Department of Medicine, University of Cincinnati, and Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45267
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ABSTRACT |
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The purpose of
the current experiments was 1) to
assess basolateral
Na+-K+-2Cl
cotransporter (NKCC1) expression and
2) to ascertain the role of cystic
fibrosis transmembrane conductance regulator (CFTR) in the regulation
of this transporter in a prototypical pancreatic duct epithelial cell
line. Previously validated human pancreatic duct cell
lines (CFPAC-1), which exhibit physiological features prototypical of
cystic fibrosis, and normal pancreatic duct epithelia (stable
recombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown to
confluence before molecular and functional studies. High-stringency
Northern blot hybridization, utilizing specific cDNA probes, confirmed
that NKCC1 was expressed in both cell lines and its mRNA levels were
twofold higher in CFPAC-WT cells than in CFPAC-1 cells
(P < 0.01, n = 3).
Na+-K+-2Cl
cotransporter activity, assayed as the bumetanide-sensitive, Na+- and
Cl
-dependent
NH+4 entry into the cell (with
NH+4 acting as a substitute for
K+), increased by ~115% in
CFPAC-WT cells compared with CFPAC-1 cells
(P < 0.01, n = 6). Reducing the intracellular
Cl
by incubating the cells
in a Cl
-free medium
increased
Na+-K+-2Cl
cotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells
and mediates the entry of
Cl
. NKCC1 activity is
enhanced in the presence of an inward
Cl
gradient. The results
further indicate that the presence of functional CFTR enhances the
expression of NKCC1. We speculate that CFTR regulates this process in a
Cl
-dependent manner.
cystic fibrosis transmembrane conductance regulator; HCO3 secretion; cystic
fibrosis
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INTRODUCTION |
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ACTIVATION OF cystic fibrosis transmembrane conductance
regulator (CFTR) by secretin enhances
Cl secretion into
pancreatic duct lumen and, as a result, depolarizes the luminal and
basolateral membranes of the duct cells (3, 6, 26, 33). In parallel
with enhanced Cl
secretion,
secretin-stimulated CFTR activation also increases HCO
3 secretion in the pancreatic duct
cells (3, 6, 16, 17, 19, 33). Several studies have demonstrated that
the Cl
concentration in the
final pancreatic ductal secretion is very low, indicating reabsorption
of Cl
along the ductal
system (3, 6). This has led to the postulation that the resulting
increase in luminal Cl
drives an apical
Cl
/HCO
3
exchanger (reviewed in Refs. 3, 6, 33). According to this model, the
indirect coupling of CFTR and the apical
Cl
/HCO
3
exchanger results in the recycling of Cl
and sustains the driving
force for Cl
and
HCO
3 secretion in the
agonist-stimulated state (3, 6, 33).
Clearly, the degree of Cl
efflux via activated CFTR depends on the availability of intracellular
Cl
(3, 6). Recent studies
demonstrate that removal of luminal Cl
or addition of DIDS only
partially inhibited secretin-stimulated HCO
3 secretion (<25%) in the guinea
pig pancreatic duct cells (16, 17), strongly suggesting that the apical
Cl
/HCO
3
exchanger does not play a major role in secretin-stimulated
HCO
3 secretion. These findings by
inference demonstrate that activation of CFTR by secretin and the
resultant HCO
3 secretion persist
despite lack of availability of luminal
Cl
. Together, these studies
indicate that a mechanism distinct from the apical
Cl
/HCO
3
exchanger is responsible for
Cl
entry into the duct cells.
A number of secretory epithelial cells express a
Na+-K+-2Cl
cotransporter on their basolateral membrane, which mediates the entry
of Cl
into the cell for
eventual secretion via apical
Cl
channels (i.e., CFTR or
a Ca+-sensitive
Cl
channel) (4, 10, 22,
24). The best example is the small intestine, in which cAMP-activated
Cl
secretion (via CFTR) is
dependent on the basolateral
Na+-K+-2Cl
cotransporter (23-25, 27). Cloning experiments have identified the
cDNA encoding the
Na+-K+-2Cl
cotransporter (called NKCC1) (12, 37). NKCC1 is expressed in a variety
of epithelial and nonepithelial cells (4, 10, 12, 15, 22-25, 37).
In nonepithelial cells such as vascular smooth muscle cells, NKCC1 is
predominantly responsible for volume regulation by the transportation
of Na+ and
Cl
into the cells (12, 15,
37). In epithelial cells such as small intestine or kidney tubules,
NKCC1 is localized on the basolateral membrane domain (12, 22-25,
34, 36, 37) and is responsible for the transport of
Cl
and
Na+ from blood to the cell.
Cl
is likely secreted into
the lumen via Cl
channels
(CFTR or Ca2+-sensitive isoform),
whereas Na+ is transported back to
the blood via the
Na+-K+-ATPase.
In addition to the basolateral cotransporter (NKCC1), an apical
Na+-K+-2Cl
cotransporter (NKCC2) has also been cloned (14). NKCC2 is exclusively expressed on the apical membranes of kidney thick limb of Henle and
is responsible for the reabsorption of
Na+ and
Cl
in this nephron segment
(14, 29). Both NKCC1 and NKCC2 are electroneutral transporters
and are sensitive to inhibition by furosemide and bumetanide (12, 14,
24, 29, 37). In addition to Na+
and Cl
, both the apical and
basolateral
Na+-K+-2Cl
cotransporters can transport NH+4, with
NH+4 substituting for
K+ (1, 2, 28, 35).
The purpose of the current experiments was to determine whether NKCC1 is expressed in cultured pancreatic duct cells. In addition, we were interested in testing whether CFTR plays any role in the regulation of this transporter. Accordingly, cultured pancreatic duct cells with or without functional CFTR were utilized. The results demonstrated that NKCC1, but not NKCC2, is expressed in pancreatic duct cells and its expression is regulated by CFTR.
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MATERIALS AND METHODS |
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Cell lines. CFPAC-1 cells were obtained from American Type Culture Collection (ATCC) and cultured as previously described (30, 31). Additional CFPAC-1 clones (1a and 1b) were developed in our laboratory. Stably transfected CFPAC-1 cells bearing functional CFTR (termed CFPAC-WT) were cultured in a similar fashion, except for the addition of G418 (1 mg/ml) to the medium (13, 31). CFPAC-WT clones (CFPAC-WT, CFPAC-WTa, and CFPAC-WTb) were a generous gift from Dr. R. Frizzell, University of Pittsburgh. Capan-1 cells were obtained from ATCC and cultured as previously described (31).
RNA isolation.
Total cellular RNA was extracted from CFPAC-1 and CFPAC-WT cells by the
method of Chomczynski and Sacchi (7). In brief, cells from three
separate flasks were homogenized at room temperature in 10 ml of
Tri reagent (Molecular Research Center, Cincinnati, OH). RNA was
extracted by phenol-chloroform, precipitated by isopropanol (7),
quantitated by spectrophotometry, and stored at
80°C.
Northern hybridization. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI) using 10× sodium chloride-sodium phosphate-EDTA as transfer buffer. Membranes were cross-linked by ultraviolet light and baked for 1 h. Hybridization was performed according to Church and Gilbert (8). The cDNA probes were labeled with [32P]deoxynucleotides using the RadPrime DNA labeling kit (GIBCO BRL). The membranes were washed twice in 40 mM sodium phosphate buffer, pH 7.2, 5% SDS, 0.5% BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM sodium phosphate buffer, pH 7.2, 1% SDS, and 1 mM EDTA for 10 min at 65°C. Thereafter, the membranes were blotted dry and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) at room temperature for 24-72 h. Densitometric scanning of the blots was performed by the PhosphorImager. The following rat PCR product fragments were used as specific probes in the Northern blot analyses: for NTCC2, nucleotides 509-3237; for NTCC1, nucleotides 906-1238 and 1631-2300. These PCR fragments have been used as specific probes (2) and under high-stringency hybridization conditions do not cross-react with one another (2).
Intracellular pH measurements. Intracellular pH (pHi) in cultured pancreatic cells was measured with the use of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as previously described (2, 5, 31). In brief, cells were grown to confluence on coverslips, loaded with BCECF, and monitored for pHi recording with the use of a Delta Scan dual-excitation spectrofluorometer (double-beam fluorometer, Photon Technology International, Brunswick, NJ) that was equipped with a water-jacketed, temperature-controlled system. Fluorescence intensity was recorded at emission wavelength at 525 nm and two excitations wavelengths at 500 and 450 nm. The fluorescence ratios (F500/F450) were converted into pHi values with use of calibration curves that were established by the KCl-nigericin method.
Measurement of
Na+-K+-2Cl
cotransporter activity.
Na+-K+-2Cl
cotransporter activity was assessed as previously described (2).
Briefly, the
Na+-K+-2Cl
cotransporter activity was measured by determining the rate of intracellular acidification caused by NH+4
entry into the cells via this transport mechanism on abrupt application of 40 mM NH4Cl (1, 2, 28, 35).
NH+4 entry into cultured cells can occur via
several transport pathways, including
Na+-dependent and
Na+-independent pathways (1, 2,
28, 35). The Na+ dependence of
NH+4 transport was determined to distinguish
this transporter from
Na+-independent pathways.
Furthermore, bumetanide, which inhibits Na+-K+(NH+4)-2Cl
cotransport, was used to distinguish this transporter from other Na+-dependent pathways (1, 2).
NKCC1 activity was therefore determined as the bumetanide-sensitive,
Na+-dependent
NH+4 entry into the cells.
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Materials. [32P]dCTP was purchased from NEN (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO BRL. BCECF was from Molecular Probes (Eugene, OR).
Statistical analysis. The data are expressed as means ± SE where appropriate. For statistical analysis of mRNA expression experiments, the PhosphorImager readings of three separate Northern hybridizations were obtained and analyzed. Statistical analysis was determined using one-way ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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Molecular expression of NKCC1 in wild type CFTR-bearing pancreatic
duct cells.
In the first series of experiments, we examined whether NKCC1 is
expressed in wild-type CFTR-bearing cultured pancreatic duct cells
(CFPAC-WT cells). Northern hybridizations utilizing a
32P-labeled probe corresponding to
nucleotides 906-1238 and 1631-2300 of rat NKCC1 identified a
6.5-kb transcript in CFPAC-WT cells (Fig. 2), consistent
with the expression of NKCC1 in the pancreatic duct cells.
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Functional expression of the
Na+-K+-2Cl
cotransporter in wild-type CFTR-bearing pancreatic duct cells.
CFPAC-WT cells were grown to confluence on glass coverslips, loaded
with BCECF, and assayed for
Na+-K+-2Cl
cotransporter activity as described in MATERIALS AND
METHODS. Figure
4A is a representative
pHi tracing that demonstrated that switching the CFPAC-WT cells to an
NH+4-containing solution that also contained
Na+ and
Cl
(solution
B, Table 1) resulted in a rapid initial cell
alkalinization, likely due to NH3
diffusion (Fig. 4A). After this
initial alkalinization, pHi
acidified back to baseline in ~10 min (Fig.
4A). This acidification represented
NH+4 entry, as induction of equivalent cell
alkalinization by acetate withdrawal (solution
E, Table 1) did not result in a significant
acidification (Fig. 4A, representative tracings). The rate of recovery from acetate withdrawal-induced alkalinization was not different from zero
(n = 3). Furthermore, the presence of
DIDS did not inhibit NH+4-induced cell
acidification, indicating that
Cl
/OH
exchange did not play a role in this process (data not shown). To
determine whether NH+4 transport in
pancreatic duct cells occurs via a
Na+-dependent pathway, NaCl was
replaced with tetramethylammonium chloride (solution
F, Table 1) and the experiments were repeated. Figure
4B indicates that
NH+4-induced cell acidification was almost
completely inhibited in the absence of
Na+. These results are consistent
with the transport of NH+4 via a
Na+-dependent pathway. To test
whether NH+4 transport in pancreatic duct
cells is mediated via NKCC1, the experiments were performed in the
presence of 500 µM bumetanide, a strong inhibitor of the
Na+-K+-2Cl
cotransporter (Na+,
Cl
, and
NH+4 were present, solution
B, Table 1). Figure 4C
is a representative pHi tracing
that indicates that the
Na+-dependent
NH+4 entry was almost abolished in the
presence of 500 µM bumetanide. These results indicate that NKCC1 is
expressed in pancreatic duct cells and is functionally active. The
results of several separate experiments on
NH+4 transport in the presence or absence of
Na+ or bumetanide is shown in Fig.
4D.
Cl
was present in all
solutions.
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NKCC1 mRNA expression and activity is enhanced in wild-type
CFTR-bearing pancreatic duct cells.
In the next series of experiments, we examined whether the mRNA
expression of NKCC1 is regulated by the CFTR. Accordingly, the
expression of NKCC1 was compared in CFPAC-1 and CFPAC-WT cells. As
indicated in Fig.
5A,
Northern hybridizations indicated that mRNA levels for the NKCC1 were
increased by approximately twofold in CFPAC-WT cells over CFPAC-1 cells
(P < 0.02, n = 3).
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Intracellular Cl depletion
potentiates NKCC1 activity only in wild-type CFTR-bearing pancreatic
duct cells.
NKCC1 is responsible for the transport of
Cl
from blood to the cell
for eventual secretion via the apical
Cl
channels. We aimed to
determine whether decreased intracellular Cl
concentration can
enhance NKCC1. Intracellular
Cl
depletion was induced by
incubating the CFPAC-WT cells in a
Cl
-free solution
(solution G, Table 1) for 15 min. As
shown in Fig.
7A,
decreasing the intracellular
Cl
concentration
significantly enhanced the rate of
Na+- and
Cl
-dependent
NH+4 entry. Figure
7B summarizes the results of four
separate experiments.
Na+-K+-2Cl
cotransporter activity was 0.051 pH units/min in normal condition in
CFPAC-WT cells and increased to 0.099 pH units/min in the
Cl
-depleted state
(P < 0.01, n = 4). These results are consistent with activation of
Na+-K+-2Cl
cotransporter activity by intracellular
Cl
depletion in CFPAC-WT
cells. Interestingly, incubation of CFPAC-1 cells in
Cl
-free medium did not
enhance the
Na+-K+-2Cl
cotransporter activity (Fig. 7, C and
D).
Na+-K+-2Cl
cotransporter activity was 0.036 and 0.040 pH units/min in normal condition and in the
Cl
-depleted state,
respectively (P > 0.05, n = 4).
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DISCUSSION |
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Cultured pancreatic duct cells (CFPAC-1 and CFPAC-WT) express NKCC1
mRNA (Fig. 1) and activity (Fig. 4). NKCC1 is responsible for the
transport of Cl from blood
to the duct cells. The mRNA expression and activity of NKCC1 is
upregulated in functional CFTR-bearing pancreatic duct cells (Figs. 5
and 6). Intracellular Cl
depletion increased the activity of NKCC1 in cells expressing functional CFTR (CFPAC-WT cells) (Fig. 7). Neither cell line expressed NKCC2 (Fig. 3).
The process of agonist stimulation of
HCO3 secretion at the apical membrane
of the pancreatic duct cells is entirely dependent on the activation of
CFTR (3, 6, 9, 19, 21, 33). This by inference indicates that the
transport of Cl
into the
pancreatic duct cells is essential for activation of CFTR in the
agonist-stimulated state. The currently accepted model of
HCO
3 transport in the pancreatic duct
cells attributes the restoration of intracellular
Cl
to the apical
Cl
/HCO
3
exchanger, which presumably recycles the luminal
Cl
back to the cell in
exchange for HCO
3 (3, 6, 33). This
model, however, is inconsistent with recent studies in which
HCO
3 efflux across the apical membrane
persisted even in the absence of luminal
Cl
, indicating that
Cl
entrance into the duct
cells occurs via a mechanism distinct from the apical
Cl
/HCO
3
exchanger (17).
One possible candidate for the transport of
Cl into the duct cells is
NKCC1. This possibility was entertained based on the fact that NKCC1 is
expressed in a number of secretory epithelial cells (4, 11, 12,
22-25). Recent studies have demonstrated that a
bumetanide-sensitive
Na+-K+-2Cl
cotransporter is located on the basolateral membranes of intestinal epithelial cells and is responsible for the transport of
Cl
from blood to the cell
for secretion via CFTR at the luminal membrane (22-25).
The present studies are the first to demonstrate the expression of
NKCC1 mRNA in the pancreatic duct cells. Our results further demonstrated the functional presence of NKCC1, indicating that this
membrane protein mediates the transport of
Cl from blood to the duct
cells. Cl
that enters the
duct cells will eventually be secreted at the apical membrane via CFTR
and to a lesser extent via other anion channels. The expression of
NKCC2, which is responsible for the reabsorption of luminal
Cl
in kidney cells (14,
29), was not detected in the pancreatic duct cells. This latter
transporter is exclusively expressed in the ascending limb of Henle in
the kidney and mediates the reabsorption of
Cl
and
Na+ from the luminal fluid (14,
29).
Various transporters, including the
Na+-nHCO3
cotransporter (5, 31) and
Cl
/HCO
3
exchanger (20), have been found to be upregulated, whereas the
Na+ channel is downregulated by
CFTR (18). The
Na+-nHCO
3
cotransporter activation is likely through membrane depolarization in
response to Cl
secretion at
the apical membrane secondary to CFTR activation (31). Decreased
activity of the Na+ channel (18)
and activation of the
Cl
/HCO
3
exchanger (20) on the other hand have been postulated to be direct and
independent of the Cl
secreting ability of CFTR.
Enhanced expression of NKCC1 in the functional CFTR-bearing pancreatic
duct cells is intriguing. It has been proposed that Cl entry across the
basolateral membrane may be the rate-limiting step in regulating the
activity of the apical Cl
channels in secretory epithelial cells (22). The current study, however, indicates that CFTR plays an important role in regulating the
activity of NKCC1. It is likely that upregulation of NKCC1 by
functional CFTR is mediated in a
Cl
-dependent manner.
According to this scheme, the presence of the functional CFTR could
increase the secretion of
Cl
and result in decreased
intracellular Cl
concentration in pancreatic duct cells. This in turn could increase the
driving force for NKCC1, leading to its enhanced activity. This is
consistent with the speculative model for the indirect regulation of
NKCC1 in intestinal epithelial cells (25). The exact mechanism by which
CFTR enhances the expression of NKCC1, of course, is unclear. Whether
it is the Cl
depletion or
an exaggerated Cl
gradient
or membrane depolarization that triggers enhanced expression of NKCC1
remains speculative. It would be unlikely that CFTR directly upregulates the expression of the basolateral cotransporter, as these
two transporters are located on two separate membrane domains, making
the possibility of direct interaction between them unlikely.
Increased activity of NKCC1 in functional CFTR-bearing duct cells
(CFPAC-WT) that are depleted of
Cl strongly suggests that
an increased inward Cl
gradient across the basolateral membrane increases the driving for this
transporter. This is consistent with published reports in intestinal
cells in which intracellular
Cl
depletion increased the
activity of NKCC1 (37). It should be mentioned that the exact mechanism
by which Cl
depletion
increases
Na+-K+-2Cl
cotransporter activity remains speculative. Intracellular
Cl
can regulate
Na+-K+-2Cl
cotransporter activity by either changing the phosphorylation state of
NKCC1 protein or by altering the driving force for ion uptake.
Activation of NKCC1 under a
Cl
-depleted state should
result in enhanced Cl
entry
into the cells across the basolateral membrane for eventual secretion
at the apical membrane. Interestingly, incubation of the mutant
CFTR-bearing duct cells (CFPAC-1) in
Cl
-free medium did not
enhance
Na+-K+-2Cl
cotransporter activity. Whether this was due to decreased cell volume
(shrinkage) or a lack of reduction in intracellular
Cl
in CFPAC-1 cells remains
speculative. Future studies aimed at measuring the intracellular
Cl
concentration in these
two cell lines should shed light on this question.
Our results indicating enhanced expression and activity of NKCC1 in
wild-type CFTR-bearing duct cells and its potentiation in the
Cl-depleted state strongly
suggest that activation of CFTR and the subsequent reduction in
intracellular Cl
increase
NKCC1 activity. This should result in enhanced entry of
Cl
into the cells and
maintain activation of CFTR and HCO
3 secretion under agonist-stimulated state. Accordingly, we propose a
model for Cl
and
HCO
3 transport in the pancreatic duct
cells, which is shown in Fig. 9. According to this
schematic diagram, secretin increases intracellular cAMP, which then
results in the activation of CFTR and secretion of
Cl
, leading to decreased
intracellular Cl
concentration. This will result in depolarization of both the luminal
and basolateral membranes. The reduction in cell
Cl
concentration activates
NKCC1, which restores the intracellular Cl
for secretion via CFTR.
The depolarization of the basolateral membrane increases the driving
force for
Na+-HCO
3
cotransporter and as a result enhances HCO
3 entry into the duct cells for
eventual secretion at the apical membrane. The identity of the
transporter that mediates the bulk
HCO
3 secretion at the apical membrane
remains speculative. With
Cl
/HCO
3
exchanger playing only a minor role and the CFTR not being able to
mediate HCO
3 secretion (32), it is
very plausible that HCO
3 secretion at
the apical membrane of the pancreatic duct cells occurs via a
HCO
3 (anion) channel. Further studies
are needed to clarify this issue.
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In conclusion, NKCC1 is expressed in pancreatic duct cells and mediates
the entry of Cl. The
presence of functional CFTR enhances the expression of NKCC1. The
results further indicate that NKCC1 activity is enhanced in the
presence of an inward Cl
gradient. We speculate that CFTR regulates this process in a Cl
-dependent manner.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent contributions of Hassane Amlal and Zhaohui Wang.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789, DK-52821, and DK-54430 and a grant from Dialysis Clinic Incorporated.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Soleimani, Dept. of Internal Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.soleimani{at}uc.edu).
Received 17 March 1999; accepted in final form 20 July 1999.
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