2 Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033; and 1 School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
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ABSTRACT |
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Using the patch-clamp technique, we studied the effects of
epidermal growth factor (EGF) on whole cell and single channel currents
in adult rat alveolar epithelial type II cells in primary culture in
the presence or absence of EGF for 48 h. In symmetrical sodium
isethionate solutions, EGF exposure caused a significant increase in
the type II cell whole cell conductance. Amiloride (10 µM) produced ~20-30% inhibition of the whole
cell conductance in both the presence and absence of EGF, such that EGF
caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that -,
- and
-subunits of rat epithelial Na+ channel (rENaC)
steady-state mRNA levels were all significantly decreased by EGF. At
the single channel level, all active inside-out patches demonstrated
only 25-pS channels that were amiloride sensitive and relatively
nonselective for cations
(PNa+/PK+
1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased
from 1 to 2. These findings indicate that EGF increases expression of
nonselective, amiloride-sensitive cation channels in adult alveolar
epithelial type II cells. The contribution of rENaC to the total
EGF-dependent cation current under these conditions is quantitatively
less important than that of the nonselective cation channels in these cells.
sodium channels; alveolar epithelium; nonselective cation channels
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INTRODUCTION |
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EPIDERMAL GROWTH FACTOR (EGF) is important to normal
lung development (2, 23, 24, 26) and appears to play a
pivotal role, in conjunction with a number of other peptide growth
factors, in recovery from lung injury (15). Although EGF provides a
potent cell proliferative signal, there is an accumulating body of
evidence to suggest that, in addition, EGF can regulate cellular
function independently of cell division. In the small intestine (an
example of an absorptive epithelium), EGF stimulates a number of
processes known to be important in transepithelial
Na+ and fluid absorption,
including Na+-glucose cotransport
(7, 8), Na+-proline cotransport
(7),
Na+/H+-exchange,
and
Na+-Cl
cotransport (11). In common with other absorptive epithelia, the mature
alveolar epithelium reabsorbs fluid via an active
Na+-linked process. In this
process, basolateral
Na+-K+-ATPase
activity energizes the vectorial movement of
Na+ through apically positioned
Na+ and cation channels.
Regulation of the composition and volume of the alveolar subphase is
crucial for effective gas exchange in the adult lung. The efficiency of
transepithelial water transport is dependent on balance of
Na+ absorption and
Cl secretion (20) and
epithelial barrier integrity. In injury states, absorptive driving
force is exceeded by fluid backflux across a damaged epithelium, and
alveolar flooding ensues. EGF and its receptor are both expressed in
alveolar epithelial cells of the postnatal lung (22), and their
expression rises dramatically in lung injury (24). It has been shown
that EGF stimulates alveolar epithelial cell migration (13) and
differentiation as well as alveolar fluid absorption (25). Therefore,
it seems likely that EGF signaling represents an important mechanism
that helps coordinate the process of recovery from lung injury by
stimulating epithelial repopulation, restoration of barrier integrity,
and clearance of alveolar fluid overload.
Recent observations that EGF treatment of cultured monolayers of alveolar epithelial cells augments both short-circuit current (Isc) (1) and Na+-K+-ATPase expression/activity (4) strongly support the notion that EGF may be capable of increasing fluid reabsorption in the mature lung, a suggestion that has now also been demonstrated in vivo (25). Although the mechanism of this upregulation of Na+ transport by EGF (in vivo and in culture) appears to be intimately linked to increased pump transcription, translation (4), and activity (1, 4) (thereby increasing the driving force), increase in the Na+-entry step is also necessary for EGF upregulation of Na+ transport (i.e., blockade of apical Na+ entry with benzamil during EGF exposure partially suppresses the EGF-evoked rise in Isc). However, to date, there have been no direct electrophysiological observations of modulation of the conductive entry step by this growth factor. The studies described herein address this directly by use of whole cell and single channel configurations of the patch-clamp technique and describe the effects of 48-h treatment of freshly isolated alveolar epithelial type II cells with EGF on amiloride-sensitive and amiloride-insensitive whole cell currents. Furthermore, it provides evidence, at the single channel level, which suggests that the mechanism for upregulation of Na+ entry with EGF treatment is via increased density of nonselective cation channels.
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MATERIALS AND METHODS |
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Isolation and Culture of Alveolar Epithelial Type II Cells
Solutions. Unless otherwise stated, all chemicals were of the highest grade available and were purchased from Sigma Chemical (St. Louis, MO). Solution I contained (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 10 HEPES, 1.0 CaCl2, and 10 D-glucose, pH 7.4 with NaOH. Solution II contained (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 10 HEPES, 1.0 EGTA, and 10 D-glucose, pH 7.4 with NaOH. Neutralization solution contained (in mM) 136 NaCl, 2.2 NaPO4, 5.3 KCl, 10 HEPES, 5.6 D-glucose, and 2 EDTA supplemented with 1% BSA and 0.1% soybean trypsin inhibitor. Minimum completely defined serum-free (MDSF) medium contained 1:1 DMEM/Ham's F-12 nutrient mix (Sigma) supplemented with 1.25 mg/ml BSA, 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 100 U/ml sodium-penicillin G, and 100 µg/ml streptomycin.Experimental. Adult male Sprague-Dawley rats were anesthetized with pentobarbital sodium, and alveolar type II cells were isolated as previously described with the use of elastase digestion and differential adhesion on IgG-coated plates (1). Briefly, alveolar macrophages were removed by 10 times lavage with the Ca2+-free solution II, and the pulmonary vascular bed was cleared of blood by transcardial perfusion with ice-cold PBS. Lungs were removed and instilled to slightly more than physiological volume via a tracheal catheter, with solution I containing 2.0-2.5 U/ml elastase (Worthington Biochemical, Freehold, NJ) and incubated for 20 min at 37°C. The lungs were then chopped finely in neutralization solution and filtered sequentially through filters of mesh sizes 100, 40, and 15 µm before being plated onto IgG-coated bacteriological plates. Contaminating cells were allowed to adhere to the plates for 1 h at 37°C before the type II cell-enriched supernatant was collected and centrifuged at 150 g. The cell pellet was then resuspended in MDSF and cells seeded, at a density of 2 × 105/cm2, onto glass coverslips and incubated in the presence or absence of added EGF (20 ng/ml) in a humidified air-CO2 mixture (19:1) at 37°C for 36-48 h. The concentration of EGF used in the present study is identical to that previously determined to maximally increase Isc across alveolar epithelial monolayers (1). Although it is above normal circulating in vivo blood levels of EGF, it is likely within the range locally attainable for alveolar epithelial cells in the setting of lung injury or by pharmacological intervention (25).
Patch-Clamp Experiments
Solutions. See Table 1 for solution compositions and abbreviations.
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Experimental. Coverslips were placed
in a perfusion bath (maximum volume = 200 µl; flow rate ~5 ml/min)
mounted on the stage of a Nikon TM-D inverted microscope and were
viewed using phase-contrast optics. Only those cells containing the
granular inclusions typical of type II cell morphology were chosen for
study. Pipettes were manufactured from thin-walled, filamented
borosilicate glass (World Precision Instruments, Sarasota, FL) using a
two-stage puller (Narishige PB-7). Pipettes used for whole cell and
single channel recording had resistances of 4-5 and 10-12
M, respectively. Voltage clamp was achieved using an Axopatch 200B
amplifier (Axon Instruments, Foster City, CA) in capacitative (single
channel recording) or resistive (whole cell recording) feedback modes.
Voltage protocols were generated and current recording/analysis was
achieved, using the pCLAMP 6.03 suite of software (Axon Instruments).
Whole cell currents were recorded in essentially symmetrical sodium
isethionate solutions during 50-ms step depolarizations (20-mV
increments) from 100 to +120 mV at 0.5 Hz. Holding potential was
0 mV. Single channel activity was recorded in the inside-out configuration and, therefore, all figures quote minus the pipette potential
(
Vp). By
convention, inward cationic current is depicted as a downward
deflection [i.e., channels open downward at negative pipette
potential
(
Vp)
values and upward at positive
+Vp
potentials].
RNA Isolation and Northern Blotting
Solutions. Hybridization buffer contained 1 M NaPO4 (pH 7.0), 7% SDS and 1% BSA.Experimental. Total RNA was isolated
from the cells by the phenol-guanidinium-chloroform method of
Chomczynski and Sacchi (3). RNA was denatured with formaldehyde,
size-fractionated by agarose gel electrophoresis under denaturing
conditions, transferred to nylon membrane (Hybond N+; Amersham Life
Science, Cleveland, OH) and immobilized by ultraviolet cross-linking.
Blots were prehybridized for 2 h and then hybridized for 16 h at
65°C in hybridization buffer with the
32P-labeled cDNA probes for -,
-, and
-subunits of rat epithelial Na+ channel (rENaC) (Dr. B. C. Rossier, University of Lausanne, Switzerland). Blots were washed with
high stringency (0.5× standard sodium citrate), visualized by autoradiography, and quanititated by densitometry. Blots
were reprobed for 18S rRNA to normalize differences in RNA loading.
Statistical Analysis
Where appropriate, data are presented as means ± SE. Comparisons between EGF-treated and EGF-untreated cells employed unpaired Student's t-test. Comparisons between current and conductance densities in the absence or presence of amiloride employed paired Student's t-test. In the current density vs. voltage plots, analysis of covariance was employed to test statistically the difference before and after addition of amiloride. Differences were considered significant at P < 0.05. ![]() |
RESULTS |
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Cell Purity and Viability
All preparations were routinely checked for cell purity and viability using tannic acid and trypan blue dye exclusion, respectively. Over 90% of cells stained positive for lamellar bodies, and viability always exceeded 90%.rENaC Northern Blotting
The Northern blot shown in Fig. 1 demonstrates clearly that exposure of cells for 2 days to EGF resulted in a significant (P < 0.01) decrease in steady-state mRNA levels of all three rENaC subunits.
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Whole Cell Currents
Two-day exposure of cultured alveolar epithelial type II cells to 20 ng/ml EGF resulted in no significant change in whole cell capacitance [
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With the use of a concentration of amiloride (10 µM) known to inhibit
maximally all classes of amiloride-sensitive
Na+ channels in tissues, isolated
cells, cell monolayers, and subcellular cell fractions, current
densities in untreated and treated cells were reduced at all test
potentials (Fig. 2, A, B, D, and
E). Conductance density of cells not
treated with EGF was significantly (P < 0.003) reduced by amiloride ~20% from 99 ± 13.1 to 78.6 ± 10.5 pS/pF (see exemplar currents in Fig. 2,
A and
B), whereas that of EGF-treated
cells was significantly (P < 0.01)
reduced ~30% from 169.6 ± 3.5 to 116.8 ± 22.4 pS/pF (see
exemplar currents in Fig. 2, D and
E). Mean current density vs. voltage
relationships are shown in Fig. 2C
(EGF) and 2F (+EGF). Analysis
of covariance showed that amiloride caused a significant
(P < 0.05) decrease in whole cell
current densities in both EGF-treated and untreated cells. The
amiloride-resistant components of current (treated vs. untreated) were
not significantly different from each other (P > 0.1), suggesting that EGF
selectively increased amiloride-sensitive channels in these cells.
Calculation of the absolute magnitude of this amiloride-sensitive
component of the whole cell currents (Fig.
2G) showed that EGF exposure
resulted in a significant (P < 0.05)
increase in the conductance density from 20.6 ± 4.8 to 52.8 ± 15.0 pS/pF (P < 0.01).
The apparently inconsistent observations that EGF treatment caused more than a doubling of the Na+ current density (Fig. 2) but about a halving of rENaC subunit mRNA expression prompted us to investigate further the nature of the currents. Substituting whole cell bath solution 1 (Na+ containing) for whole cell bath solution 2 (K+ containing) resulted in only a mild reduction of the inward currents (data not shown), which suggested that the majority of the channels underlying the whole cell currents discriminated poorly between cations. To examine the possibility that a nonspecific cation conductance is a major cation-selective permeability pathway in these cells, we employed excised, inside-out membrane patches from both control and EGF-treated cells.
Single Channel Currents
Nonselective cation channels have been described previously in fetal and postnatal alveolar epithelial type II cells (5, 21). However, long-term regulation of these channels has not been observed to date. We investigated the possibility that EGF increases the magnitude of amiloride-sensitive whole cell currents via changes in the biophysical properties of these single channels.With the use of the single channel bath solution and single channel
pipette solution (see Table 1), the most common channel type observed
in inside-out, excised membrane patches from both treated and untreated
cells (Fig.
3A) was
similar to that described previously (5). In these symmetrical
Na+-containing solutions, the
channel had linear current-voltage relationships (Fig.
3B), with a unitary conductance of
24.7 ± 0.8 pS (n = 7) in treated
cells and 24.1 ± 0.7 pS in untreated cells
(n = 10). No channel activity could be
recorded when the pipette solution contained 10 µM amiloride
(n = 8, data not shown). In
untreated cells, exchanging the intracellular (single channel bath)
solution for solutions where Na+
had been substituted isosmotically by different monovalent cations (Cl salts; Fig.
4A,
n = 4) resulted in small positive
perturbations in reversal potentials and decreases in the magnitude of
the outward currents (Fig. 4B).
These changes were consistent with the channel being selective for
cations. This maneuver allowed calculation of a permeability sequence
for the channel as Na+ > Cs+ > Rb+
K+ > Li+ (with
Na+ permeability being unity, the
ratio was 1.0:0.72:0.56:0.48:0.40). Similarly, in EGF-treated cells,
substitution with CsCl resulted in a positive shift in reversal
potential and a calculated
PNa+/PK+ of 1.0:0.72 (Fig. 4C,
n = 3). Channels from both cohorts
(±EGF) were insensitive to the effects of the classical anion
channel blocker DIDS, even at 100 µM (data not shown). In addition to the almost identical conductance and selectivity of the channels observed in EGF-treated and untreated cells, there was also a strong
similarity between open state probability
(Po) at
potentials similar to the predicted membrane potential in intact cells.
At
60 mV
(
Vp),
EGF-treated cells had a mean
Po of 0.42 ± 0.07 (Fig. 5,
C and
D), whereas in untreated cells
Po was 0.38 ± 0.12 (Fig. 5, A and
B). Because of the low probability
of recording only one channel per patch in the EGF-treated group (see
below), further rigorous kinetic comparison was not attempted.
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The major difference between single channels recorded in the two groups
of cells was the observation that EGF-treated cells had a higher
channel density than untreated cells (Fig.
6). More than one-third of the patches
obtained from untreated cells contained no channel activity. In
contrast, only 15% of patches from EGF-treated cells were quiescent.
Furthermore, the modal number of channels per patch increased from one
to two with EGF exposure. In EGF-treated cells, 70% of the patches
contained two or more channels, whereas this was reduced to <20% in
untreated cells. The observation that nonspecific cation single channel
density is increased by EGF treatment is consistent with the whole cell
data in Fig. 2. Furthermore, taken together with the Northern blot data
in Fig. 1, these data suggest that the EGF-evoked increase in
Na+ current is not likely due to
the increased functional expression of classical trimeric rENaC but
rather to the expression of 25-pS, nonselective cation
channels.
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DISCUSSION |
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We have previously shown that EGF increases short-circuit current (Isc) across alveolar epithelial cell monolayers over a relatively delayed time course, beginning at 10-12 h and becoming maximal at 24-36 h (1). Although EGF-induced signal transduction events occur within minutes (28), changes in Na+ pump and channel expression occur over hours and immediately precede Isc changes (4). The present study was designed to investigate the mechanisms by which this chronic effect, which appears to require changes in gene and protein expression, occurs in response to EGF.
This study demonstrates that EGF causes an increase in total alveolar
epithelial type II cell whole cell cation current and that
~20-30% of this current is amiloride sensitive. The largest component of the whole cell conductance in both treated and untreated cells was insensitive to blockade by amiloride. This is not the first
observation in alveolar epithelial type II cells of significant amiloride insensitivity in whole cell currents (18). It has been
suggested that the residual current may be carried by the anion species
employed. Indeed, in many studies, glutamate has been used as the major
charge-carrying anion (e.g., see Ref. 18). Based on the observation
that at least one of the Cl
conductances found in the alveolar epithelium (10) demonstrates significant permeability to glutamate (14), we chose to employ isethionate as the substitute for
Cl
in this study.
Isethionate is also the least permeable anion tested in the fetal
alveolar apical membrane vesicle experimental system (6). With the use
of isethionate, the whole cell currents were essentially nonrectifying
[in contrast to the previous report (18)] but still
contained a sizable amiloride-sensitive component. This component was
not investigated further in this study.
Figure 2G shows that EGF more than
doubled the amiloride-sensitive whole cell current in alveolar
epithelial type II cells. Although it is generally accepted that rENaC
underlies Na+ currents in fetal
alveolar epithelial type II cells, its contribution to adult cell
currents is less well established. The Northern analysis in Fig. 1
shows clearly that mRNA encoding -,
-, and
-rENaC are all
present in primary cultured cells at 48 h. However, it seems unlikely
that EGF increases whole cell Na+
currents by inducing rENaC transcription and translation, since steady-state mRNA for all three subunits is markedly decreased by EGF.
Taken together, the electrophysiological recordings and rENaC Northern
analysis suggests three possibilities:
1) the EGF-dependent increase in
amiloride-sensitive current results from an increase in the probability
of
/
ENaC dimers carrying this component of the current,
2) the available rENaC trimer
function is posttranslationally upregulated by EGF, or
3) another cation channel is being
upregulated by EGF. The first two possibilities seem unlikely, since
recombinant
/
-ENaC has been reported to have a single channel
conductance of 5.1 pS and a higher permeability to
Li+ than
Na+ (19), whereas the vast
majority of single channel recordings that we obtained (see below) did
not demonstrate channels with conductances small enough to represent
classical rENaC subunits (4-6 pS). Therefore, it appears
that the classical rENaC pathway may not be the major mechanism
contributing to the EGF-induced increase of the conductance in these
cells. Because the previous Isc data show
that EGF is an important modulator of electrogenic Na+ flux (and, therefore, fluid
absorption), an amiloride-sensitive, nonselective cation channel
becomes the likely EGF-responsive Na+ entry pathway. Kizer et al.
(12) showed that expression of
-rENaC alone results in formation of
a 24-pS channel that cannot distinguish between
Na+ and
K+, whereas Jain et al. (9) have
recently suggested that
-rENaC alone (or in combination with
proteins other than
- or
-rENaC) forms the major cation-selective
channel in adult alveolar epithelium. These observations are compatible
with our electrophysiological findings, although the Northern analysis
data would require that
-rENaC is more efficiently
translated than
- or
-rENaC in both EGF-treated and untreated
cells. Thus, while we recognize the possibility that expression of
-rENaC alone could result in formation of nonselective 24-pS cation
channels (12), a decrease in all three rENaC subunit mRNA levels has
not been associated with increased rENaC channels in any setting
reported to date. Until further data are available concerning
rENaC subunit protein abundance in these cells and/or the
molecular identity of the nonselective cation channel is available,
however, the issue will remain unresolved.
With the use of a cytosolic (bath) solution that contained the same low intracellular Ca2+ concentration ([Ca2+]i) as that used in the whole cell recording experiments, only 2/37 patches demonstrated single channel events. These events had a calculated conductance of <10 pS and may have been rENaC. However, event frequency was too low for systematic study, and activity rapidly ran down. EGF treatment did not appear to increase the likelihood of observing these infrequent and small events (data not shown). When a solution with 1 mM [Ca2+]i was employed, single channels were seen in >60% and 85% of patches from EGF-untreated and EGF-treated cells, respectively.
Figures 3, 4 and 5 show that the major cation channel in the plasma
membrane of adult alveolar epithelial type II cells has a unitary
conductance of ~25 pS, selects poorly among monovalent cations, and
has a Po of
~0.4 at quasiphysiological membrane potential. EGF exposure does not
result in any significant alteration in conductance, selectivity, or
Po but causes
increased expression/membrane insertion of these channels, as evidenced
by the increased density shown in Fig. 6. The biophysical properties of
the nonselective cation channels observed in this study closely
resemble those found by Feng et al. (5) in adult type II cells, which
had a linear slope conductance of 20.4 pS in a symmetrical NaCl
solution similar to that used in the current study. These channels were approximately equally permeable to
Na+ and
K+
(PK+/PNa+ = 1.15) and were highly selective for cations
(PCl/PNa+ < 0.05), as we similarly found. Channel activity was
Ca2+ dependent, requiring at least
10 µM Ca2+ on the cytosolic side
of an inside-out patch to activate the channel. Similar channels have
also been reported in fetal alveolar type II epithelial cells (21),
which were reversibly inactivated when the bath was exchanged with a
Ca2+-free solution. As noted in
Table 1, the bath solution used for single channel patch experiments
reported herein (single channel bath solution) contained 1 mM
CaCl2. This solution bathes the cytosolic face of the inside-out membrane patch and contains sufficient Ca2+ to fully activate the
channels. Although this is out of the range normally seen in living
cells, similar channels become almost maximally activated (to
Po
0.6) by
2-agonists at 1 µM
Ca2+ when intracellular
Cl
concentration
([Cl
]i)
is reduced to between 40 and 20 mM (17). This suggests that the
activity that we record at the artificially elevated
[Cl
]i
and supramaximal
[Ca2+]i
is of physiological significance. We did not specifically test whether
lowering bath Ca2+ would
inactivate the channels, which would definitively show these channels
to be identical to those described above. However, their relative
nonselectivity between Na+ and
K+ indicates that they are in some
respects different from those described by Yue and Matalon (27), which
were seven times more permeable to
Na+ than to
K+. Importantly, a similar
nonspecific cation channel in fetal alveolar epithelial type II cells
appears to be the major
2-agonist
regulatable conductance (16).
In summary, we have shown that exposure of cultured, subconfluent adult alveolar epithelial type II cells to EGF causes a marked increase in whole cell cation current and nonselective cation channel density. Under these conditions, it appears that the amiloride-sensitive component of the whole cell current is <30% of the total current and is carried by ionic channels that select poorly among cation species.
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ACKNOWLEDGEMENTS |
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We thank Drs. Cecilia Canessa and Bernard Rossier for Na+ channel subunit cDNAs. We note with appreciation the expert technical support of Li Ma, Martha Jean Foster, and Susie Parra.
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FOOTNOTES |
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This work was supported, in part, by a Human Frontiers Science Program Fellowship to P. J. Kemp, by the American Lung Association, the American Heart Association, by National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, HL-38658, HL-51928, HL-38658, and HL-46943, and by the Hastings Foundation.
E. D. Crandall is Hastings Professor of Medicine and Kenneth T. Norris, Jr., Chair of Medicine.
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: P. J. Kemp, School of Biomedical Sciences, Worsely Medical and Dental Bldg., Univ. of Leeds, Leeds LS2 9JT, UK (E-mail: p.z.kemp{at}leeds.ac.uk).
Received 25 February 1999; accepted in final form 30 August 1999.
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