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Vol. 297, Issue 2, 804-810, May 2001
Department of Medicine, University of Sydney, Sydney, Australia (M.M., H.H.R.); Department of Cardiology, Royal North Shore Hospital, Sydney, Australia (M.M., A.S.M., H.H.R.); Department of Cardiology, Westmead Hospital, Sydney, Australia (M.M.); and Department of Pathology, St. Vincents Hospital, Sydney, Australia (A.W.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Cyclosporine A (CsA) and FK506, important immunosuppressants, have been shown to inhibit the enzymatic equivalent of the Na+-K+ pump (Na+, K+-ATPase) in renal tissue. A similar effect in the heart may contribute to the adverse effects of these agents that include calcification, contractile dysfunction, and altered calcium handling. However, inhibition of the pump has not been demonstrated in cardiac myocytes. We isolated single ventricular myocytes from control rabbits and from rabbits administered CsA or FK506 for 1 week. Na+-K+ pump current (Ip) was measured using the whole-cell patch-clamp technique. When patch pipettes contained Na+ in a concentration ([Na]pip) near physiological intracellular levels mean Ip of cardiac myocytes from rabbits with serum CsA levels within the therapeutic range was significantly lower than mean Ip of cardiac myocytes from controls. Treatment had no effect on Ip measured using a [Na]pip expected to nearly saturate intracellular binding sites. The CsA-induced inhibition of Ip was dependent on the K+ concentration in pipette solutions. Mean Ip in myocytes from rabbits with serum levels of FK506 within the therapeutic range was similar to mean Ip in myocytes from controls, whereas FK506 in a dose inducing serum levels severalfold above the therapeutic range caused significant pump inhibition. Using ion-sensitive microelectrodes we showed the intracellular Na+ activity in papillary muscles isolated from rabbits treated with CsA was significantly higher than in papillary muscles from control rabbits, indicating that CsA causes pump inhibition in intact myocytes with a physiological intracellular milieu.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cyclosporine
A (CsA) and FK506 are used for immunosuppression in organ
transplantation and in the treatment of autoimmune diseases. Of the two
drugs CsA is used most widely. Their usefulness is restricted by
significant adverse effects, including nephrotoxicity, cardiotoxicity,
hyperkalemia, and in regards to CsA in particular, the induction of
hypertension. It has been speculated that the adverse effects in part
are the results of CsA- or FK506-induced inhibition of membrane ion
transport systems, including the
Na+-K+ pump (Tumlin and
Sands, 1993
). The enzymatic equivalent of the Na+-K+ pump,
Na+-K+-ATPase, is inhibited
when renal cells are exposed to CsA (Tumlin and Sands, 1993) and FK506
(Lea et al., 1994) in vitro. Two studies on vascular smooth muscle
cells and cardiac myocytes, respectively, have shown that CsA
(Bokemeyer et al., 1994) and FK-506 (McCall et al., 1996) induced an
increase in the intracellular Ca2+ concentration.
The increase was consistent with impaired efflux of
Ca2+ via the
Na+-Ca2+ exchange
mechanism. Because Na+-Ca2+
exchange-mediated Ca2+ efflux ultimately derives
its energy from the transmembrane gradient for
Na+ the impaired Ca2+
efflux might be due to inhibition of the
Na+-K+ pump. However,
sarcolemmal pump inhibition could not be demonstrated (Bokemeyer et
al., 1994; McCall et al., 1996).
Functional properties of the sarcolemmal
Na+-K+ pump are complex.
Sodium binds to the pump at three intracellular sites, two near the
cytoplasmic surface and at a third site inside the membrane dielectric
(Or et al., 1996). Binding at the sites near the cytoplasmic surface
occurs in competition with K+ and is independent
of membrane voltage. This
K+/Na+ antagonism is
particularly pronounced in the heart (Therien and Blostein, 1999).
Binding at the third site is selective for Na+.
However, because of location of the site within the electrical field of
the membrane, binding is voltage-dependent (Hansen et al., 2000 and
references therein). The
Na+-K+ pump in cardiac
myocytes can be regulated in a manner consistent with effects at either
the sites near the cytoplasmic surface (Buhagiar et al., 1999) or the
site inside the membrane dielectric (Bewick et al., 1999; Hansen et
al., 2000). The methods used in the previous studies on the effect of
CsA and FK506 on the sarcolemmal Na+-K+ pump (Bokemeyer et
al., 1994; McCall et al., 1996) did not take into account the voltage
dependence and Na+/K+
interaction at intracellular sites. We have used the whole-cell patch-clamp technique to measure
Na+-K+ pump current
(Ip). This technique allows control of membrane voltage and the concentration of the transported ligands on both sites
of the membrane.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Treatment Protocols.
Male New Zealand White rabbits
(2.5-3.0 kg, age 12-14 weeks) were given CsA or FK506 for 7 days. A
separate group of untreated rabbits served as controls. CsA was
dissolved in polyoxyethylated castor oil and ethanol and administered
by subcutaneous injections. This route of administration has been shown
to provide reliable levels and adequate bioavailability (Wassef et al.,
1985). The controls did not receive ethanol injections because previous
work from our laboratory (Mihailidou et al., 2000a) has shown that similar doses of ethanol had no effect on
Na+-K+ pump activity. FK506
was included in capsules and administered by oral gavage. Toxicity
studies have demonstrated a wasting syndrome during CsA treatment
(Gratwohl et al., 1986) and in our initial dose-finding studies we
found that there was a marked reduction in weight when CsA levels were
in the toxic range. To avoid inducing severe toxicity we charted the
weight of all rabbits daily at the same time each day. If a decrease
occurred during a 24-h period we omitted the next scheduled dose of
drug. No rabbit had drugs omitted for more than 1 day and all received
the same weight-adjusted total dose. Because CsA is used more commonly
than FK506 we predominantly studied CsA.
Measurement of Ip and Intracellular Na+
Activity (aiNa).
We anesthetized rabbits
with ketamine (50 mg kg
1) and xylazine
hydrochloride (20 mg kg1) given intramuscularly
after 7 days of treatment. Single myocytes were isolated from either
ventricle (Whalley et al., 1993) and placed in a tissue chamber
perfused with modified Tyrode's solution. The solution contained 140 mM NaCl, 5.6 mM KCl, 2.16 mM CaCl2, 0.44 mM
NaH2PO4, 10 mM glucose, 1.0 mM MgCl2, and 10 mM HEPES and was titrated to a
pH of 7.40 ± 0.01 at 35°C with 1 M NaOH. The solution also
contained 16 mg/l gentamicin to inhibit bacterial growth and 0.5%
bovine serum albumin. Cardiac myocytes were superfused with this
solution until the whole-cell configuration was established. The
myocytes were voltage clamped with wide-tipped (4-5 µm) patch pipettes. For measurement of Ip at a fixed test
potential (Vm) of 
40 mV we varied the
concentration of Na+
([Na]pip) or K+
([K]pip) in pipette solutions. The solutions
with variable Na+ contained 70 mM potassium
glutamate, 1 mM KH2PO4, 5 mM HEPES, 5 mM EGTA, 2 mM MgATP, 10 or 80 mM sodium glutamate, and 80 or 10 tetramethylammonium chloride. The solutions were titrated to a pH
of 7.05 ± 0.01 at 35°C with 1 M KOH. Solutions with variable K+ concentrations contained 9 mM sodium
glutamate, 1 mM NaH2PO4, 5 mM HEPES, 5 mM EGTA, 2 mM MgATP, 0 to 140 mM potassium chloride and
titrated to a pH of 7.05 ± 0.01 at 35°C with 1 M
tetramethylammonium hydroxide. For measurement of
Ip at variable Vm, pipettes
were filled with a solution containing 10 mM sodium glutamate, 1 mM KH2PO4, 5 mM HEPES, 5 mM
EGTA, 2 mM MgATP, 60 mM tetramethylammonium chloride, 20 mM
tetraethylammonium chloride, 70 mM cesium chloride, and 50 mM aspartic
acid. The solution was titrated to a pH of 7.05 ± 0.01 at 35°C
with 1 M HCl. Filled pipettes had resistances of 0.9 to 1.1 M
. When
the whole-cell configuration had been established we switched to a
superfusate that was identical to the Tyrode's solution used
initially, except that it was nominally Ca2+-free
and contained 0.2 mM CdCl2 and 2 mM
BaCl2. All superfusates were warmed to 35°C.
80 mV. The charge
transferred during each voltage pulse was derived by integrating the
capacitive current with respect to time. Membrane capacitance was then
calculated by dividing the charge transferred by the voltage step of 10 mV.
We measured aiNa in papillary
muscles isolated from the right ventricle using ion-sensitive
microelectrodes. Details have been described (Mihailidou et al., 1998).
Ouabain was purchased from Sigma Chemical Co. (St. Louis, MO). All
other chemicals were "analytical" grade and purchased from BDH
Laboratory Supplies (Melbourne, Australia).
Statistical Analysis. Results are expressed as mean ± S.E. Comparisons between the different treatment groups are made using one-way analysis of variance followed by Dunnett's test. Differences are regarded as significant when P < 0.05. Comparisons using unpaired Student's t test were followed by the Bonferroni adjustment. Body weight, serum levels of K+, and creatinine before and after treatment were compared using paired Student's t test.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Description of in Vivo Model.
To evaluate the clinical
relevance of our treatment protocols we measured body weight, serum
levels of K+, creatinine, CsA, and FK506 and
studied the effect of treatment on renal histology. Clinically relevant
levels of CsA (Keogh et al., 1995) were achieved with a dose of 10 mg
kg 1
day1 and FK506
(Radermacher et al., 1998) in a dose of 1 mg
kg1 day1 (Fig.
1). Table 1 shows the
mean body weights for rabbits treated with the two different dosing
schedules for CsA and FK506. There was no significant change in
weight during treatment. The table also shows serum levels for
K+ and creatinine. The drugs had no significant
effect on these levels.
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1
day1 were examined. All showed focal glomerular
arteriolar and interstitial changes. These changes ranged from minor
arteriolar thickening only through to patchy areas of tubular
interstitial inflammation. No hyaline vascular changes were observed.
Representative micrographs are shown in Fig.
2. In contrast, renal histology of three
rabbits treated with the highest dose of FK506 showed no significant
changes relative to controls. Rabbits given CsA in a dose of 10 mg
kg1 day1 had an
increase in blood pressure by 14 ± 1 mm Hg. This was
significantly higher than a change in blood pressure of 2 ± 1 mm
Hg in control rabbits. However, the blood pressure of rabbits given CsA
in a dose of 5 mg kg1
day1 and blood pressures of rabbits given FK506
according to both dosage schedules were not significantly different
from the blood pressure of control rabbits. We conclude our dosage
schedules induced effects similar to those seen in the clinical use of
the drugs studied.
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Effect of CsA and FK506 on Ip.
To examine the
effect of CsA and FK506 on pump activity when intracellular
Na+ is near physiological levels we voltage
clamped cardiac myocytes at 40 mV using a
[Na]pip of 10 mM. A typical recording of
membrane currents during measurement of Ip is
shown in Fig. 3A.
Ip was defined as the shift in holding current
(Ih) induced by ouabain. We identified
Ih before and after superfusion of ouabain with an electronic cursor as described previously (Hemsworth et al., 1997).
To normalize Ip for cell size we measured the
membrane capacitance. Mean levels for the normalized
Ip have been summarized in Fig. 3B. Treatment
with CsA in a dose inducing clinically relevant drug levels (10 mg
kg1 day1; Fig. 1) was
associated with a statistically significant 43% reduction in mean
Ip. In contrast, FK506 administered in a dose (1 mg kg1 day1) inducing
clinically relevant levels had no effect on Ip.
Only levels well above the upper limit used in clinical practice in humans (Fig. 1) was associated with a statistically significant 40%
reduction in mean Ip. Because CsA and FK506 have
been reported to have effects on cardiac hypertrophy we have included
membrane capacitance separately in Table 1. There was no statistically significant difference between the membrane capacitance of myocytes from control rabbits and rabbits treated with the drugs.
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Dependence of CsA-Induced Pump Inhibition on [Na]pip
and [K]pip and Membrane Voltage.
To examine whether
CsA has an effect on maximal pump rate or on the sensitivity of the
pump to intracellular Na+ we voltage clamped
myocytes using a [Na]pip ranging from low, rate-limiting levels to a level expected to nearly saturate
intracellular binding sites. The myocytes were isolated from controls
and from rabbits given 10 mg kg1
day1 CsA. The relationship between
[Na]pip and Ip is shown
in Fig. 4A. Mean Ip
measured using a [Na]pip of 5 or 10 mM was
significantly lower for myocytes from rabbits treated with CsA than for
myocytes from controls. However, there was no significant effect of CsA on Ip measured using a
[Na]pip of 35 or 80 mM. These findings suggest
that CsA induces a change in the pump's sensitivity to intracellular
Na+ rather than a change in maximal rate.
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). We have previously
demonstrated [K]pip-dependent changes of
Ip induced by in vivo blockade of
angiotensin-converting enzyme or angiotensin II receptors (Buhagiar et
al., 1999). To examine whether the CsA-induced decrease in
Ip depends on [K]pip we
voltage clamped myocytes at 40 mV using a
[Na]pip of 10 mM and a
[K]pip of 0, 35, 70, or 140 mM. Mean values of
Ip for myocytes isolated from controls and from
rabbits given 10 mg kg1
day1 CsA are shown in Fig. 4B. Mean
Ip was similar for myocytes isolated from rabbits
treated with CsA and from controls when [K]pip
was 0 or 140 mM. However, treatment with CsA induced a significant decrease in the mean Ip measured using a
[K]pip of 35 or 70 mM. This suggests that CsA
enhances the antagonistic effect of K+ on
activation of the pump by Na+ at sites near the
cytoplasmic surface.
To examine whether there is an effect of CsA on cytosolic binding sites
inside the membrane dielectric we determined the
Ip-Vm relationship for
myocytes from rabbits treated with CsA and from controls. We used a
[Na]pip of 10 mM. Pipette solutions and
superfusates were designed to eliminate time- and voltage-dependent
nonpump currents (Gray et al., 1997). The voltage clamp protocol and
recordings of membrane currents are illustrated in Fig.
5A. We identified Ih at the different test potentials as described
(Gray et al., 1997). Figure 5B shows a summary of
Ip-Vm relationships of
myocytes from control- and CsA-treated rabbits. The
Ip-Vm relationships have
been normalized to the Ip recorded at 0 mV to
facilitate comparison of slopes (Gadsby and Nakao, 1989). The
normalized relationships were similar.
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Effect of CsA on aiNa.
To examine
whether the effect of CsA on Ip demonstrated in
patch clamped myocytes is reflected by an increase in
aiNa in intact myocytes with a
physiological ionic composition of the intracellular compartment we
directly measured aiNa in intact
right papillary muscles using ion-sensitive microelectrodes. We
measured aiNa in papillary
muscles isolated from six control rabbits and from six rabbits treated
with CsA in a dose of 10 mg kg1
day1. The mean
aiNa was 7.9 ± 0.1 mM in
papillary muscles isolated from control rabbits and 9.7 ± 0.5 mM
in papillary muscles from rabbits treated with CsA. The difference was
statistically significant.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of CsA and FK506 on the Sarcolemmal
Na+-K+ Pump.
In this study we used an ex
vivo experimental protocol. Rabbits were treated with drugs in vivo and
Ip was subsequently measured in isolated myocytes
not exposed to the drugs in vitro. Because we typically measured
Ip ~3 to 6 h after isolation of myocytes the inhibition of Ip we demonstrated is likely to
reflect chronic changes in pump function. Such a chronic change induced
by the drugs may contribute to the discrepancy between our study and two previous studies examining the effect of CsA and FK506 on the
sarcolemmal Na+-K+ pump
(Bokemeyer et al., 1994; McCall et al., 1996).
exposed vascular smooth muscle to CsA in a
concentration ~100-fold higher than serum levels achieved in our
study. However, the tissue was exposed to CsA in vitro only. In
addition the assay of pump function used may not have allowed the
detection of CsA-induced changes. Pump function was measured in cells
suspended in a markedly hyperosmolar solution. This may have confounded
results because extracellular osmolarity is an important determinant of
pump activity (Whalley et al., 1993; Bewick et al., 1999). McCall et
al. (1996) exposed the isolated enzymatic equivalent of the
Na+-K+ pump,
Na+-K+ ATPase, to FK506 in
a concentration much higher than the serum levels we achieved. Any
effect of FK506 dependent upon cellular mechanisms involved in
long-term regulation of the pump is not expected to be detectable with
such a protocol. It should also be noted that the effect of FK506 on
Na+-K+ ATPase was examined
under conditions of maximal enzymatic activity. In our studies on CsA
we could not detect an effect on Ip measured under conditions expected to induce nearly maximal activity.
Mechanism for CsA-Induced Na+-K+ Pump
Inhibition.
Because the CsA-induced decrease in
Ip is dependent on [K]pip
(Fig. 4B) and because it is independent of voltage (Fig. 5) it is
reasonable to consider that treatment with CsA alters the K+/Na+ antagonism at sites
near the cytoplasmic surface. This antagonism can be quantified on the
basis of the ratio of apparent affinity constants in kinetic models
fitted to Na+ activation profiles of a single
isoform of Na+-K+-ATPase at
various concentrations of K+ (Therien and
Blostein, 1999). However, it is highly likely that Ip measured in our study reflects the activity of
more than one isoform in the heart (Sweadner, 1989).
Ip reflects the net forward pump rate of all
isoforms and does not allow discrimination between their different
Na+ activation profiles and/or
K+/Na+ antagonism. In
addition, it would not be possible to ascertain that
Na+ activation is the rate-limiting step in the
pump cycle at all levels of [Na]pip we used to
measure Ip. This would preclude a meaningful
quantitative analysis based on the fit of a specific kinetic model to
the data. However, our study is nevertheless consistent with the
conclusion that treatment with CsA enhances the antagonistic effect of
K+ on activation of the pump by
Na+ at sites near the cytoplasmic surface.
; Hansen et al., 2000) and noncardiac (Ragolia et
al., 1997) myocytes, whereas protein phosphatase 2B has not been linked
to the sarcolemmal Na+-K+
pump. CsA is best known as an inhibitor of calcineurin. However, it
must be recognized that it has a multitude of other actions that alone
or in combination may mediate the effect on
K+/Na+ antagonism at
cytosolic Na+-K+ pump
sites. These include effects on protein kinases, hormones, and direct
effects of CsA on membranes. This diversity would make it very
difficult to firmly establish a causal relationship between cellular
processes affected by CsA and
Na+-K+ pump function.
However, we will briefly consider some of the mechanisms that might be involved.
CsA can activate protein kinase C both in vitro (Haller et al., 1991)
and in vivo (Demeule et al., 1994). Protein kinase C, in turn, can
inhibit the sarcolemmal
Na+-K+ pump in vivo
(Mihailidou et al., 2000b). In vivo treatment with CsA results in an
increase in levels of angiotensin II (Müller-Schweinitser, 1988)
and aldosterone (Siegl et al., 1983). These hormones inhibit the rabbit
sarcolemmal Na+-K+ pump in
vivo (Hool et al., 1996; Mihailidou et al., 2000a). Although effects of
CsA usually are attributed to an interaction with intracellular messenger mechanisms a direct effect on membrane structure and function
should also be considered. CsA is markedly lipophilic and readily
partitions into membranes. This influences the lateral organization of
lipids and hence the fluidity of membranes in vitro (Söderlund et
al., 1999). Such physical effects may influence Na+-K+ pump function.
Implications of Na+-K+ Pump Inhibition
during Treatment with CsA and FK506.
Figure 4B suggests the
CsA-induced pump inhibition saturates when
[K]pip approaches levels for intracellular
K+ found in quiescent cardiac tissue (Baumgarten
et al., 1981). However, our studies on intact, quiescent papillary
muscles indicate that a CsA-induced increase in
aiNa does occur. In the beating
heart the intracellular Na+ concentration is
expected to be higher than in the quiescent heart (Cohen et al., 1982),
whereas the converse should apply for the concentration of
K+. The effect of CsA on
Na+-K+ pump function may
therefore be more pronounced in vivo than our measurements of
aiNa suggest.
). Such down-regulation may contribute to a CsA-induced
reduction in cytosolic Ca2+ transients and
contractility in cardiac tissue. A decrease in sarcoplasmic reticulum
Ca2+ content due to sustained CsA-induced
Ca2+ leakage may also contribute to this negative
inotropic effect of CsA (Janssen et al., 2000). However, the multiple
nonspecific effects of CsA on cellular processes suggest that other
mechanisms of importance for cellular Ca2+
handling and contractility also contribute to effects of CsA on contractility.
It has been proposed that the calcineurin-inhibiting effect of CsA may
be beneficial in the treatment of cardiac hypertrophy (Olson and
Molkentin, 1999). However, a CsA-induced increase in the intracellular
Na+ concentration in the heart may contribute to
the increase in intracellular Ca2+ levels that
are believed to play a role in the cardiotoxicity of CsA (Park et al.,
1999, and references therein). Pump inhibition also activates reactive
oxygen species and key growth-related genes in isolated cardiac
myocytes (Kometiani et al., 1998). This may contribute to myocyte
hypertrophy (Huang et al., 1997). Pump inhibition can also have adverse
effects in intact animals. Treatment with cardiac glycosides in vivo
has been shown to accentuate histological abnormalities in the
myocardium in an experimental model of cardiomyopathy (Ahmad and Bloom,
1989).
If CsA were to be used in the treatment of cardiac hypertrophy adverse
effects of pump inhibition might counterbalance any beneficial effects
on the hypertrophic process. In support of these reservations,
prevention of hypertrophy by CsA is not a universal finding (Walsh,
1999) and may even enhance hypertrophy in vivo (Semsarian et al., 2000)
and enhance the susceptibility to congestive heart failure (Meguro et
al., 1999). It has been suggested that FK506 may be a useful
alternative to CsA in the treatment of cardiac hypertrophy. However,
although FK506 at the serum levels used for immunosuppression did not
induce Na+-K+ pump
inhibition in our study it should be kept in mind that doses proposed
to be used for the prevention of cardiac hypertrophy are much higher
(Crabtree, 1999). Possible adverse effects related to
Na+-K+ pump inhibition may
therefore be similar to those encountered with CsA.
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Acknowledgments |
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Novartis Pharmaceuticals (AG Basel, Switzerland) and Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan) kindly provided CsA and FK506, respectively.
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Footnotes |
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Accepted for publication January 23, 2001.
Received for publication October 19, 2000.
This study was supported by the North Shore Heart Research Foundation. M.M. was a recipient of a Postgraduate Medical Research Scholarship from the National Heart Foundation of Australia. Part of this work is presented in abstract form in Circulation (1999) 100 (Suppl):I-223.
Send reprint requests to: Dr. Mahidi Mardini, Department of Cardiology, Royal North Shore Hospital, Pacific Highway, St. Leonards, Sydney, New South Wales, Australia 2065.
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Abbreviations |
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CsA, cyclosporine; Na+-K+-ATPase, enzymatic equivalent of Na+-K+ pump; Ip, Na+-K+ pump current; Vm, test potential; [Na]pip, pipette Na+ concentration; [K]pip, pipette K+ concentration; Ih, holding current; Ip-Vm, pump current-voltage relationship.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-subunit isoform gene expressions in cardiac myocytes by ouabain and other hypertrophic stimuli.
J Mol Cell Cardiol
29:
3157-3167[Medline].
regulates cardiac Na+-K+ pump function in hyperaldosteronemia (Abstract).
Circulation
102 (Suppl II):
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) and rabbits treated with CsA (
)
is shown. Error bars for some measurements are contained within the
symbols. The numbers in parentheses indicate the number of cells.
Asterisks indicate statistically significant differences (unpaired
Student's t test followed by Bonferroni procedure).
).