1 Departments of Molecular and Cellular Physiology, and 2 Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 3 Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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The Na/H exchanger isoform 3 (NHE3) is expressed in the proximal
tubule and thick ascending limb and contributes to the reabsorption of
fluid and electrolytes in these segments. The contribution of NHE3 to
fluid reabsorption was assessed by micropuncture in homozygous
(Nhe3/
)
and heterozygous
(Nhe3+/
)
knockout mice, and in their wild-type (WT,
Nhe3+/+)
littermates. Arterial pressure was lower in the
Nhe3
/
mice (89 ± 6 mmHg) compared with
Nhe3+/+ (118 ± 4) and
Nhe3+/
(108 ± 5). Collections from proximal and distal tubules
demonstrated that proximal fluid reabsorption was blunted in both
Nhe3+/
and
Nhe3
/
mice (WT, 4.2 ± 0.3;
Nhe3+/
,
3.4 ± 0.2; and
Nhe3
/
,
2.6 ± 0.3 nl/min; P < 0.05).
However, distal delivery of fluid was not different among the three
groups of mice (WT, 3.3 ± 0.4 nl/min;
Nhe3+/
,
3.3 ± 0.2 nl/min; and
Nhe3
/
,
3.0 ± 0.4 nl/min; P < 0.05). In
Nhe3
/
mice, this compensation was largely attributable to decreased single-nephron glomerular filtration rate (SNGFR): 10.7 ± 0.9 nl/min in the
Nhe3+/+ vs. 6.6 ± 0.8 nl/min in the
Nhe3
/
,
measured distally. Proximal-distal SNGFR differences in
Nhe3
/
mice indicated that much of the decrease in SNGFR was due to activation
of tubuloglomerular feedback (TGF), and measurements of stop-flow
pressure confirmed that TGF is intact in
Nhe3
/
animals. In contrast to
Nhe3
/
mice, normalization of early distal flow rate in
Nhe3+/
mice was not related to decreased SNGFR (9.9 ± 0.7 nl/min), but rather, to increased fluid reabsorption in the loop segment
(Nhe3+/+, 2.6 ± 0.2;
Nhe3+/
,
3.6 ± 0.5 nl/min). We conclude that NHE3 is a major Na/H exchanger isoform mediating Na+ and fluid
reabsorption in the proximal tubule. In animals with NHE3 deficiency,
normalization of fluid delivery to the distal tubule is achieved
through alterations in filtration rate and/or downstream transport processes.
kidney; single-nephron glomerular filtration rate; sodium/proton exchange; proximal tubule; tubuloglomerular feedback
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INTRODUCTION |
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SODIUM/HYDROGEN EXCHANGE is a widespread and fundamental mechanism for the translocation of these ions across membranes and represents an important component of transepithelial ion transport in a variety of tissues. In the mammalian kidney, four isoforms of the Na/H exchanger (NHE1, NHE2, NHE3, and NHE4) have been identified. Isoform 3 (NHE3) has been identified as the major Na/H exchanger in the apical membrane of the S1 and S2 segments of the proximal tubule and in the apical membrane of cortical thick ascending limb of the loop of Henle (2, 3, 7, 9, 17). More than 85% of the filtered load of NaHCO3 is reabsorbed in the proximal tubule, and NHE3 contributes up to ~60% of the total reabsorption of this segment (1, 7). Because of its distribution and functional role, one would predict that NHE3 exerts a major influence on overall fluid and electrolyte balance by mediating the bulk reabsorption of sodium and water in the early segments of the proximal tubule as well as in the thick ascending limb of Henle's loop.
The present study uses a recently developed mouse model in which the gene encoding NHE3 (Nhe3, gene symbol Slc9a3) has been ablated using gene-targeting strategies (22). Initial studies evaluating this mouse revealed that the homozygous null mouse has a decreased arterial blood pressure and a modest acidosis. Homozygous null animals also have increased levels of kidney renin mRNA and circulating aldosterone, suggesting that they are somewhat volume depleted. Experiments performed in perfused proximal tubule segments in these mice demonstrated that fluid reabsorption and absorptive HCO3 flux are both decreased by more than 60% (22). In addition to its expression in the kidney, NHE3 is also heavily expressed in intestinal epithelial cells, and in accordance with this, Nhe3 null mice display a marked intestinal phenotype consisting of modest diarrhea, enlarged intestinal tract, and markedly alkaline intestinal contents (22). This intestinal phenotype likely contributes to the presumed volume depletion in these animals.
The purpose of the present study was to evaluate single-nephron function in situ in NHE3-deficient mice. Experiments were conducted in heterozygous knockouts as well as in homozygous and wild-type animals to evaluate the possible quantitative contribution of NHE3 to proximal tubular and loop of Henle function. Late proximal and early distal collections were used to evaluate proximal tubule and loop of Henle fluid transport, as well as the possible contribution of macula densa-mediated processes on nephron filtration rate, which together may contribute to the overall sodium balance achieved in these animals. Our results indicate that NHE3 deficiency is associated with the predicted reduction in proximal tubule fluid reabsorption, but that reductions in single-nephron glomerular filtration rate (SNGFR) and/or increases in loop of Henle transport prevent major increases in fluid delivery into the collecting duct system.
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METHODS |
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Animals. Wild-type, heterozygous, and homozygous NHE3-deficient mice were obtained from an established colony that was generated by targeted gene disruption as previously described (22). All animals were derived from heterozygous crosses, and genotypes were determined by polymerase chain reaction analysis of DNA from tail biopsies. Mice were maintained on normal rodent chow and tap water in a barrier facility until the time of the experiment.
Free-flow micropuncture experiments.
Male mice of each genotype
(Nhe3+/+,
n = 5;
Nhe3+/,
n = 4; and
Nhe3
/
,
n = 4) weighing between 30 and 40 g
were prepared for micropuncture according to conventional techniques
modified for use in the mouse as previously described (16, 21). Mice
were anesthetized with separate intraperitoneal injections of ketamine
(50 µg/g body wt) and thiobutabarbital (Inactin, 100 µg/g body wt;
Research Biochemicals International, Natick, MA) and placed on a
thermally controlled surgical table. Following tracheostomy, the right
femoral artery and vein were cannulated with polyethylene tubing
hand-drawn to a fine tip over a flame (0.3-0.5 mm OD). The
arterial catheter was connected to a fixed-dome pressure transducer
(model CDXIII; COBE Cardiovascular, Arvada, CO) for measurement of
arterial blood pressure, and the venous catheter was connected to a
syringe pump for infusion. The bladder was also cannulated with flared
PE-10 polyethylene tubing for the collection of urine.
Blood pressure and heart rate were monitored throughout the experiment
using a MacLab data acquisition system (AD Instruments, Boston, MA) with a sampling rate of 200 samples/s. In some experiments, data were
collected using a conventional chart recorder. Body temperature was
maintained at 37.5°C, and animals were provided with a steady stream of 100% O2 to breath. The
left kidney was then exposed via a flank incision, carefully dissected
free of adherent fat and connective tissue, placed in a Lucite cup, and
covered with mineral oil. A 4 µl/g body wt bolus infusion of isotonic
saline containing 2.25 g/100 ml BSA and 1.00 g/100 ml glucose was then administered, followed by a maintenance infusion of the same solution at 0.2 µl · min
1 · g
body wt
1. The infusate
contained either 0.75 g/100 ml of FITC-inulin (Sigma) or
[125I]iothalamate
at an activity of ~20 µCi/ml (Glofil; Cypros Pharmaceutical, Carlsbad, CA). A previous study from our laboratory (16) demonstrated that these two markers yield nearly identical measurements of SNGFR and
tubular fluid/plasma ratios [subsequently, this ratio will be
referred to as (TF/P)In].
After a 30- to 45-min equilibration period, micropuncture collections and urine collections were begun. Surface convolutions of the same nephron were identified by injecting a small volume of stained saline (0.25% Fast Green dye, Sigma) into a random proximal segment. Late proximal puncture sites were identified as the last surface segment to fill with green dye before it disappeared into the loop of Henle. In a small population of nephrons (10-20%), an early distal puncture site could be identified when the green dye returned to the kidney surface. During two consecutive clearance periods lasting 30-60 min, at least seven timed proximal collections were made, and usually two to three paired distal collections were made. In those nephrons having both proximal and distal collection sites, the distal tubule collection was performed before the proximal collection. In these nephrons, late proximal flow rate (Vprox) in the presence of flow to the macula densa was estimated from the distal measurement of SNGFR (SNGFRdist) and the proximal measurement of (TF/P)In [(TF/P)In prox] using the following equation: Vprox = SNGFRdist/(TF/P)In prox. Blood samples (5-10 µl) were taken in heparinized tubes before and after each clearance period. Sharpened glass micropipettes used for dye injection were 2-3 µm in diameter, and those used for fluid collection were 6-7 µm. At the end of each experiment, tubular fluid samples were transferred individually to 1-µl constant-bore microcaps for determination of volume and FITC-inulin concentration or [125I]iothalamate activity as described previously (16, 21). Blood samples were centrifuged, and plasma aliquots were transferred into 1-µl microcaps for inulin or iothalamate determination. Urine samples were also evaluated for inulin or iothalamate for the determination of whole kidney GFR.
Stop-flow pressure measurements.
Separate male
Nhe3+/+
(n = 4) and
Nhe3/
(n = 3) mice weighing 30-40 g
were prepared, and proximal tubule segments for micropuncture were
identified as described above. Identified tubules were blocked with
wax, and a micropipette attached to a nanoliter infusion pump was
inserted into the last superficial proximal segment for loop of Henle
perfusion. Another micropipette, attached to a servo-null pressure
device (WPI, New Haven, CT), was then inserted into an early proximal
segment recognizable from the widening of the tubular lumen. When
stop-flow pressure (Psf)
stabilized, loop of Henle perfusion rate was altered from 0 to 45 nl/min, and responses in Psf were
recorded. Perfusion fluid contained (in mM) 136 NaCl, 4 NaHCO3, 4 KCl, 2 CaCl2, 7.5 urea, and 100 mg/ml
Fast Green.
Statistics. Statistical analysis was performed by ANOVA using a single factor design or a mixed factorial design with repeated measures on the second factor. Where necessary, individual comparisons of group means were accomplished using individual contrasts. Data are expressed as means ± SE, and differences are regarded as significant at P < 0.05.
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RESULTS |
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Table 1 summarizes whole animal data
obtained from free-flow micropuncture experiments on wild-type
(Nhe3+/+),
heterozygous
(Nhe3+/),
and homozygous knockout mice
(Nhe3
/
).
As previously reported (22), mean arterial blood pressure was
significantly lower in
Nhe3
/
mice compared with
Nhe3+/+ mice.
Furthermore, blood pressure in the
Nhe3
/
mice was also significantly lower than in the
Nhe3+/
mice. Although the pressure in the
Nhe3+/
was
somewhat lower than in the
Nhe3+/+ mice, the
difference was not significant (P = 0.18). These data suggest a direct correlation between NHE3 expression
and mean arterial blood pressure. Whole kidney GFR was highest in the
Nhe3+/+ and
lowest in the
Nhe3
/
,
but none of the differences between groups reached significance. Although body weight and kidney weight did not differ among the three
groups of mice, the kidney weight-to-body weight ratio was consistently
and significantly lower in
Nhe3
/
mice compared with the other two groups, which did not differ from each
other. Urine flow rate was significantly reduced in the
Nhe3
/
mice compared with the other two groups, but urine osmolality was not
different among any of the groups.
|
The results from late proximal micropuncture collections from these
experiments are shown in Fig. 1. Fluid
reabsorption in the proximal segment was found to be directly related
to the gene copy number: reabsorption was significantly lower in
Nhe3+/
mice compared with
Nhe3+/+mice, and
lower still in
Nhe3
/
mice. Fractional reabsorption showed a similar pattern
(Nhe3+/+, 42 ± 1%;
Nhe3+/
, 33 ± 1%;
Nhe3
/
,
28 ± 2%; P < 0.001). Despite these differences in reabsorption, fluid
delivery to the late proximal tubule was not significantly different in
the
Nhe3
/
animals compared with wild type. Interestingly, proximal fluid delivery
in the
Nhe3+/
animals was significantly higher than in the
Nhe3+/+ animals.
The normalization of late proximal flow rate in the Nhe3
/
mice was primarily due to a significantly lower SNGFR in these animals
compared with both
Nhe3+/+ and
Nhe3+/
(P < 0.05). It should be noted that
these collections were made during complete blockade of loop of Henle
flow rate and therefore with interrupted tubuloglomerular feedback
(TGF). Thus the measured values for SNGFR are probably artificially
elevated to some degree.
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We found that, on average, only one in four to five nephrons on the
kidney surface had an early distal convolution that was accessible for
micropuncture. Therefore there were markedly fewer early distal
collections obtained in these experiments than proximal collections.
Measurements from these collections are summarized in Fig.
2. In contrast to the measurements from
proximal collections, distally measured values of SNGFR were
significantly lower in the
Nhe3/
mice compared with both
Nhe3+/+ and
Nhe3+/
mice, which were not different from each other. Early distal flow rate,
however, was not different among any of the three groups. When the
distal measurement of SNGFR and the proximal measurement of
(TF/P)In from the same tubule were
used to estimate flow rate to the late proximal tubule in the presence
of an intact TGF mechanism, late proximal flow rate was found to be
significantly increased in the
Nhe3+/
mice compared with the other two groups. Counteracting this increased delivery, fluid reabsorption in the loop segment was significantly elevated in the
Nhe3+/
mice compared with the other two groups.
|
The above observations suggest that the TGF mechanism is largely intact
in mice with reduced or absent NHE3 expression and that it plays a
major role in maintaining normal fluid flow to the distal nephron.
Analysis of SNGFR measurements from proximal and distal collection
sites in the same nephron further suggest active participation of the
TGF mechanism, as illustrated in Fig. 3. In
all three groups of mice, distal measurements of SNGFR were lower than
proximal measurements (P < 0.001).
Interestingly, in Nhe3/
mice, the distal/proximal ratio of 70 ± 9% was significantly reduced compared with the other two groups
(P < 0.05).
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To confirm operation of the TGF system in these animals, we performed a
separate series of experiments in
Nhe3+/+ and
Nhe3/
mice to directly evaluate Psf
during loop of Henle perfusion. These data are summarized in Fig.
4A, which
shows the individual Psf responses
to changing loop of Henle flow rate from 0 to 45 nl/min. In both
Nhe3+/+ and
Nhe3
/
mice, there was a significant and consistent decrease in
Psf in response to increased loop
of Henle perfusion rate. The mean values are shown by the bold lines
and indicate that the magnitude of the response was not different among
groups. A representative set of tracings from an
Nhe3
/
mouse is shown in Fig. 4B.
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DISCUSSION |
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Consistent with previously published results from proximal tubules
perfused in situ (22), we found that proximal fluid reabsorption in
free-flowing nephrons was decreased by about one-half in
Nhe3/
compared with
Nhe3+/+ mice.
Furthermore, these results show that
Nhe3+/
mice also demonstrate a clear deficit in proximal tubule reabsorption, suggesting that even modest alterations in the expression of this protein can influence overall sodium and water balance. Although the
amount of NHE3 protein expression in
Nhe3+/
mice has not been evaluated, Northern analysis indicated that NHE3 mRNA
is reduced by about one-half in the kidneys of
Nhe3+/
animals (22). Our results also demonstrate that the TGF mechanism is
largely intact in NHE3-deficient animals and accounts for a considerable portion of the compensation that occurs in these animals
to achieve sodium balance.
In the present study, mean arterial blood pressure was significantly
lower in the
Nhe3/
compared with both
Nhe3+/+ and
Nhe3+/
mice. This hypotension is consistent with the notion that these animals
are significantly volume depleted, as suggested in our original report
(22). Since NHE3 is a major pathway for electrolyte and water
reabsorption in both the kidney and small intestine, volume depletion
is likely due to a combination of reduced dietary NaCl absorption and
increased renal NaCl excretion. Furthermore, it would seem that the
lower pressure is a necessary adaptive response, over and above other
salt-conserving mechanisms (such as activation of the
renin-angiotensin-aldosterone axis), that enables these animals to
achieve sodium balance. Interestingly, we found in the present studies
that
Nhe3+/
also showed a tendency for lowered blood pressure. Whether the more
modest decrement in NHE3 expression seen in the heterozygous animals
can directly influence blood pressure remains to be determined. Along
these lines, it is important to note that neither serum aldosterone nor
renal renin mRNA appears to be elevated in these animals (22),
suggesting that intrarenal mechanisms are able to largely compensate
for the decreased proximal reabsorption observed in these animals.
The proximal micropuncture measurements presented in this study
directly evaluated the capacity of the proximal tubule to transport
fluid in NHE3-deficient mice. However, to evaluate proximal tubule
function in the context of more distal compensatory mechanisms, such as
TGF, proximal-distal differences must be analyzed. We found that in
Nhe3/
mice, the decreased proximal transport was almost completely compensated through a decrease in SNGFR, such that delivery to the
distal tubule was not different from that observed in
Nhe3+/+ mice.
Distal micropuncture measurements indicated that SNGFR was ~40%
lower in
Nhe3
/
mice compared with
Nhe3+/+ mice,
whereas corresponding proximal measurements indicated that SNGFR was
only ~20% lower in
Nhe3
/
than in Nhe3+/+
mice. These data suggest that about one-half of the compensatory decrease in SNGFR in these mice is attributable to activation of TGF.
That the TGF mechanism is indeed intact, and even robust in
Nhe3
/
animals, is confirmed by the measurements of
Psf. It should be cautioned,
however, that due to the substantially different volume and pressure
conditions which almost certainly exist in these two groups of animals,
quantitative interpretation of these TGF responses cannot be reliably
made. One could argue, for instance, that volume depletion, as well as
the associated increase in the renin-angiotensin system observed in
Nhe3
/
animals, might be expected to significantly increase the magnitude of
the TGF response in these animals.
The possible role of Na/H exchange in macula densa signaling has
recently been addressed in a series of studies from Bell and coworkers
(5, 13, 19). Using the isolated perfused loop of Henle with attached
macula densa, these investigators have demonstrated apical and
basolateral Na/H exchange activity in macula densa cells, and they have
shown that both are responsive to low concentrations of angiotensin II.
They hypothesize, therefore, that the modulation of macula
densa-mediated signaling events (such as TGF and renin secretion) by
angiotensin II may be in part due to its influence on Na/H exchange. If
this is indeed the case, then one could speculate that the lack of TGF
potentiation observed in volume-depleted
Nhe3/
animals in the present study may be due to the absence of angiotensin II-sensitive NHE3 in macula densa cells. Although there are conflicting data regarding the identity of the Na/H exchangers in macula densa cells, available functional evidence from the rabbit (13) and immunohistochemical evidence from the rat (2) suggests that NHE3 is
expressed in the apical membrane. There is also evidence, however, that
NHE2 is expressed in the cortical thick ascending limb up to and
perhaps including the macula densa (10). Also, NHE1 appears to be
ubiquitously expressed in the basolateral membrane of most nephron
segments (8).
In an interesting contrast to
Nhe3/
mice,
Nhe3+/
mice showed little or no increase in SNGFR in compensation for reduced
proximal reabsorption. Like the
Nhe3
/
mice, distal delivery was completely normalized in
Nhe3+/
mice, but this was accomplished largely through an increased reabsorption in the loop segment of the nephron (see Fig. 2). Although
the mechanisms underlying this compensatory response remain to be
elucidated, a number of possibilities exist. First, it is well
established that loop of Henle reabsorption is a flow-dependent process, with increased transport at higher flow rates (4, 20). It is
possible that there is sufficient transport reserve in the loop of
Henle to accommodate the modest increase in late proximal delivery seen
in Nhe3+/
animals. In contrast, late proximal flow rate in
Nhe3
/
mice may approach levels (if left unchecked) that would overload loop
transport mechanisms, in turn activating TGF. It is also possible that
ion transporters in the loop segment are upregulated in the
Nhe3+/
mice. There are numerous studies demonstrating that Na/H exchange activity and or NHE3 mRNA expression can be significantly altered in
response to changes in NaCl intake and acid-base balance (18). For
example, chronic metabolic acidosis has been shown to increase NHE3
mRNA levels, NHE3 protein expression, and Na/H transport activity in
medullary thick ascending limb (15). Whether alterations in Na/H
exchange in the loop of Henle can significantly influence net fluid and
NaCl reabsorption remains to be seen. Other loop of Henle transporters
may also be altered in these animals as a result of NaCl depletion or
acidosis. Indeed, it has been shown that high dietary NaCl intake is
associated with increased NH+4 absorption
(14), as well as increased BSC1 protein expression (12), suggesting
that increases in NaCl and fluid delivery to the loop of Henle
upregulate the activity of the Na-K-2Cl cotransporter. Increased
activity of the Na-K-ATPase has also been reported in salt-loaded rats
(24). Unfortunately, the relevance of these findings on NHE3-deficient
animals may be difficult to predict, since they have the unusual
circumstance of being volume depleted (usually associated with NaCl
restriction) and having increased delivery to the loop of Henle
(usually associated with NaCl loading).
The proximal tubule micropuncture results presented here bear a strong
resemblance to those in previous studies in rats (11, 23) and mice (16)
evaluating the effects of carbonic anhydrase inhibition on proximal
tubular function. The Na/H exchange-dependent pathway for fluid
reabsorption in the proximal tubule is mediated by a process requiring
carbonic anhydrase activity (6). Inhibition of carbonic anhydrase by
either acetazolamide or benzolamide has been shown to inhibit proximal
tubule fluid reabsorption in the rat by 50-60% or more (11, 23).
Furthermore, in a recent study, we showed that carbonic anhydrase
inhibition also reduces proximal fluid reabsorption in mice by ~40%
(16). These values are consistent with the present findings that
proximal fluid reabsorption is 41% lower in
Nhe3/
than in Nhe3+/+
mice. Furthermore, the high degree of similarity between these two sets
of data in the mouse suggest that there is little, if any, upregulation
of other proximal tubule transport pathways in
Nhe3
/
mice to compensate for the lack of Na/H exchange. It is also interesting to note that, in these previous investigations of the rat
and the mouse, carbonic anhydrase inhibition results in a decrease in
SNGFR of ~30%, which is consistent with the decrease seen in distal
collections from
Nhe3
/
mice.
In summary, proximal fluid reabsorption in situ was lower in
Nhe3+/
mice than in
Nhe3+/+ mice, and
lower still in
Nhe3
/
mice. The decrements in proximal tubule function appear to be directly
proportional to the level of NHE3 mRNA expression. Despite these
observed decreases in proximal reabsorption, the delivery of fluid to
the early distal tubule was not different among the three groups of
animals. In
Nhe3
/
animals, this compensation appeared to be primarily due to
TGF-dependent and -independent decreases in SNGFR. Although TGF was
shown to be intact in
Nhe3
/
animals, more studies will be required to determine whether modulation of the TGF signaling pathway is altered in the absence of NHE3. The
TGF-independent decrease in SNGFR may be related to volume depletion in
these animals. In contrast, compensation in
Nhe3+/
animals seemed to be related more to increased transport in the loop of
Henle segment.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50594.
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FOOTNOTES |
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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: J. N. Lorenz, Dept. of Molecular & Cellular Physiol., Univ. of Cincinnati, P.O. Box 670576, Cincinnati, OH 45267-0576 (E-mail: lorenzjn{at}uc.edu).
Received 23 February 1999; accepted in final form 19 May 1999.
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