a trace of kallikrein activity was detected.
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It is possible that the transfected cells did not secrete prokallikrein but instead released active kallikrein that was complexed with an inhibitor susceptible to proteolysis by the activator enzyme. To rule this out, media from prokallikrein-transfected cells or control MDCK cells were incubated with recombinant kallikrein that had been activated with trypsin followed by SBTI inhibition. No inhibition by the media from either transfected or control MDCK cells was seen (not shown), proving that the expressed enzyme was prokallikrein and not an inhibited form of active kallikrein.
The ratio of apical to basolateral release of prokallikrein from the cells was approximately 4:1, and this ratio stayed the same in samples taken at various time intervals (Fig 1). When the medium was collected separately from the apical and basolateral sides, 99.5% or 96.5% of the enzyme secreted apically or basolaterally was prokallikrein, as established after activation with trypsin.
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Prokallikrein in MDCK Cells
To exclude the possibility that only prokallikrein was released by
transfected cells because active kallikrein was retained in a
sequestered form, ExPKK and control cells were homogenized
and subcellular fractions were separated by differential
centrifugation. None of the fractions had significant
amounts of active kallikrein, indicating that the lack of activation,
and not sequestration of the active enzyme, was responsible (Table 3). Kallikrein activity in the ExPKK
homogenate was 2250±401 (U per mg protein) after
activation, while it was only 125±22 in the homogenate
without activation (Table 3). Homogenates of
cont-MDCK and ExPC cells had low activity (73±15 and 125±40) even
after activation. The highest activity after activation was found in
the final supernatant (S3) obtained from the ExPKK cell
homogenate (4478±681), while the high-speed sediment,
membrane-enriched fraction was less active (618±142). In the
control cells, the level of activated kallikrein was low in all
the fractions tested.
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Identity of Kallikrein
Substrate hydrolysis by the activated prokallikrein was
inhibited by aprotinin in a dose-dependent manner; 10 U inhibited
90% of the activity in the reaction mixture.
Polyclonal antiserum to rat urinary kallikrein precipitated 87±4.4% of the activatable kallikrein activity from the culture medium of ExPKK cells and 88±3% of the activity in the homogenates of the same cells.
Immunocytochemistry
ExPKK cells, immunostained for prokallikrein, showed
immunoreactive structures dispersed in the apical cytoplasm, as judged
by focusing through the cells from base to apex (Fig 2), and in juxtanuclear aggregates
against one side of the cell nucleus (Fig 3). These patterns of staining suggest
the localization of prokallikrein in elements of the Golgi complex and
in vesicles of the apical cytoplasm in ExPKK cells. These sites usually
are associated with glycosylation and other posttranslational
processing of proteins before their release.46 47 There
was no immunoreactivity to antiserum in the ExPC or MDCK cells or to
nonimmune rabbit serum in ExPKK cells (Fig 4).
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Activation of Prokallikrein
In contrast to rat urinary kallikrein, which is mostly in the
active form,1 25 the kallikrein in the
homogenized transfected cells or released by them into the
medium was almost entirely prokallikrein. It was therefore necessary to
establish whether or not the MDCK cells transfected with rat kallikrein
cDNA lacked the enzyme or enzymes to activate
prokallikrein.
Homogenized ExPKK cells activated the added prokallikrein from the medium at either pH 5.5 or 7.4. One microgram of protein in the homogenate produced 24.2 U at pH 7.4 and 20.8 U at pH 5.5 in 2 hours of incubation. This activation was partially inhibited by both catheptic and serine protease inhibitors at either pH. The inhibition by E64, TLCK, and LBTI at pH 7.4 was 40%, 54%, and 66%, respectively (see Table 4). Thus, serine protease inhibitors were more effective than the cathepsin inhibitor.
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Because high concentrations of renal kallikrein have been detected in the Golgi apparatus in previous studies,23 24 we investigated the activation of rat prokallikrein by a rat liver Golgi membrane fraction and, as a control, by an ER membrane fraction. Both the ER and the highly enriched Golgi membrane fraction (about 300-fold; K.J.C., unpublished data, 1995) activated prokallikrein, but the Golgi fraction was much more effective: The ratio of specific activity of Golgi versus ER was 38:1 (Table 5).
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The activation of prokallikrein by the enzymes in the Golgi fraction (Table 6) was inhibited by several compounds; inhibitors of trypsin-type serine proteases were the most effective, with diisopropyl fluorophosphate (DFP) inhibiting the activation at pH 7.4 by 84%. To ensure that the DFP used under our conditions would not inhibit the resulting active kallikrein, we added DFP to thermolysin in the same ratio. Thermolysin still activated kallikrein measured after DFP was removed by dialysis as above. LBTI and urinary trypsin inhibitor inhibited 42% and 63%. Other inhibitors of catheptic enzymes and metalloproteases were less effective (N-ethylmaleimide, E64, o-phenanthroline).
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Cathepsin B
To determine whether or not cathepsin B, a potential
activator of proenzymes, was present in the extracts, a
Golgi membrane–enriched fraction of the rat liver and the
homogenized MDCK cells were tested with the cathepsin B
substrate Z-Arg-Arg-AMC. The cleavage as measured in
fluorescence units per minute per 0.5 mL was 17.7±3 (Golgi)
and 9.8±1 (MDCK cells) and in the presence of added cathepsin
inhibitor E64, was decreased to 1.2±0.1 and 0.7±0.1,
respectively.
Western Blot Analysis
The immunoreactivity and molecular mass of prokallikrein and
trypsin-activated kallikrein released into the medium of
transfected cells and of rat urinary kallikrein were compared (Fig 5). The medium from transfected cells
yielded a single band, reacting strongly with anti-rat urinary
kallikrein antiserum (Fig 5B, lane 4). The single
reactive band from rat urine was the same size (Fig 5B, lane 1). Media from cont-MDCK and ExPC cells
yielded no detectable bands (Fig 5B).
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Fig 5A shows trypsin-activated (lane 1) and unactivated (lane 2) media from ExPKK cells. Both lanes gave single bands of apparently identical molecular mass, indicating that trypsin activation, as expected from the small size of the propeptide,41 resulted in an undetectable change in the enzyme molecular mass.
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These experiments showed that MDCK cells transfected with rat tissue kallikrein cDNA (ExPKK) contained and secreted prokallikrein from both the apical and basolateral aspects of the cells at an approximately 4:1 apical:basolateral ratio. The cells that were not transfected or were transfected with cDNA ligated in the inverted direction contained or released into the medium only traces of kallikrein. In the homogenates of transfected cells, prokallikrein appeared mainly in the final supernatant fraction.
In rat urine, kallikrein released from the kidney is mainly in an active form,1 25 whereas rabbit and human urine contain substantial levels of prokallikrein.31 41
Prokallikrein is activated by the release of a short propeptide ending in arginine, and as in serine proteases, the newly revealed N-terminus of active kallikrein forms an internal link with an aspartic acid residue, thereby rendering the active center accessible to the substrate.41 Rat tissue prokallikrein is activated by hydrolysis of the Arg-1-Val+1 bond. This can be accomplished by an enzyme having a specificity similar to trypsin (such as plasmin32 or a thiol protease), cleaving at the C-terminus of a basic amino acid, or by an enzyme such as thermolysin that hydrolyzes at the N-terminus of a hydrophobic amino acid.41 In contrast to studies with human kallikrein,41 in which thermolysin was about three times more active than trypsin, it was a less effective activator of the rat prokallikrein than trypsin was in these studies. This may be due to differences in the N-terminal sequence of rat and human prokallikrein, the rat having Val+1-Val+2-Gly+3,35 while the human N-terminus is Ile+1-Val+2-Gly+37 ; thus, the N-terminal amino acid is more hydrophobic in humans than in rats. This variance in the +1 residue could affect the efficiency of thermolysin-catalyzed activation of the proenzyme.
Although kallikrein in rat urine is almost entirely active,1 25 MDCK cells transfected with rat kallikrein cDNA contained or released essentially only prokallikrein. Thus, we considered whether or not the MDCK cells would have the enzyme or enzymes that can activate prokallikrein. Because prokallikrein lacks the repeating pairs of basic amino acid sequence necessary for cleavage by mammalian Kex2-like prohormone convertases,47 48 it would not be processed by them. When MDCK cells were homogenized, the homogenate added to prokallikrein activated both at an acid and a neutral pH. This result indicated that MDCK cells contain enzymes, a serine protease–type enzyme and possibly cathepsin B, which can activate prokallikrein. It is apparent, however, that during the intracellular processing of prokallikrein, it did not come in contact with activator enzymes, probably because of differences in the compartmentalization of the proenzyme and the activators. Other reasons for the lack of activation of transfected rat prokallikrein in MDCK cells could be either the inability of the dog Golgi enzymes to be effective activators of a rat proenzyme or the high level of expression and rapid secretion, allowing insufficient time for activation. Thus, a multiplicity of factors can affect intracellular processing of proproteins.49 In vivo, it is possible that kallikrein is synthesized and released as the proenzyme, which is then activated by a different cell type than the one that produced it.
Renal kallikrein (or prokallikrein) has been detected in high concentration in the Golgi apparatus,50 51 a putative site for the activation of prokallikrein. A highly enriched Golgi membrane fraction, isolated from rat liver, indeed contains a membrane-associated serine protease and some other enzymes that can activate prokallikrein at both acid (5.5) and neutral (7.4) pH values by cleaving after a single basic residue. The activation was blocked effectively by added trypsin inhibitors and less by reagents that react with SH enzymes, such as the inhibitors E64 or N-ethylmaleimide. Thus, a cathepsin B–type enzyme could be responsible for only about one third of the enzymatic activation of prokallikrein, judging from inhibition studies (Table 6). Homogenized rat kidney also contains an enzyme that activates prokallikrein and is inhibited by SBTI.52
In the present results, light microscopy of MDCK cells transfected with kallikrein cDNA (ExPKK) shows punctate immunostaining in the apical and juxtanuclear cytoplasm. This pattern of staining suggests the localization of prokallikrein in elements of the classic rough ER–Golgi apparatus–secretory vesicle pathway for glycoprotein synthesis.23 24 50 Absent from present findings was a localization of immunoreactivity to the apical or basal plasma membrane of the ExPKK cells. This lack of staining, based on positive controls (not shown), is not an artifact of specimen preparation. It is correlative evidence that the final destination of the ExPKK kallikrein is secretion rather than integration into the plasma membrane of the transfected cells.
The results of the present investigation are consistent with two other reports in which kallikrein cDNA was transfected into either insect cells or AtT-20 cells and found to be secreted in the prokallikrein form without any evidence of membrane binding.53 54 This is in contrast to findings in homogenized kidney, where kallikrein has been recovered in part as prokallikrein that sedimented with membrane fragments.20 22 34 52 55 56 It has even been suggested that kallikrein is an ectoenzyme.57
This is not only a feature of kallikrein in vivo, as a study on the renal cell line A6 from Xenopus laevis showed that kallikrein was present in membrane-bound form on both the basolateral and apical surfaces and that secretion occurred only from the apical side, after the enzyme had reached the plasma membrane.58 The mechanism of membrane binding of renal prokallikrein is unknown, as the prokallikrein sequence lacks an obvious hydrophobic transmembrane spanning region or a signal for attachment to a glycosylphosphatidylinositol anchor. One possibility is that membrane-bound kallikrein is lipid modified, eg, by palmitoylation.59 Myristoylation and prenylation are less likely, as the former requires an N-terminal glycine residue and the latter a cysteine at or near the C-terminus,59 both of which are lacking in the sequence of rat kallikrein.6 Alternatively, kallikrein may not be directly bound to the membrane but instead might interact with a membrane-associated receptor or binding protein. Thus, the lack of membrane binding of recombinant kallikrein in transfected cells might be due to the lack of synthesis of a specific binding protein by the cells.
The finding that prokallikrein is secreted from both the apical and basolateral sides of renal epithelial cells is consistent with studies on perfused kidneys33 and in vivo work and suggests a function for the enzyme in the tubular fluid and/or urine as well as in blood. For example, urinary kinins, released by kallikrein in the distal nephron, are thought to participate in the autoregulation of the kidney by enhancing Na+ and water excretion and the release of PGE2.30 60 Thus, kinins filtered by the glomerulus are eliminated by kininase II–type enzymes such as angiotensin I–converting enzyme or neutral endopeptidase 24.1120 61 on the proximal tubular brush border. In the distal tubules, as judged from experiments with cultured MDCK cells, membrane carboxypeptidase M is the major kininase of this portion of the nephron and is excreted in the urine as well.36 62 Kallikrein, present on the basolateral membrane of renal cells as we reported first,21 34 may enter the renal circulation; thus, it may be the source of kallikrein that appears when the isolated kidney is perfused.33 Renal kinins can be involved in the regulation of papillary blood flow and in the synthesis of NO and prostaglandins.1 60 Other experiments suggest additional roles for kallikrein in the basal part of kidney cells; for example, when kinins are applied to the serosal side of isolated epithelial cells of the colon, they enhance Cl- secretion from the apical side.63
In conclusion, MDCK cells transfected with rat kallikrein cDNA release prokallikrein into the culture medium from both the apical and basolateral sides. These experiments also showed that Golgi membrane fraction is rich in a serine protease with the specificity to activate prokallikrein.
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These studies were partially supported by grants HL-36473 and HL-36081 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. The editorial assistance of Sara Thorburn is gratefully acknowledged. We also thank Dennis Riley for the radioimmunoassay of bradykinin, Dr Raymond J. MacDonald for useful discussions, and Claudie Hecquet for her cooperation.
Received May 2, 1995; first decision June 6, 1995; accepted August 24, 1995.
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