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*
Department of Physiology and Biophysics, University of Southern California
Keck School of Medicine, Los Angeles, California
Department of Medical Physiology, The Panum Institute, Copenhagen,
Denmark.
Correspondence to Dr. Alicia A. McDonough, Department of Physiology and Biophysics, University of Southern California School of Medicine, 1333 San Pablo Street, Los Angeles, CA 90033. Phone: 323-442-1238; Fax: 323-442-2283; E-mail: mcdonoug{at}hsc.usc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chou and Marsh (3,4) addressed the conceptual difficulty in understanding how acute hypertension could lead to increased NaCl delivery to the macula densa in the absence of a change in GFR or NaCl filtration rate. Using micropuncture of rat renal tubules, they discovered that acute hypertension rapidly inhibits proximal tubule (PT) volume reabsorption, increasing end PT fluid velocity 40%, which could provide the error signal for TGF, and verified that the response occurred in the absence of a measurable change in GFR. An acute decrease in salt and volume reabsorption in the PT in the face of constant GFR can be due to either (1) a change in activity per ion transporter in the plasma membrane (apical or basolateral) or (2) redistribution of ion transporters from surface to internal stores or both. We have been addressing these cellular issues and have determined that 5-min acute hypertension increases volume flow out of the PT, redistributes apical Na/H exchanger (NHE-3) and Na-Pi cotransporter (NaPi2) from the villi to intermicrovillar cleft and subapical endosomal stores, and inhibits basolateral Na,K-ATPase activity (5,6,7).
Another conceptual difficulty in understanding the generation of the TGF error signal at the macula densa is that the thick ascending limb of the loop of Henle (TALH) has a load-dependent response to reabsorb more salt when more is delivered, which is thought to be important in normalizing salt delivery to the distal nephron. This issue was also addressed by Chou and Marsh (3) who demonstrated that the 40% increase in flow leaving the PT during acute hypertension was reduced to a 10% increase in volume entering the early distal tubule and that the 35% increase in Cl- load leaving the PT became a 35% increase in Cl- delivery to the macula densa and early distal tubule over what was delivered in the control period. Volume and Cl- reabsorption rates between the late proximal and early distal collection sites, estimated from their measurements, increased 86% and 34%, respectively, during acute hypertension, which provides evidence for a downstream shift in salt and volume reabsorption from the PT to the loop of Henle during acute hypertension. The aim of this study was to determine whether there are rapid increases in sodium pump activity in the outer medulla during acute hypertension, a region enriched in TALH, which are themselves enriched in Na,K-ATPase, and if so, to determine whether changes are due to the increased volume flow to the region or dependent on hypertension per se. We provide evidence that the downstream shift in sodium and volume reabsorption described by Chou and Marsh (3,4) is accompanied by a downstream shift in Na,K-ATPase activity and provide evidence that it is likely mediated by the increased volume flow to the region.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three protocols were used in the acute studies (n = 4 to 5 rats in each group): (1) control: control sham operated; (2) acute hypertension: BP was increased 60 mmHg over basal level for 5 min by increasing total peripheral resistance by constricting superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery, as adapted from the method of Roman and Cowley (8) and used in our previous studies (5,6,7), (3) benzolamide treatment: the carbonic anhydrase inhibitor benzolamide (obtained from R. Blantz, University of California San Diego), which inhibits PT Na+ reabsorption, was infused at 50 µl/min (2 mg/kg in 300 mM NaHCO3) over 5 to 7 min, as described previously (5, 9).
Studies were also performed in spontaneously hypertensive rats (SHR) before and after the development of chronic hypertension, as recently described (10). Four groups of male rats were compared: (1) young Sprague Dawley (YSD), 3 to 4 wk old, 79 ± 7 g body wt; (2) young SHR (YSHR), 3 to 4 wk old, 76 ± 2 g body wt; (3) adult Sprague Dawley (SD), 12 wk old, 356 ± 6 g body wt; and (4) adult SHR (SHR), 12 wk old, 286 ± 5 g body wt. In this series, all rats were anesthetized by intraperitoneal injection with Nembutal (30 mg/kg-1) (Abbott Laboratories, Abbott Park, IL). Polyethylene catheters were placed into the carotid artery for monitoring BP and into the right jugular vein for infusion of 4% bovine serum albumin in 0.9% NaCl at 50 µl/min during the experimental period to maintain euvolemia. Mean arterial pressures were monitored for at least 30 min before removal of kidneys and sacrifice by overdose injection of Nembutal. The reason to compare SHR to SD rather than Wistar-Kyoto rats (WKY) (discussed in reference 10) is that there is now a very high degree of divergence between WKY and SHR, the mean arterial pressures in SHR and SD were not different at 3 to 4 wk of age, and the SHR developed a significantly higher arterial pressure by 12 wk of age, while there was no change in BP in the SD between 3 and 12 wk of age.
Urine Production Rate and Endogenous Lithium Clearance
Rate of urine production was assessed by collecting urine from the ureter
catheter, and volume was determined gravimetrically. A blood sample was
collected after the kidneys were removed. The concentrations of endogenous
lithium in blood and urine samples were measured by flameless atomic
absorption spectrophotometry (5100PC; Perkin-Elmer, Norwalk, CT) as described
previously (11,
12). Endogenous lithium
clearance, an inverse measure of volume flow from the PT, was calculated from
blood and urine lithium concentrations as [Li]u x
V/[Li]p.
Homogenization, Differential Sedimentation, and Density Gradient
Centrifugation
Kidneys were cooled in situ by flushing the abdominal cavity with
ice-cold phosphate-buffered saline (PBS) solution to lower temperature to a
point that blocks membrane trafficking. Kidneys were excised, the renal outer
medulla and cortex were rapidly dissected in isolation buffer (5% sorbitol,
0.5 mM Na2EDTA, 5 mM histidineimidazole buffer with 0.2 mM
phenylmethyl sulfonyl fluoride, 9 µg/ml aprotinin, pH 7.5). Outer medulla,
which is enriched in TALH, and cortices, enriched in PT, were homogenized and
fractionated on sorbitol density gradients and stored as described in detail
previously
(5,6).
So refers to the low-speed supernatant, which contains total
membranes and soluble proteins that were analyzed directly or applied to the
sorbitol gradients.
Na,K-ATPase and Enzymatic Marker Measurements
Na+,K+-ATPase activity was measured by two methods.
The K+-dependent p-nitrophenyl phosphatase reaction
(K+-pNPPase, expressed as µmol Pi/mg protein per h)
(13) was used to measure
sodium pump activity in membrane fractions. A constant volume of each fraction
from experimental and paired control samples was assayed under identical
conditions, and activity was expressed as the pNPPase activity measured in
each fraction divided by the total protein recovered in the 12 fractions (to
correct for differences in the amount of protein applied to the two
gradients). In addition, the distribution of pNPPase activity in the gradient
was expressed as the pNPPase activity in each fraction as percentage of the
total activity measured in all 12 fractions. Ouabain-sensitive Na,K-ATPase
activity (expressed as µmol Pi/mg protein per h) was measured in
So samples after deoxycholic acid (0.1%) permeablization, as
described previously (14)
under Vmax conditions with the modification that samples were
preincubated with 2.5 mM ouabain for 30 min. Standard assays used for protein
concentration (15) and
alkaline phosphatase activity
(16) have been implemented in
related studies
(5,6).
Immunoblot Analysis and Antibodies
A constant amount of So protein from control and treated samples
or a constant volume of sample from each gradient fraction was prepared in
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer (final concentration: 2% SDS, 1% ß-mercaptoethanol, 0.25 mM
Na2EDTA, and 2.5 mM H2PO4-HPO4
buffer, pH 7.0), incubated for 30 min at 37°C, resolved on 7.5% SDS
polyacrylamide gels, and transferred to polyvinylidene fluoride membranes
according to standard methods. The antibody incubation protocol has been
detailed previously
(5,6).
Three anti-Na,K-ATPase 
1 subunit monoclonal antibodies were used: McK1
(provided by K. Sweadner, Harvard University)
(17), 464.6 (also known as 6H)
(provided by M. Kasgarian, Yale University)
(18), and Fam5
(provided by D. Fambrough, Johns Hopkins University)
(19), and FPß1, a rabbit
anti-rat ß1 polyclonal generated in this lab and used previously in
related studies
(5,6).
Antibody-antigen complexes were detected with 125I-labeled Protein
A (ICN) after incubation with rabbit anti-mouse secondary antibody where
appropriate. The resulting autoradiographic signals were quantified with a
Bio-Rad imaging densitometer with Molecular Analyst software (Hercules, CA).
Multiple amounts of protein or volumes of density gradient fractions were
analyzed, and multiple exposures of autoradiograms were analyzed to ensure
that signals were within the linear range of the film.
Statistical Analyses
Data are expressed as mean ± SEM. For analysis of density gradient
fractions, ANOVA was applied to determine whether there was a significant
effect of treatment on the overall fractionation pattern of a given parameter.
For So samples, effect of treatment was assessed by two-tailed
t test for paired samples, and differences were regarded significant
at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Response of Renal Outer Medulla Na,K-ATPase to Acute
Hypertension
We aimed to determine whether the increased solute and volume flow out of
the PT during acute hypertension would increase Na,K-ATPase activity in TALH.
We and others have demonstrated that Na,K-ATPase activity and abundance is
highest in the TALH and very low in other tubular elements present in the
outer medulla
(22,23).
The outer medulla is anatomically enriched in TALH; therefore, we use outer
medullary Na,K-ATPase activity to estimate changes in TALH Na,K-ATPase
activity. After 5-min acute hypertension, renal outer medulla membranes were
fractionated and assayed for Na,K-ATPase activity by the pNPPase activity
assay (Figure 2A). The broad
peak of pNPPase activity, indicative of basolateral membranes, is located
between fraction 6 to 9. Activity was significantly increased after acute
hypertension (P = 0.03 by ANOVA). The 5-min stimulus is not long
enough to increase the total pool size of sodium pumps; therefore, this change
represents an increase in enzymatic activity per transporter. The distribution
pattern of the pNPPase activity was not altered by acute hypertension
(Figure 2B), indicating that
the increase in activity occurs throughout the fractionated membranes, rather
than by activation in a discrete region of the gradient. To assess the
possibility that the increase in pNPPase activity was accompanied by a
redistribution of pumps from one region of the gradient to another, the
relative abundance of the sodium pump 1 catalytic and ß1
glycoprotein subunit immunoreactivity was measured in a single paired set in
which the increase in pNPPase activity was the largest. As shown in
Figure 2C, the control 1
and ß1 subunits had very similar distribution patterns that mimicked that
of % pNPPase activity, focused in fraction 5 to 10. With acute hypertension,
there was no evidence for a shift of 1 or ß1 subunits into the
peak activity fractions. 1 was detected with two monoclonal antibodies
that recognize distinct epitopes: McK1 was made against a peptide in the
N-terminus (17), and
Fam5 recognizes an epitope in the central portion of the 1
subunit (19).
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The previous observation that acute hypertension caused a rapid decrease in PT and renal cortical Na,K-ATPase activity (5,6) coupled with the current observation that it causes a rapid increase in outer medullary Na,K-ATPase activity illustrates that there are region-specific changes in Na,K-ATPase activity. This leads to the prediction that within a given kidney exposed to acute hypertension, the ratio of Na,K-ATPase activity in medulla to cortex will increase from what is measured at baseline. This was tested in So fractions (the low-speed supernatants containing both membranes and soluble proteins). Ouabain-sensitive Na,K-ATPase activity, assayed under Vmax conditions, increased significantly in medulla to 1.27 ± 0.04—fold above paired controls (P = 0.04) and decreased significantly in cortex to 0.60 ± 0.04—fold below paired control (P = 0.004) (Figure 3). The ratio of activity in medulla relative to cortex increased from 1.81 ± 0.33 in controls to 3.88 ± 1.15 after 5-min hypertension, evidence for a simultaneous decrease driving force for sodium reabsorption in the cortex and an increase driving force for sodium reabsorption in the medulla.
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Because some investigators have found that the dose response of
phosphorylation of Na,K-ATPase 1 at Ser18 in kidney cortex slices is
associated with the dose response of inhibition of Na,K-ATPase activity
(23,24)
while others are not (17). The
So samples were also probed with two antibodies that recognize
different epitopes on Na,K-ATPase 1: McK1, a monoclonal that loses its
epitope recognition when the protein kinase C (PKC) site on Ser18 is
phosphorylated (17) and 464.6
(also known as 6H), which has not been reported to be sensitive to this
phosphorylation (24). We
tested whether the recognition with McK1 would decrease in cortex and/or
increase in medulla during acute hypertension when constant amounts of
So protein were compared. The results, which are summarized as
arbitrary densitometry units normalized to the mean control value = 1.0 in
Table 1, show that there was no
statistically detectable difference in the immunoreactivity of the samples
between control and pressure with either anti-1 antibody. These are the
same samples in which there was a significant difference in Na,K-ATPase
activity, the results suggest that either acute hypertension does not provoke
changes in the phosphorylation state of Ser18 or that such a small fraction of
the pumps in the So fraction are altered at Ser18 that the effect
is masked by the unaffected pumps. This finding confirms our previous finding
of no difference in 1 total abundance in renal cortex after acute
hypertension using McK1 (before it was known that the antibody reacted only
with unphosphorylated pumps)
(5). The immunoblot results in
Figure 2C and 2D also support
this conclusion; the density distribution pattern of 1 detected with
either McK1 or Fam5, which recognizes that the cytoplasmic loop of
Na,K-ATPase 1 was not altered by acute hypertension.
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During acute hypertension, solute and volume delivery to the TALH are increased. To test the hypothesis that the change in Na,K-ATPase activity in the outer medulla is secondary to the increased volume flow to the region and independent of the hypertension per se, benzolamide was infused (2 mg/kg in 300 mM NaHCO3 at 50 µl/min for 5 to 8 min) as discussed under Physiologic Responses. We have previously demonstrated that benzolamide infusion does not have any effect on pNPPase activity or distribution in the cortex (5). As summarized in Figure 4A, pNPPase activity increased significantly in outer medulla after benzolamide infusion (P = 0.0002 by ANOVA), and the overall distribution pattern of pNPPase activity was not significantly altered (Figure 4B). The response of outer medulla Na,K-ATPase activity to benzolamide (Figure 2A) is statistically indistinguishable from the response to acute hypertension (P = 0.77 by ANOVA). From this finding, we conclude that the increase in medullary activity during acute hypertension can be attributed, in part or in whole, to the increase in volume flow to the region, secondary to decreased sodium and volume reabsorption in the PT.
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Our previous studies in cortex demonstrated that acute hypertension also depressed the activity of the apical membrane marker alkaline phosphatase; therefore, we studied the effect of acute hypertension or benzolamide infusion on alkaline phosphatase activity in outer medulla. There is a broad peak of activity in fractions 6 to 9 (Figure 5), which was about one tenth of that measured in cortex (5,6). Nonetheless, peak alkaline phosphatase activity doubled after either acute hypertension or benzolamide (P < 0.001), which suggests that solute and volume flow to the outer medulla increases activity of this apical microvillar enzyme (Figure 5, A and B).
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In a recent report, we compared the effects of acute hypertension on
proximal tubule Na,K-ATPase to the effects of developing chronic hypertension
in YSHR and discovered that there was a similar decrease in sodium pump
activity in both situations
(10); these results are
redrawn in Figure 6A.
Specifically, Na,K-ATPase activity in prehypertensive YSHR is significantly
higher than that in YSD rats, and as hypertension develops, activity of the
SHR falls to that seen in the agematched SD. In this study, we tested the
hypothesis that the stimulation of outer medullary Na,K-ATPase activity during
acute hypertension is mimicked during the development of chronic hypertension,
secondary to the fall in PT sodium transport.
Figure 6A summarizes
experiments that were conducted simultaneously on sets of one each of the four
groups: YSHR, YSD, SHR, SD. Outer medulla Na,K-ATPase activity was more than
30% lower in the YSHR compared with YSD, and as hypertension developed in SHR,
Na,K-ATPase activity in the outer medulla increased significantly to a level
not different from that in the age matched SD; there was no age-related change
in Na,K-ATPase activity in SD in cortex or medulla. Protein expression of
1 in this set of samples is summarized in
Figure 6B. There appears to be
a near doubling of 1 protein abundance in both SHR and SD between 3 and
12 wk of age. The change was statistically significant in adult compared with
young SHR (P = 0.04) but not SD (P = 0.09) because of the
wide variability in the increase with age. Nonetheless, the changes in
1-protein levels are greater than the changes in activity during this
period in the four sets that were assayed, which suggests inactive or less
active 1 subunits in the medullae of 12-wk animals. Like Na,K-ATPase
activity, as hypertension developed in the SHR, alkaline phosphatase activity
in the outer medulla increased 70% from 0.84 ± 0.24 (YSHR) to 1.44
± 0.29 (SHR) µmol Pi/h per mg. These findings demonstrate that for
the specific case of SHR, the changes in sodium transporters that occur during
chronic hypertension mimic those seen during acute hypertension in SD: a
downstream shift in sodium reabsorption and Na,K-ATPase activity from PT to
TALH. The physiologic impact in both cases would be to limit the increase in
delivery of sodium chloride and volume to the distal nephron.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The changes in Na,K-ATPase activity during the onset of chronic hypertension in the SHR mimic the responses to acute hypertension in the SD: in 12-wk hypertensives versus 3- to 4-wk prehypertensives, there is a decrease in Na,K-ATPase activity in the cortex and an increase in the outer medulla (Figure 6). Garg et al. (25) did a similar comparison of YSHR versus WKY rats and measured lower Na,K-ATPase activity in TALH of YSHR and no significant differences in the adults of these strains. Our comparisons in the SHR versus SD rat complement these observations. Consistent with the pattern during acute hypertension, Roman et al. (28) analyzed chloride and volume transport along the nephron by micropuncture in the hypertensive Dahl salt sensitive (SS) versus salt resistant (SR) strains on low-salt diets where BP was slightly higher in the SS strain. The results revealed that in the SS strain the percent of the filtered Cl- and volume reabsorbed in the PT was lower and the percent of Cl- and volume reabsorbed in the loop of Henle was higher compared with the SR strain, physiologic evidence for a downstream shift in ion and volume reabsorption in this strain that mimics the response to acute hypertension. Thus, the hypertension-related elevation in outer medulla Na,K-ATPase activity and abundance in the SHR and salt and water reabsorption in the Dahl SS rat are both indicative of a compensation to an increase in flow to the TALH.
An exception to the observation that an increase in BP (whether acute or
chronic) is generally associated with a downstream shift in Na,K-ATPase
activity and salt and water transport is seen in the young Milan hypertensive
rat (MHS), where outer medulla Na,K-ATPase activity and 1 abundance is
higher than in the normotensive strain (MNS), even before hypertension
develops. Subsequently, activity increases significantly in both strains by 11
to 12 wk, and activity and 1 abundance are elevated in adult MHS
versus MNS in both outer medulla and cortex
(26,27).
We suggest that a compensatory downstream shift is not observed in the MHS as
hypertension develops because the lesion driving sodium reabsorption and
development of hypertension may reside in the TALH itself.
We adjusted a dose of benzolamide to acutely increase volume flow out of the PT to the same extent as we observed with acute hypertension. Both designs increased CLi three- to four-fold, and interestingly, both benzolamide and acute hypertension evoked a very similar three- to four-fold increase in urine output, which suggests that inhibition of luminal carbonic anhydrase along the nephron—found primarily in the PT but also in intercalated cells and OMCD (29)—reduces volume reabsorption to the same extent as acute hypertension, which suggests that pressure-diuresis may be strongly influenced by the decrease in proximal sodium reabsorption.
Na,K-ATPase is the second step in NaCl reabsorption from the luminal
compartment; the first step is apical transport. Can we determine whether
changes in Na,K-ATPase or apical transport are driving the increased loop
sodium and volume reabsorption measured previously
(3,4)?
The benzolamide experimental results demonstrate that it is sufficient to
increase salt and volume delivery to the TALH, which increases apical NaCl
transport, to immediately increase basolateral Na,K-ATPase activity. Although
this is strong evidence, it does not establish a causal link between increased
apical and basolateral sodium transport. Related studies have also focused on
the effects of a chronic increase in salt delivery to the loop. Feeding rats a
high-salt diet for 5 d leads to an increase in the relative abundance of both
the apical NaK2Cl transporter and an increase in the basolateral Na,K-ATPase
1 (30); the increase in
TALH Na,K-ATPase activity can be prevented by treating rats with the NaK2Cl
inhibitor furosemide
(31,32),
evidence for a causal link between chronic salt delivery to the TALH
stimulating increased apical transport (by NaK2Cl) and driving an increase in
basolateral sodium pump activity. Is there any adaptive rationale for an
animal on a high-salt diet to reabsorb more salt in the loop of Henle?
High-salt diet provokes a decrease in proximal tubule sodium reabsorption that
is mediated by the production of dopamine and associated with endocytosis of
Na,K-ATPase (33). This
response, like acute hypertension, increases the volume and salt load entering
the loop of Henle, and the load-dependent properties of the loop prevent the
distal nephron from receiving an overwhelming load of salt and volume. Unlike
salt loading, chronic treatment with benzolamide over 24 h did not lead to a
detectable increase in NaK2Cl abundance
(21).
The sodium pump abundance and activity in the TALH is so high that the
renal outer medulla is the standard tissue for purification of the 1
and ß1 subunits for protein studies. High sodium pump activity is
necessary to extract sodium from the lumen and concentrate it in the
basolateral interstitium, a process critical for both urinary dilution and
concentration. Even so, activity increases further when volume flow is
increased, which suggests that there is a tightly regulated match between
sodium delivery and ATP-driven sodium transport that would provide energy
efficiency to a process that requires a high rate of ATP consumption. What
potential mediators could stimulate Na,K-ATPase activity in the TALH? Only a
few studies have addressed this question. The chronically elevated chloride
transport in the Dahl SS versus SR rat has been attributed to
differentially lower expression of cytochrome P450 4A2 and resultant lower
production of 20-HETE, a known inhibitor of TALH sodium transporters
(34). 20-HETE, in turn, has
been shown to activate PKC in the kidney and inhibit Na,K-ATPase activity
(35); therefore, turning down
signaling through this cascade could stimulate Na,K-ATPase activity. Whether
acute hypertension treatment turns down an inhibitory pathway and/or activates
a stimulatory pathway remains to be determined. The results with benzolamide
suggest that a simple increase in apical Na+ entry may be
sufficient to drive the increase in Na,K-ATPase activity.
In the current experiments, Na,K-ATPase activity was measured enzymatically
(rather than as Na+ or Rb+ transport) and the activation
of sodium pump activity persisted through tissue disruption and membrane
isolation, indicating an increase in the activity of the total cellular pump
pool. Whether there is also a realignment of pumps from intracellular pools to
the plasma membrane remains to be further investigated in a subcellular
fractionation scheme that does a better job at separating different
populations of membranes. The increase in Vmax activity/transporter
suggests covalent modification or an allosteric interaction. The study of
Kiroytcheva et al.
(36) demonstrates an
association between the cAMP-driven increase in 1 phosphorylation and
increase in Na,K-ATPase activity in the TALH, and the experiments of Li et
al. (24) in renal
cortical slices and tubules demonstrated an association between PKC-driven
increase in 1 phosphorylation and inhibition of Na,K-ATPase activity.
The direct assays of the effects of PKC phosphorylation on Na,K-ATPase
activity conducted by Fenschenko and Sweadner
(17) add to the complexity of
interpreting these associations. After cellular Na,K-ATPase was phosphorylated
by stimulation of endogenous PKC (to stoichiometry of 0.9 at Ser18), there was
no effect on Na,K-ATPase Vmax activity or on apparent affinity for
Na+, which lead these investigators to conclude that any effects of
PKC on Na,K-ATPase activity are lost on cell disruption and not directly due
to phosphorylation of Ser18. Complementing this conclusion is that from the
careful study in renal proximal tubule cells of Chibalin et al.
(37) that analyzes the
molecular mechanisms responsible for depressing Na,K-ATPase—mediated
transport during dopamine stimulation, which is mediated at least in part by
PKC and accompanied by increased phosphorylation at Ser11 and Ser18. Activity
and trafficking regulation of sodium pump alpha subunit were analyzed in
parallel. The results demonstrate that phosphorylation of Ser18, not Ser11
mediates internalization and resultant fall in activity of plasma membrane
ATPase activity and that prevention of endocytosis blocked the inhibitory
effect of dopamine on activity, which supports the notion that phosphorylation
of either site does not change Na,K-ATPase Vmax activity. In this
article we do provide evidence for an increase in Na,K-ATPase Vmax
activity that persists through membrane isolation. The results of these
studies
(17,37)
concur with our failure to detect a change in phosphorylated 1 with the
antibody sensitive to Ser18 phosphorylation
(Table 1 and
Figure 2D) during acute
hypertension and suggest that other mechanisms such as covalent modification
at other sites or allosteric regulation should be investigated.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß subunits along the rat nephron: Isoform specificity and
response to hypokalemia. Am J Physiol267
: C901-C908,1994 and ß subunit mRNA and protein isoforms in the rat
nephron. Am J Physiol 266:F240
-F245, 1994This article has been cited by other articles:
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  A. Just Mechanisms of renal blood flow autoregulation: dynamics and contributions Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R1 - R17. [Abstract] [Full Text] [PDF]
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E. A. Dos Santos, A. J. Dahly-Vernon, K. M. Hoagland, and R. J. Roman Inhibition of the formation of EETs and 20-HETE with 1-aminobenzotriazole attenuates pressure natriuresis Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2004; 287(1): R58 - R68. [Abstract] [Full Text] [PDF]
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 L. E. Yang, H. Zhong, P. K. K. Leong, A. Perianayagam, V. M. Campese, and A. A. McDonough Chronic renal injury-induced hypertension alters renal NHE3 distribution and abundance Am J Physiol Renal Physiol, May 1, 2003; 284(5): F1056 - F1065. [Abstract] [Full Text] [PDF]
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L. E. Yang, P. K. K. Leong, S. Ye, V. M. Campese, and A. A. McDonough Responses of proximal tubule sodium transporters to acute injury-induced hypertension Am J Physiol Renal Physiol, February 1, 2003; 284(2): F313 - F322. [Abstract] [Full Text] [PDF]
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 C. Barlassina, C. Lanzani, P. Manunta, and G. Bianchi Genetics of Essential Hypertension: From Families to Genes J. Am. Soc. Nephrol., November 1, 2002; 13(90003): S155 - 164. [Abstract] [Full Text]
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