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Department of Medicine B, The Heart Centre, Rigshospitalet, National University Hospital, DK-2100 Copenhagen, Denmark
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Bundgaard, Henning, Thomas A. Schmidt, Jim S. Larsen, and
Keld Kjeldsen. K+
supplementation increases muscle
[Na+-K+-ATPase]
and improves extrarenal K+
homeostasis in rats. J. Appl. Physiol.
82(4): 1136-1144, 1997.
Effects of
K+ supplementation (~200 mmol
KCl/100 g chow) on plasma K+,
K+ content, and
Na+-K+-adeonsinetriphosphatase
(ATPase) concentration
([Na+-K+-ATPase])
in skeletal muscles as well as on extrarenal
K+ clearance were evaluated in
rats. After 2 days of K+
supplementation, hyperkalemia prevailed
(K+-supplemented vs.
weight-matched control animals) [5.1 ± 0.2 (SE) vs. 3.2 ± 0.1 mmol/l, P < 0.05, n = 5-6], and after 4 days
a significant increase in K+
content was observed in gastrocnemius muscle (104 ± 2 vs. 97 ± 1 µmol/g wet wt, P < 0.05, n = 5-6). After 7 days of
K+ supplementation, a significant
increase in
[3H]ouabain binding
site concentration (344 ± 5 vs. 239 ± 8 pmol/g wet wt,
P < 0.05, n = 4) was observed in gastrocnemius
muscle. After 2 wk, increases in plasma
K+,
K+ content, and
[3H]ouabain binding
site concentration in gastrocnemius muscle amounted to 40, 8, and 68%
(P < 0.05) above values observed in
weight-matched control animals, respectively. The latter change was
confirmed by K+-dependent
p-nitrophenyl phosphatase activity
measurements. Fasting for 1 day reduced plasma
K+ and
K+ content in gastrocnemius muscle
in rats that had been K+
supplemented for 2 wk by 3.1 ± 0.3 mmol/l
(P < 0.05, n = 5) and 15 ± 2 µmol/g wet wt
(P < 0.05, n = 5), respectively. After induction of anesthesia, arterial plasma K+
was measured during intravenous KCl infusion (0.75 mmol
KCl · 100 g body
wt
1 · h1).
The K+-supplemented fasted group
demonstrated a 42% (P < 0.05) lower plasma K+ rise, associated with a
significantly higher increase in
K+ content in gastrocnemius muscle
of 7 µmol/g wet wt (P < 0.05, n = 5) compared with their control
animals. In conclusion, K+
supplementation increases plasma
K+,
K+ content, and
[Na+-K+-ATPase]
in skeletal muscles and improves extrarenal
K+ clearance capacity.
plasma potassium; muscle potassium; potassium adaptation; sodium-potassium-adenosinetriphosphatase concentration
INTRODUCTION IT IS WELL KNOWN that long-term
K+ supplementation reduces the
toxic effect of an acute high K+
intake. In animal models of K+
supplementation, this adaptation has been associated with an upregulation of both renal (11, 26, 33) as well as colon villus
epithelial
Na+-K+-adeosinetriphosphatase
(ATPase) in animals (10) and humans (12). During the last decade, a
number of reports (3, 4, 14, 19) have focused on the importance of
Na+-K+-ATPase
in skeletal muscle for extrarenal
K+ homeostasis. Changes in
Na+-K+-ATPase
activity in skeletal muscles have been shown to have the potential to
induce extreme hypo- or hyperkalemia within seconds to minutes.
Furthermore, the
Na+-K+-ATPase
concentration in skeletal muscles is subject to major regulations in
animals as well as in humans (3, 4, 19). For example, after
K+ depletion,
Na+-K+-ATPase
concentration in skeletal muscles is downregulated by as much as 78%
in animals (23, 28) and 18% in humans (7, 8).
Na+-K+-ATPase
regulation in skeletal muscles in association with high K+ intake has been less well
studied. In rats exposed for 10 days to
K+-enriched chow, increases of 100 and 64% have been reported (2) in
[3H]ouabain binding
capacity and
Na+-K+-ATPase
activity in microsome fractions of skeletal muscle, respectively. On
the other hand, no changes in
Na+-K+-ATPase
activity in microsomes of diaphragm were found after 7 days of
K+ supplementation (33). However,
the methods applied for
Na+-K+-ATPase
determinations in these studies include purification procedures. Therefore, identical recovery of enzyme in the groups compared cannot
be ensured (15-17). Furthermore, it seems confusing that, contrary
to the conclusion in one of the former studies (2), K+ balance calculations indicate a
reduced K+ uptake in skeletal
muscles in K+-supplemented rats
despite a reported increase in
Na+-K+-pump
number. A preliminary study of
[3H]ouabain binding to
intact muscle samples from rats given
K+-enriched chow showed an
upregulation (21), whereas no effect was found in a study with moderate
K+ supplementation (23).
On this basis, the aims of the present study were to determine in a
large-scale study whether K+
supplementation is associated with a general
Na+-K+-ATPase
upregulation in skeletal muscles as assessed by using vanadate-facilitated
[3H]ouabain binding to
intact muscle samples and, furthermore, to evaluate whether such an
increase in total skeletal muscle
Na+-K+-pump
number might influence extrarenal
K+ homeostasis, evaluated in
functionally nephrectomized rats by measuring plasma
K+ and
K+ content in skeletal muscles in
response to KCl infusions.
MATERIALS AND METHODS Animals
Instrumentation
Animals were anesthetized by pentobarbital sodium (0.05 mg/g body wt ip; Mebumal, 50 mg/ml, Nycomed DAK). Anesthesia was supplemented during procedures by pentobarbital sodium (0.005-0.01 mg/g body wt) if the animal showed any limb movements. Anesthesia was maintained until animals were decapitated. With animals in the supine position, the left jugular vein and right carotid artery were located through an anterior midline incision, dissected free, and ligated. Under a microscope, polyethylene catheters with a diameter of 1 mm were inserted caudally into the vein and artery and subsequently used for infusion and blood sampling, respectively. Catheter patency was ensured by using a heparinized (170 IU/ml) isotonic NaCl solution (heparin, Sygehus Apotekerne). On the basis of rectal temperature measurements, body temperature was maintained between 36.5 and 37.5°C by using a heating lamp above the animal. Fifteen minutes after completion of surgical procedures, the first blood sample was drawn and infusion of 1.5 ml · 100 g body wt1 · h1
of a solution of 0.5 mol/l KCl (i.e., 0.75 mmol
KCl · 100 g body wt · 1 · h1)
was initiated. The continuous infusion was delivered by an electric infusion pump (Perfusor Secura, B. Braun, Messungen, Germany). Blood
samples were drawn at 5- to 15-min intervals during the infusion
period. The syringes were heparinized in a standardized manner by
flushing with a heparin-NaCl solution. Before sampling, a volume of
0.05-0.10 ml blood was drawn into a separate syringe to avoid
dilution of the sample by the solution in the catheter. Blood sampling
was then performed by drawing 0.15 ml blood each time. Immediately
after sampling, the volume obtained initially to avoid dilution was
reinjected. In addition, 0.15 ml of the heparinized isotonic NaCl
solution was injected to obtain volume compensation. Infusion was
continued until animals demonstrated respiratory and cardiac arrest, as
visually observed. KCl infusion was immediately stopped, and the
infusion time was registered. Then animals were decapitated and hind
leg soleus, gastrocnemius, and extensor digitorum longus (EDL) muscles
were excised and immediately taken for measurements. In experiments
with special focus on extrarenal K+ homeostasis, renal
K+ clearance was eliminated by
bilateral ligation of the renal vessels, i.e., functional nephrectomy.
This procedure was performed through a midline laparotomy. After
successful ligation of the vessels, an ischemic blue discoloration of
the kidneys ensued. Care was taken not to compromise suprarenal blood
supply. After 15 min of recovery, the first blood sample was drawn and
KCl infusion was immediately started. Animals taken only for
[3H]ouabain binding or
K+ content measurements in
skeletal muscles were not catheterized but decapitated immediately
before excision of muscles.
Plasma K+
Arterial blood samples were immediately analyzed for plasma K+ concentration by a K+-sensitive electrode using a KNA 2 (Radiometer, Copenhagen, Denmark). During infusions, adequate ventilation was monitored by measurements of arterial PO2, PCO2, and oxygen saturation by using an OSM3 (Radiometer). Hemoglobin concentrations were measured by using an OSM3 and pH by using an ABL 510 (Radiometer).Skeletal Muscle and Chow K+ and Na+ Content
K+ and Na+ contents were measured by flame photometry by using a FLM3 (Radiometer) with lithium as an internal standard. A sample of ~25 mg wet weight was dissolved in 1 ml of 30% H2O2, and the suspension was placed at 90°C for 12 h to allow complete evaporation. After addition of 2 ml of trichloroacetic acid (TCA; 5% wt/vol), 0.5 ml of the solution was used for flame photometry after final addition of further 0.5 ml 5% TCA and 1.5 ml of 5 mmol/l of LiCl. Measurements were performed in duplicate and expressed as micromoles per gram wet weight of muscle and as millimoles per 100 g wet weight of chow.[3H]Ouabain Binding
Measurement of [3H]ouabain binding was performed as previously described in detail for intact skeletal muscle samples (18, 29). In brief, all procedures were performed by using freshly made vanadate (Merck, Darmstadt, Germany) buffer containing (in mmol/l) 10 tris(hydroxymethyl)aminomethane (Tris) · HCl, 250 sucrose, 3 MgSO4, and 1 vanadate. pH was adjusted to 7.3 with Tris · HCl. Samples in the wet weight range 2-4 mg were cut from the original specimens and prewashed in unlabeled buffer at 0°C for 20 min (2 × 10 min). In the standard assay, incubations took place at 37°C in buffer containing [3H]ouabain (2.1 µCi/ml) (Amersham International, Buckinghamshire, UK) and ouabain (Sigma Chemical, St. Louis, MO) added to a final concentration of 1 × 106 mol/l for
2 h (2 × 1 h). Hereafter, a washout at 0°C in unlabeled buffer for 2 h (4 × 30 min) was performed to reduce the amount of
[3H]ouabain in the
extracellular space, thereby enhancing the precision of the method.
After washout samples were blotted, weighed, and soaked overnight in
vials containing 0.5 ml of 5% wt/vol TCA. Thereafter, 2.5 ml
Opti-flour scintillation mixture (Packard Instruments, Downers Grove,
IL) were added, and
[3H]activity in
samples and incubation medium was assayed by liquid scintillation
counting (Tri-Carb, 1600TR, Packard Instruments). On the basis of
sample wet weight,
[3H]activity in the
incubation medium, and
[3H]activity retained
in the samples, the concentration of
[3H]ouabain binding
sites in the samples was calculated and expressed as picomoles per gram
wet weight. To evaluate possible sources of error associated with the
standard [3H]ouabain
assay, special kinetic experiments were performed as earlier described
in detail (18). In brief, the effect of ouabain concentration for
[3H]ouabain binding
was evaluated in saturation experiments during which samples were
incubated in buffer with final
[3H]ouabain and
ouabain concentrations of 1 × 108, 5 × 108, 1 × 107, 5 × 107, 1 × 106, and 5 × 106 mol/l. Nonspecific
uptake and retention of
[3H]ouabain were
measured after unlabeled ouabain was added to a final concentration of
1 × 103 mol/l.
Release of specifically bound
[3H]ouabain during
washout was evaluated in experiments prolonging washout to 12 × 30 min, with subsequent measurements of
[3H]activity in
samples as well as in washout media.
Muscle water content was determined as the relative reduction in weight after the samples are heated at 90°C until weight stabilization. Protein content was determined after the method of Lowry (25) and was expressed as milligrams per gram wet weight of muscle. For calculations of total [3H]ouabain binding sites per muscle, weights of soleus and EDL muscles were determined immediately after they were dissected out and tendons had been removed.
K+-dependent p-nitrophenyl phosphatase ( pNPPase) activity was determined in crude homogenates as previously described in detail (24). In brief, muscle samples were weighed and homogenized by using an Ultra-Turrax T25 homogenisator (Janke & Kunkel, Staufen, Germany) at 0°C in a buffer containing (in mM) 30 histidine, 2 EDTA, and 250 sucrose (pH 7.2). Subsequently, the tissue homogenate was further homogenized at 0°C by using a glass homogenisator with a rotating Teflon pestle (B. Braun, Messengen, Germany). From the final homogenate, with a tissue concentration of 10 mg/ml, the K+-dependent pNPPase activity was determined by incubating 100 µl homogenate at 37°C in 800 µl buffer and calculating the difference in measurements obtained by using buffer containing (in mmol/l) 25 histidine, 15 MgCl2, and 100 NaCl (pH 7.4) vs. buffer containing (in mmol/l) 25 histidine 15 MgCl2, and 50 KCl (pH 7.4). The phosphatase reaction was started by addition of 100 µl of 100 mmol/l pNPP (Sigma Chemical) and stopped after 30 min by further addition of 2 ml ice-cold buffer containing 500 mmol/l Tris and 55 mmol/l EDTA. The liberated p-nitrophenyl was determined by absorption spectrophotometry at a wavelength of 410 nm by using a Photometer 4020 (Hitachi, Tokyo, Japan).
Statistics
Results are given as means ± SE. Statistical significance among groups was ascertained by Student's two-tailed t-test for unpaired observations. Bonferroni's correction was applied to correct for multiple comparisons. For example, for three comparisons P is multiplied by three. Only corrected P values are given. Corrected P values <0.05 were considered significant. Linear regression analysis was performed by using the method of the least squares. The squared correlation coefficient (r2) and the coefficient of inclination (
) are given.
RESULTS
Animals
K+-supplemented animals demonstrated a reduced body weight gain. Thus body weights in rats K+ supplemented for 2 wk and standard control animals were 150 ± 3 and 169 ± 3 g (P < 0.05, n = 5), respectively. Body weight of weight-matched control rats was 143 ± 2 g (n = 5). Between rats K+ supplemented for 2 wk and weight-matched control animals, no significant weight difference was seen (P > 0.10).Plasma K+
K+ supplementation for 2 days increased arterial plasma K+ by 66% (P < 0.05, n = 4) (Fig. 1). Plasma K+ remained stable at this level for the rest of the K+-supplementation period. After 2 wk of K+ supplementation, plasma K+ was 1.5 mmol/l, i.e., 40% above (P < 0.05, n = 5) the level observed in weight-matched control animals. Fasting for 1 day reduced plasma K+ in rats K+ supplemented for 2 wk by 3.1 ± 0.3 to 2.2 ± 0.2 mmol/l, i.e., by 58% (P < 0.05, n = 6), whereas fasting had no significant effect on plasma K+ in control animals (Fig. 1).
)
and weight-matched control rats (
). Animals were either allowed free
access to K+-enriched chow
(K+ supplemented) or restricted
access to standard chow (weight control), ensuring comparable body
weights between experimental and control animals. Dashed lines, values
for animals fasted from 14th to 15th day. Values are means ± SE;
n = 4-6 animals at each point. * Significantly different
K+-supplemented rats vs.
weight-matched control animals, P < 0.05.
Muscle K+
Before introduction of any special diet, K+ content in gastrocnemius muscle was 100 ± 1 µmol/g wet wt (n = 5). After 4, 7, and 14 days of K+ supplementation, K+ content in gastrocnemius muscle had increased to 104 ± 2, 104 ± 1, and 108 ± 1 µmol/g wet wt (P < 0.05, n = 5 in each group), respectively. Corresponding values obtained in weight-matched control animals were 97 ± 1, 96 ± 1, and 100 ± 1 µmol/g wet wt (n = 5 in each group), respectively, revealing no significant changes. Thus K+ content in gastrocnemius muscle was, throughout, significantly higher in K+-supplemented rats compared with weight-matched control animals (P < 0.05, n = 5). Equivalent changes were observed in both soleus and EDL muscles. Between standard control animals and weight-matched control animals, no significant differences in K+ content in skeletal muscles were found. Na+ content in skeletal muscles was generally reduced equivalently to the increase observed in K+ content. Gastrocnemius muscle K+ content relative to dry weight was calculated to be 444 ± 4 and 414 ± 4 µmol/g dry wt in rats K+ supplemented for 2 wk and weight-matched control animals, respectively. This corresponds to an increase by K+ supplementation of 7% (P < 0.05, n = 5), i.e., in the same order of magnitude as found by using wet weight as a reference. This indicates that the observed changes were not the mere outcome of changes in muscle water content. Free access to normal chow for 1 day after K+ supplementation for 1 wk reduced K+ content in gastrocnemius muscle to between 89 and 96 µmol/g wet wt, i.e., 8-14% (P < 0.05, n = 2) below the level observed in animals that were maintained on chow with high K+ content. Reductions in a similar order of magnitude were seen in soleus and EDL muscles. Fasting for 1 day of rats K+ supplemented for 2 wk caused a significant decrease in K+ content in soleus, EDL, and gastrocnemius muscles of 14, 10, and 14%, respectively (P < 0.05, n = 5). However, fasting for 1 day had no significant effect on K+ content in skeletal muscles in control animals.[3H]Ouabain Binding
Initially, the influence of the duration of K+ supplementation on [3H]ouabain binding site concentration was assessed in the gastrocnemius muscle (Fig. 2). A gradual increase in [3H]ouabain binding site concentration was seen in K+-supplemented animals ( = 6.1 pmol · g wet
wt1 · day1,
r2 = 0.73, P < 0.01, n = 8). In weight-matched control
animals, a corresponding decrease was observed ( = 
6.0
pmol · g wet
wt1 · day1,
r2 = 0.81, P < 0.01, n = 8). Thus a significant difference
between the two groups developed (P < 0.001). After ~1 wk of K+
supplementation, a maximum increase in
[3H]ouabain binding
site concentration seemed to have occurred. In separate studies,
increases of 42% (P < 0.05, n = 4) and 68% (P < 0.01, n = 6) were found in gastrocnemius
muscle after 1 and 2 wk of K+
supplementation, respectively, compared with weight-matched control levels. The most pronounced upregulation after 2 wk of
K+ supplementation was thus
observed in the gastrocnemius (mixed fiber), followed by EDL (white
fiber) with 48% (P < 0.01, n = 6) and soleus (red fiber) muscle
with 26% (P < 0.01, n = 6) (Fig. 3). The observed increase in
Na+-K+-ATPase
concentration may be the combined outcome of upregulation due to
K+ supplementation and
downregulation due to semistarvation. Thus it was also of interest to
compare rats K+ supplemented for 2 wk with standard control animals allowed free access to chow. This
showed upregulations in gastrocnemius, EDL, and soleus muscles of 25, 24, and 10% (P < 0.05, n = 5-6), respectively. When
weight-matched control animals were compared with standard control
animals, downregulations of 26, 16, and 12%
(P < 0.05, n = 5-6) were seen as a result of
semistarvation in the respective muscles.
) and in
weight-control rats (). After 1 wk, a fraction of rats from each
group was allowed free access to standard chow (dashed lines; 
,
refed K+-supplemented rats; x,
refed weight-control rats). Each point, mean of 3-5 observations
on 1 animal, except initial (t = 0)
value, which is mean of 3-5 observations on 5 animals. See text
for statistics.
Reversibility of the observed changes in [3H]ouabain binding site concentration was also assessed (Fig. 2). When animals again were allowed free access to normal chow after 1 wk of K+ supplementation or chow restriction, respectively, similar gastrocnemius muscle [3H]ouabain binding site levels were obtained in the 2 groups within 3 days. Fasting for 1 day caused only a tendency to a reduction in the effect of K+ supplementation from 68 to 58% (P > 0.2, n = 5-6) in [3H]ouabain binding site concentration in gastrocnemius muscle. Putative age-related [3H]ouabain binding site concentration changes during the experimental period were assessed in standard control animals with free access to standard chow. Thus after 2 wk, an age-dependent decrease from 269 ± 11 to 210 ± 16 pmol/g wet wt (P < 0.05, n = 5) was observed in gastrocnemius muscle.
To ensure that differences observed in
[3H]ouabain binding
were not merely the outcome of variations in binding kinetics between the groups, special kinetic experiments were performed. Nonspecific [3H]ouabain uptake and
retention in gastrocnemius muscle samples amounted, in standard assays,
to 2.8 ± 0.8 and 6.2 ± 0.9% of total uptake and retention in
rats K+ supplemented for 2 wk and
weight-matched control animals, respectively (P < 0.05, n = 5). Analysis of saturability in a
Scatchard-type plot revealed a linear distribution of gastrocnemius
muscle [3H]ouabain
binding values in both groups
(r2 = 0.94 and
0.94 in 2-wk
K+-supplemented rats and
weight-matched control animals, respectively, P < 0.05, n = 5), indicating a single major
population of
[3H]ouabain binding
receptors. The affinity constants for
[3H]ouabain binding to
gastrocnemius muscle samples from rats
K+ supplemented for 2 wk and
weight-matched control animals were 9.2 ± 0.1 and 9.7 ± 1.2 × 108 mol/l,
respectively (P > 0.70, n = 6). In a semilogaritmic plot analysis of values obtained in prolonged
[3H]ouabain washout
experiments using samples from the gastrocnemius muscle from rats
K+ supplemented for 2 wk and
weight-matched control animals, linear courses of
[3H]ouabain release
were seen after an initial steep falloff for 1.5 h. Half-life times for
the late slow phase of the washout curves were 2.0 and 2.4 h in
gastrocnemius muscle samples from rats
K+ supplemented for 2 wk and
weight-matched control animals, respectively (P > 0.8, n = 5). On the basis of the
above-determined values and as earlier described in detail (18), values
for [3H]ouabain
binding obtained in the standard assay were corrected for nonspecific
uptake and retention, incomplete saturation, and loss of specifically
bound [3H]ouabain
during washout. This revealed a 70% increase
(P < 0.05, n = 5) in
[3H]ouabain binding
site concentration in gastrocnemius muscle from rats
K+ supplemented for 2 wk compared
with weight-matched control animals. Taken together, differences in
[3H]ouabain binding
observed between groups by using the standard assay were not the
outcome of differences in binding kinetics.
To ensure that the changes in [3H]ouabain binding site concentration were not merely the outcome of changes in either muscle water or in protein content, these parameters were determined. In gastrocnemius muscle, [3H]ouabain binding site concentration expressed relative to dry weight was 69% (P < 0.05, n = 5) higher in rats K+ supplemented for 2 wk compared with weight-matched control animals (Table 1). Correspondingly, gastrocnemius muscle [3H]ouabain binding site concentration expressed relative to protein content was 55% (P < 0.05, n = 5) higher in rats K+ supplemented for 2 wk compared with weight-matched control animals. To ensure that the upregulation in skeletal muscle [3H]ouabain binding site concentration in K+-supplemented animals was not the simple outcome of loss of muscle mass relative to membranes, [3H]ouabain binding site number per muscle was calculated; i.e., the [3H]ouabain binding site concentration (pmol/g wet wt) was multiplied by the wet weight (g wet wt) of the muscle. These calculations showed upregulations of the entire amount of Na+-K+-ATPase per muscle of 15 and 39% (P < 0.05, n = 5) in soleus and EDL muscles, respectively, in rats K+ supplemented for 2 wk compared with weight-matched control animals.
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For further evaluation of the [3H]ouabain binding measurements, K+-dependent pNPPase activity was determined in crude homogenates. Thus increases in K+-dependent pNPPase activity of 51% (P < 0.05, n = 5), 45% (P < 0.05, n = 4), and 18% (P = 0.19, n = 4) were observed in gastrocnemius, EDL, and soleus muscles, respectively, in rats K+ supplemented for 2 wk compared with weight-matched control animals. These changes are in accord with the increases found in [3H]ouabain binding site concentrations. Thus the observed changes in [3H]ouabain binding site concentrations reflect changes in total Na+-K+-ATPase concentration.
KCl Infusions
Effects on plasma K+. First, the effect of KCl infusion on plasma K+ was assessed in animals with preserved renal function (Fig. 4A). During KCl infusion, an initial steep rise in plasma K+ was generally followed by an attenuated and almost linear increase. No significant differences in hemoglobin and pH values were observed between groups during infusions. Between K+-supplemented animals and weight-matched control animals, no significant difference in plasma K+ values was observed after 5 min of infusion. However, after 15 min plasma K+ had increased only 3.4 ± 0.4 mmol/l in K+-supplemented rats compared with an increase of 4.7 ± 0.3 mmol/l in the control animals (P < 0.05, n = 4). The purpose of experiments with fasting was to assess the effect of KCl infusion in animals with increased Na+-K+-ATPase concentration in skeletal muscles but without simultaneously increased plasma K+ and K+ content in skeletal muscles. After fasting for 1 day, rats K+ supplemented for 2 wk showed a 26% (P < 0.05, n = 5) lower plasma K+ rise after 15 min of KCl infusion compared with values obtained without fasting. From linear regression analysis of values for plasma K+ and KCl infusion time, it was found that fasting of rats K+ supplemented for 2 wk caused a 30% (P < 0.05, n = 5) reduction in the inclination of the dose-response curve.
1 · h1.
Experiments were performed without
(A) or after functional nephrectomy obtained by ligation of renal vessels
(B). After surgical procedures were
finished animals were allowed a 15-min recovery period before blood
sampling and infusion were started. In experiments with fasting, only
water was administered for 1 day before experimental procedures. Values
are means ± SE; n = 4-7 animals. Each point, means of single determinations of
plasma K+ values; bars, SE; end
point of each curve, mean duration (5-min intervals) of KCl infusion
before respiratory and cardiac arrest occurred.
A: values for plasma
K+ from group of fasted
K+-supplemented animals (
) were
significantly different throughout from those from nonfasted
K+-supplemented animals () as
well as from weight-matched control animals ().
B: values for plasma
K+ from the group of fasted,
functionally nephrectomized, and
K+-supplemented animals () were
significantly different from those from nonfasted, nephrectomized, and
K+-supplemented animals () as
well as from their control animals ().
Second, the effect of KCl infusion on plasma K+ was assessed in functionally nephrectomized animals (Fig. 4B). Functional nephrectomy did not significantly change initial plasma K+ values. However, after 15 min of KCl infusion, plasma K+ values had increased additionally 3.1 ± 0.5 mmol/l (P < 0.05, n = 5) after functional nephrectomy in rats K+ supplemented for 2 wk and 2.7 ± 0.6 mmol/l (P < 0.05, n = 5) in weight-matched control animals. After they fasted for 1 day, a 42% (P < 0.05) lower plasma K+ rise was observed after 15 min of KCl infusion in rats K+ supplemented for 2 wk compared with weight-matched control animals. In this K+-supplemented group, fasting caused a significant attenuation of 31% (P < 0.05, n = 5) in the inclination of the dose-response curve. In weight-matched control animals, fasting had no significant effect on plasma K+ in response to KCl infusion. Effect on skeletal muscle K+. Further assessment of the extrarenal K+ homeostasis was obtained by measurements of changes in K+ content in skeletal muscles in response to KCl infusion (Table 2). After they fasted for 1 day, KCl infusion caused a 19% (P < 0.05, n = 5) increase in K+ content in gastrocnemius muscle in functionally nephrectomized rats that had been K+ supplemented for 2 wk, whereas only a tendency to an increase of 5% (P > 0.10, n = 6) was seen in the control animals. The final K+ contents obtained in gastrocnemius and EDL muscles after K+ infusion were 7 and 10 µmol/g wet wt higher, respectively, in rats K+ supplemented for 2 wk than in weight-matched control animals (P < 0.05, n = 5) (Table 2). In soleus muscle, a tendency to a higher increase in K+ content of 6 µmol/g wet wt (P = 0.15, n = 5) was observed in the K+-supplemented group compared with weight-matched control animals. It is noteworthy that these effects were found even though K+-supplemented, fasted, and functionally nephrectomized rats throughout had a plasma K+ level below the control animals (Fig. 4B).
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DISCUSSION
The major importance of the present study is the establishment of the relationship between upregulation of Na+-K+-ATPase concentration in skeletal muscles and improved capacity for extrarenal K+ handling in K+-supplemented experimental animals. A relationship between Na+-K+-ATPase concentration changes and active transmembrane K+ transport capacity changes in skeletal muscles has previously been demonstrated in intact fibers after Na+ loading (5) and by measurements of electrically stimulated active K+ uptake (9). In humans, the relationship is evident from a lower exercise-induced plasma K+ rise in association with upregulation of Na+-K+-ATPase concentration in skeletal muscles in response to physical training (13, 27). Furthermore, inhibition of a fraction of the Na+-K+ pumps in skeletal muscles by digoxin has been found to increase exercise-induced plasma K+ rise (30). Thus the present study is in accord with the evolving concept of Na+-K+-ATPase concentration in skeletal muscles as an important factor for extrarenal K+ homeostasis.
After 2 wk of K+ supplementation, plasma K+ had increased to 5.3 ± 0.2 mmol/l, i.e., 40% above the level observed in weight-matched control animals. This is considerably higher than the increases previously reported of 0.3 (2) and 0.6 mmol /l (33, 35), respectively, after K+ supplementation. The discrepancy may be due to unintended fasting in some of the earlier studies. This is likely because an earlier (35) as well as the present study have demonstrated a plasma K+ decrease in K+-supplemented animals after fasting for 1 day, to a level even below that seen in control animals. Furthermore, it is in accord with the observation that, in K+-supplemented animals, increased renal K+ excretion is maintained for at least 40 h after removal of K+ from the chow despite intervening hypokalemia and K+ depletion (35). Taken together, the presently observed plasma K+ increase in K+-supplemented animals is in accord with a plasma K+ value of 5.5 ± 0.2 mmol/l (n = 5) found in K+-supplemented animals that were not fasted before plasma K+ measurements (32).
Two weeks of K+ supplementation significantly increased K+ content in all skeletal muscle types examined by up to 8%. This is at variance with other studies of K+-supplemented rats, in which the K+ content in the gracilis muscle did not change significantly (2), and a study in which K+ content in soleus muscle showed no increase (23). Unintended fasting might, at least in part, also explain these findings. This is likely because fasting of animals for 1 day in the present study reduced K+ content in skeletal muscles by as much as 14%. However, in a more recent study a significant 7% increase in K+ content in skeletal muscle was found in K+-supplemented rats (1), and a tendency to an increase of 5% was observed in the diaphragm of K+-supplemented rats that were not fasted (33). The magnitude of K+ loss from skeletal muscle due to fasting in K+-supplemented animals is in accord with a previous report of a 9% reduction below control level observed in K+-supplemented animals after 24 h on a K+-free diet (35).
K+ supplementation was found to induce a progressive and reversible increase of up to 70% in gastrocnemius muscle [3H]ouabain binding capacity. Significant although smaller increases were seen in muscles predominantly consisting of red and white fibers, indicating that the magnitude of increase might be muscle or muscle fiber specific. It is noteworthy that the upregulation seems to be a general skeletal muscle feature in response to K+ supplementation. The use of weight- and age-matched control animals in the present study was necessary because K+ supplementation caused a reduction in body weight gain, and semistarvation has been associated with a 25% decrease in Na+-K+-ATPase concentration in skeletal muscles (20). Therefore, comparing K+-supplemented animals to age-matched control animals only would underestimate the effect of K+ supplementation. Evaluation of [3H]ouabain binding kinetics in K+-supplemented and weight-matched control animals showed no major differences. Furthermore, the increase in [3H]ouabain binding site concentration observed with K+ supplementation was of the same order of magnitude as the increase observed in K+-dependent pNPPase activity in crude muscle homogenates. This is of importance because alterations, if any, in the number of low-affinity ouabain binding sites might not have been detected by the standard [3H]ouabain binding assay. Thus the presently observed increases in [3H]ouabain binding can be recognized as increases in total Na+-K+-ATPase concentrations, and the upregulation could not be the outcome of quantitative changes between Na+-K+-ATPase subtypes. It was verified that the upregulation in Na+-K+-ATPase concentration in skeletal muscles was not the outcome of altered water content and that it was selective in relation to general protein synthesis. Furthermore, when calculated per muscle, a significant upregulation was also observed. This indicates that the upregulation is due to an increased synthesis of the Na+-K+-ATPase enzyme and not the outcome of reduced muscle mass relative to membranes. The discrepancy between the upregulation observed in the present study and the earlier reports of no change (23) and of a 36% upregulation (21) may be related to the intensity of K+ supplementation. Thus in the first study (23) chow K+ content was 135 mmol KCl/100 g, i.e., ~5 times above normal K+ content compared with seven- to eightfold in the present study. In the first study (23), only soleus muscle was evaluated, and in the present study the less-pronounced upregulation was actually seen in this muscle. Furthermore, the aspects of semistarvation (20) were not addressed in any of the former studies (2, 21, 23, 33). The trend toward a decline in [3H]ouabain binding site concentration seen after the first week in each of the four groups of animals studied (Fig. 2) is in accord with the well-known age-dependent downregulation (22).
The relative rise in plasma K+ in response to KCl infusion was attenuated in K+-supplemented animals compared with weight-matched control animals independently of renal function. Fasting caused a reduction in relative plasma K+ rise in response to KCl infusion in K+-supplemented rats, whereas no effect of fasting was seen in weight-matched control animals. This attenuated plasma K+ increase was probably, in part, due to reduced intra- and extracellular K+ concentrations. However, the important observation is that the curve for plasma K+ rise as a function of duration of KCl infusion has a lower inclination in K+-supplemented, fasted, and functionally nephrectomized rats compared with their control animals. This indicates that the difference is not the mere outcome of different initial plasma K+ levels but a result of increased K+ uptake in the skeletal muscles.
Fasting of K+-supplemented rats for 1 day increased K+ content in skeletal muscles by up to 19% in response to KCl infusion, whereas no increase was observed in weight-matched control animals. Between the two groups of animals, the most pronounced K+ content differences in skeletal muscles in response to KCl infusion were obtained in EDL and gastrocnemius muscles. This is of interest because the highest [3H]ouabain binding site upregulation was also seen in these two muscles, indicating an overall muscle-specific regulation. The relatively higher increase in K+ content in skeletal muscles in K+-supplemented rats might, in part, be ascribed to the lower initial value. Moreover, generally a high plasma K+ level will be associated with a high K+ uptake in skeletal muscles. Therefore, it is noteworthy that the K+ content in muscles from K+-supplemented, fasted, and functionally nephrectomized rats increased to a level above that obtained in weight-matched control animals and that this occurred even though plasma K+ level in the K+-supplemented group was continuously lowest. In the present study, a total of ~0.6 and 0.4 mmol KCl was infused in fasted and functionally nephrectomized rats K+ supplemented for 2 wk and their weight-matched control animals, respectively. On the basis of body weight, plasma K+, and K+ content in skeletal muscles, it was calculated that a total of 0.2-0.3 mmol more KCl could be recovered in K+-supplemented rats than in their weight-matched control animals after KCl infusions. When the putative sources of error associated with these calculations are taken into account, the present findings seem to be in good accord. The increased K+ uptake in skeletal muscles is not likely to be the outcome of increased Na+-K+-pump activity induced by higher insulin or catecholamine levels (6) because such possible hormonal changes induced by hyperkalemia might vanish with fasting-induced hypokalemia. Plasma insulin was not measured in the present study. However, hyperkalemia has the capability to increase plasma insulin level (6), and a high insulin level has been found to be associated with increased Na+-K+-ATPase concentration in skeletal muscles (31). Therefore, in K+-supplemented animals a putative increase in plasma insulin level may be associated with increased Na+-K+-ATPase concentration in skeletal muscles.
It is interesting that the Na+-K+-ATPase concentration changes in skeletal muscles after K+ supplementation and reversibility hereof when chow K+ was normalized were of similar time courses and magnitudes, although in opposite directions, as the changes previously observed after K+ depletion and subsequent K+ repletion (23, 28). It should be noted that these major changes have been obtained with K+-intake changes exceeding what is to be expected to occur physiologically. Thus the effects may be more moderate with more lenient changes in chow K+ content. Because other modulators of Na+-K+-ATPase in skeletal muscles first described in rodents later have been demonstrated also in humans, this might also be expected regarding increased K+ intake or persisting hyperkalemia.
Because of the large renal capacity for K+ excretion, a physiological increase in K+ intake in human subjects may not be of major importance for the ability to clear an acute K+ load. However, in patients with impaired renal function, hyperkalemia might induce an upregulation of the Na+-K+-ATPase concentration in skeletal muscles, and a resulting increase in K+ clearance capacity in skeletal muscles might serve as an important compensatory mechanism for the handling of an acute K+ load. A more physiological effect of increased skeletal muscle Na+-K+-pump number because of K+ supplementation or hyperkalemia may be a modulation of the ongoing K+ fluctuations across the muscle plasma membranes. Thus changes in Na+-K+-ATPase concentrations in skeletal muscles because of high or low K+ intake may be of special clinical importance in relation to K+ administration, when drugs influencing Na+-K+-pump activity are administered as well as during exercise during which changes in fluctuations of interstitial and plasma K+ levels may affect muscle function (34) and heart impulse generation and conduction. In this context, it is of interest that a number of studies in humans have associated high K+ intake with prevention or reduction of cardiovascular diseases (37). A major objection against these findings has been the lack of a correlation between the claimed effects and plasma K+. However, the highly dynamic character of plasma K+ and K+ content in skeletal muscles observed in the present study suggests that more reliable measurements than standard plasma K+ determinations are needed. It may be ventured that studies of extrarenal K+ clearance, K+ content, and Na+-K+-ATPase concentration measurements in skeletal muscles might be more reliable correlates.
ACKNOWLEDGEMENTS
We thank Grete Simonsen for skilled technical assistance and Stig Haunsø for valuable discussions.
FOOTNOTES
Address for reprint requests: H. Bundgaard, Dept. of Medicine B 2142, The Heart Centre, Rigshospitalet, National Univ. Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
Received 12 March 1996; accepted in final form 25 November 1996.
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