Journal of Applied Physiology
Vol. 82, No. 4, pp. 1136-1144, April 1997
EXERCISE AND MUSCLE

K⁺ supplementation increases muscle [Na⁺-K⁺-ATPase] and improves extrarenal K⁺ homeostasis in rats

Henning Bundgaard, Thomas A. Schmidt, Jim S. Larsen, and Keld Kjeldsen

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 wt¹ · 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

1

1

1

1

Plasma K⁺

Skeletal Muscle and Chow K⁺ and Na⁺ Content

[³H]Ouabain Binding

6

8

8

7

7

6

6

3

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

Animals

Plasma K⁺

Muscle K⁺

[³H]Ouabain Binding

1

1

1

1

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.

Table 1. [3H]ouabain binding sites expressed relative to wet weight, dry weight, and protein content of gastrocnemius muscle and to total mass of extensor digitorum longus (EDL) muscle Weight-Control Rats K+-Supplemented Rats Wet weight, pmol/g 209 ± 16  356 ± 16* Dry weight, pmol/g 867 ± 83  1465 ± 66* Protein content, pmol/mg 1.36 ± 0.1  2.11 ± 0.1* Muscle mass (EDL), pmol/muscle 15.2 ± 0.5  21.2 ± 0.7* Values are means ± SE; n = 5 rats. Values were obtained in rats after 2 wk of K+ supplementation with free access to K+-enriched chow (K+ supplemented) or restricted access to standard chow (weight control). EDL, extensor digitorum longus. * Significantly different, P < 0.05.

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

1

1

Table 2. K+ content in soleus, EDL, and gastrocnemius muscles KCl Infusion K+ Content, µmol/g wet wt Weight-control rats K+-supplemented rats Soleus Without 80 ± 3  75 ± 2  After 100 ± 4* 106 ± 2* EDL Without 102 ± 3  95 ± 1  After 112 ± 1  122 ± 1* Gastrocnemius Without 99 ± 4  93 ± 1  After 104 ± 1  111 ± 2* Values are means ± SE; n = 5-6 animals in each group. Values were obtained in K+-supplemented and weight-control rats. After 2 wk on respective diets, rats were fasted for 1 day before measurements. Functional nephrectomy was obtained by renal vessel ligation. Values are obtained without and after intravenous infusion of 0.5 mol/l KCl at a rate of 1.5 ml · 100 g body wt1 · h1. * P < 0.05 between animals without and after KCl infusion. P < 0.05 between K+-supplemented and weight-control rats.

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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