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J Am Soc Nephrol 14:1435-1442, 2003
© 2003 American Society of Nephrology

Cyclosporine A Amplifies Ca2+ Signaling Pathway in LLC-PK1 Cells through the Inhibition of Plasma Membrane Ca2+ Pump

Vincenzo Calderaro*, Mariarosaria Boccellino*,{dagger}, Giovanni Cirillo*, Lucio Quagliuolo*,{dagger}, Domenico Cirillo* and Alfonso Giovane*,{dagger}

*Institute of Internal Medicine and Nephrology, and {dagger}Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy.

Correspondence to Dr. Vincenzo Calderaro, Institute of Internal Medicine and Nephrology, 2nd University of Naples, Nuovo Policlinico, Pad. 17, Via S. Pansini 5, 80123 Naples, Italy. Phone: 81-566-6667; Fax: 81-566-6666;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca2+ signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1; the assay of the in vitro activity of the plasma membrane Ca2+ pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 µM did not change the basal level of intracellular calcium ([Ca2+]i), whereas it affected the [Ca2+]i response to thapsigargin (TG), a powerful inhibitor of microsomal Ca2+ pump. In control studies, 5 µM TG produced a biphasic response: [Ca2+]i peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca2+]i, which remains above basal. However, it became evident that CsA strengthened the Ca2+ response to TG because the addition of 5 µM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca2+]i = 156 ± 4.84 versus 130 ± 3.28 nmol, mean ± SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 µM calmodulin (CaM), a significant decrease of plasma membrane Ca2+ pump (PMCA) activity (46.96 ± 0.26 versus 53.48 ± 1.96 nmol · mg of protein-1 · min-1, n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immunosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca2+ export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca2+]i response after exposure to agents that trigger intracellular Ca2+ release. The increased cell sensitivity during Ca2+ signaling events ensuing from the impairment of this "defense system" may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA. E-mail: vincenzo.calderaro@unina2.it


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. The importance of cyclosporine-induced nephrotoxicity has increased as the drug, originally introduced as an immunosuppressive agent for organ transplantation, has gained wider acceptance as a treatment for a variety of diseases, including some nephropathies, lupus, and other autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity, arterial hypertension, and cancer progression (4,17). Angiotensin II was first implicated as a prime mediator of CsA nephrotoxicity in 1991 (22); since then, a large array of experimental data has shown that the renin angiotensin system plays an important role in the development of CsA-induced toxicity (2,37). Interestingly, other studies emphasized the potential role of different mediators, prostaglandins, endothelins, nitric oxide, TGF-{beta}, etc. (13,21,24,29,34). However, despite these efforts, the information gathered at the present from the various experimental approaches cannot be assembled in an unifying view of the CsA-induced nephrotoxicity.

A growing volume of evidence suggests that the immunosuppressive effects as well as the main side effects of CsA might reflect extensive changes of cell calcium pathway. For example, antigen presentation to the T cell receptor triggers a cascade of Ca2+-dependent signal-transduction events that culminate in lymphokine-gene expression and T cell proliferation (10). This hypothesis becomes more consistent when considering the large distribution and physiologic roles of calcineurin/nuclear factor of activated T cells (NF-AT) system and the emerging role for the cyclophilins. Expression of the antigen-regulated, cyclosporine A-sensitive NF-AT, in fact, is not restricted to lymphoid cells as initially thought, because in cultured vascular muscle cells phospholipase C-coupled receptors stimulate NF-AT-mediated transcription (6). However, our knowledge of the molecular basis underlying the CsA effects on Ca2+ signaling pathway is incomplete and complicated by the observation that CsA may affect all the branches of the Ca2+ messenger system. An early contribution to the recognition of the role of Ca2+ signaling pathway in the generation of toxic effects of CsA came from the demonstration that CsA augmented the receptor stimulated calcium fluxes in isolated hepatocytes (27) and increased [Ca2+]i in mesangial cells (39). CsA may also affect the extracellular domain of Ca2+ signaling pathway; the incubation of different cell types with therapeutic doses of CsA is associated with the potentiation of the calcium response upon Ca2+-coupled receptor engagement (21,24), probably through an upregulation of receptor transcription (2). In addition, CsA may alter the control of intracellular Ca2+ release through ryanodine binding modifications in rat heart (30), and it may stimulate or inhibit the release of Ca2+ from IP3 -sensitive stores (14,26). This is not surprising if we consider that cyclophilin, the intracellular target of CsA, is a retained component of endoplasmic reticulum (1) that, once bound to CsA, may influence the proper conformation of IP3 receptors. Lastly, CsA can inhibit the permeability of the transition pore (PTP) of inner mitochondrial membrane, which participates in the regulation of intracellular Ca2+ signaling through a slow Ca2+ release (12).

The present study, carried out in intact cells and membranes of LLC-PK1 cell line, examined the role of CsA on Ca2+ signaling pathway. The results provided evidence that: (1) CsA did not affect the resting [Ca2+]i levels, but it amplified and strengthened the [Ca2+]i response after the exposure to intracellular Ca2+ mobilizing agents; (2) this effect was associated with a reduction of the Ca2+ export mediated primarily by the inhibition of PMCA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Line
LLC-PK1 cells (porcine proximal tubule cells; American Type Culture Collection, Rockville, MD) were cultured in plastic six-well tissue culture plates (Costar, Cambridge, MA) in minimal essential medium alpha supplemented with 10% fetal bovine serum (Life Technologies BRL, Gaithersburg, MD) under a humidified 95% air-5% CO2 atmosphere at 37°C.

LLC-PK1 Membrane Preparation
The preparation of membrane was carried out using the procedure described by Wiener et al. with minor modifications (44). Briefly, 100 to 120 x 106 cells were homogenized at 4°C by 40 strokes in 20 ml of HEPES-MEM containing 0.2 M sucrose, 100 µg/ml soybean trypsin inhibitor, and 100 µg/ml leupeptin, in a Potter apparatus at 1000 rpm using a Teflon pestle (tube volume, 10 to 50 ml). The homogenate was subjected to low-speed differential centrifugation at 800 x g for 15 min. Centrifugation (at 70,000 x g for 35 min) of the supernatant containing the crude membrane fraction produced a soluble fraction and the pellet containing the crude membrane fraction, which was further subfractioned by isopycnic centrifugation. The crude membranes were resuspended at 5 to 10 mg of protein/ml in 250 mM sucrose, 5 mM HEPES-Tris, pH 7.4, by 40 strokes in a Dounce homogenizer. Preparative subfractionations were obtained with a step gradient of 8 ml of 16% sucrose, 25 ml of crude membrane fraction made up to 38% by adding 43% sucrose solution, and 6 ml of 43% sucrose cushion, centrifuged for 14 hr at 75,000 x g. The fraction collected at 38/43% interface was further purified by centrifugation at 70,000 x g in Ficoll 400 for 20 min. Each tube contained a 3-ml sample layer, 2 ml of 6% (wt/vol) Ficoll 400 in 250 mM sucrose, 5 mM HEPES-Tris, pH 7.4, and a 5-ml 25% sucrose cushion. After centrifugation, the 3-ml sample and a small band at the sample/Ficoll interface (containing endoplasmic reticulum and Golgi membranes, as assessed by marker enzyme analysis) were discarded. The membrane fraction trapped within the Ficoll barrier was collected, diluted fivefold with medium, and centrifuged at 70,000 x g for 35 min. The pellet, resuspended in medium to 12 to 15 mg of protein/ml by repeated passage through a 25-gauge needle, was frozen in liquid nitrogen and stored at -80°C until used. Protein concentration was measured by the method of Bradford (7), using bovine serum albumin as the standard.

Galactosyl transferase activity was measured by the method described by Bergeron et al. (5) to evaluate the presence of Golgi apparatus, and the extent of contamination with endoplasmic reticulum was judged on the basis of NADPH-cytochrome c oxidoreductase activity measured in freshly prepared fractions by the method of Beaufay et al. (3). The identification of membrane domain was performed by assaying the Na+/K+-ATPase activity as the rate of inorganic phosphate released in the medium as reported by Mircheff and Wright (25). Table 1 summarizes the specific activities of marker enzymes in membrane vesicles, compared with those measured in whole-cell homogenates.


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  		Table 1. Marker enzyme activities of LLC-PK1 membranesa
 
PMCA Assay
The Ca2+-dependent ATPase activity of membrane was assayed after the procedure previously described with modifications (8). Briefly, membrane vesicles (1.5 mg of protein/ml) were incubated at 25°C for 4 min in medium (final volume, 1 ml) containing 100 mM KCl, 0.5 mM MgCl2, 0.5 mM MgATP, 20 mM HEPES, pH 7.5, 0.2 mM NADH, 1.1 mM EGTA, 10 µM TG, 10-4 mM ouabain, CaCl2 in appropriate amounts (approximately 0.8 mM total CaCl2) to yield a free Ca2+ concentration of 7.5 µM (36), 2 µM A23187, 1 mg/ml digitonin, 10 mM phosphoenolpyruvate, 18 IU/ml purified pyruvate kinase, and 18 IU/ml lactate dehydrogenase (Sigma). Assays were started by addition of 0.5 mM MgATP, and the rate of ATP hydrolysis was calculated from spectrophotometric recordings of the oxidation of NADH at 340 nm, using the molar absorption coefficient of NADH of 6.22 x 103 M-1 · cm-1 (38). The enzymatic activity was measured in the presence and absence of 10 µg/ml bovine brain calmodulin. PMCA activity was expressed as nmol · mg of protein-1 · min-1. Basal ATPase (or Mg2+-ATPase) activity was measured in assay medium containing 4 mM EGTA without added Ca2+; the Ca2+-dependent ATPase was determined from the difference between total ATPase (7.5 µM Ca2+ in the assay medium) and basal ATPase activities. Pilot experiments showed that under these conditions the addition of Triton X-100 (0.005%) did not affect Ca2+-ATPase activity.

Single Cell [Ca2+]i Measurement
LLC-PK1 cells grown on coverslips were loaded for 60 min at room temperature with the incubation medium containing 8 µM fura-2/AM and 0.025% Pluronic F-127 dissolved in DMSO, final concentration of which was 0.25%. After the incubation period, the coverslips with attached cells was mounted in a Teflon chamber (bath volume, 1.5 ml; flow rate, 9 ml/min to assure a rapid exchange of applied solutions) and perfused continuously on the stage of a Nikon Diaphot epifluorescence microscope (Nikon, Tokyo, Japan) equipped with a 40x Neofluor objective (1.3 NA) coupled to a Spex Fluorolog II dual excitation spectrofluorometer (Spex Industries Inc., Edison, NJ). The excitation wavelengths were 340 and 380 nm, and fluorescence emission was monitored at 510 nm. All experiments were performed at room temperature, and experimental agents were applied by exchanging the contents of the microscope chamber. Cells were calibrated for Ca2+ measurement by obtaining a maximum signal using the calcium ionophore ionomycin (final concentration, 10 µM), followed by a minimum signal using EGTA (final concentration, 4 mM) and Ca2+ calculated; after correction for autofluorescence (<10% of absolute photon counts), fluorescence intensity ratios (340/380) were converted to Ca2+ as described by Grynkiewicz et al. (16).

Assessment of Ca2+ Influx
Entry of Ca2+ was estimated by using the fura-2 fluorescence Mn2+ quenching technique. Briefly, cells were loaded as for [Ca2+]i measurements and fura-2 fluorescence monitored at excitation wavelength of 360 nm, at which level Ca2+ changes did not alter fura-2 fluorescence; fluorescence changes were therefore caused only by Mn2+ quenching. Then, 0.5 mM MnCl2 was added and its entry into cells measured as rate of fluorescence decrease during the first 2 min after its addition.

Reagents
All the chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Fura-2/AM and Pluronic F-127/AM were purchased from Molecular Probes (Eugene, OR). Cyclosporine A and H were gifts of Novartis Pharma (Basel, Switzerland). Stock solutions of CsA and CsH were prepared at a concentration of 10-2 M in ethanol.

Statistical Analyses
Student’s paired t test was used for paired-matched controls; otherwise, Student’s unpaired t test was used. A P value of < 0.05 was assumed to be significant. All values are expressed as mean ± SEM unless otherwise specified.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CsA did not have effects on resting [Ca2+]i levels at CsA concentrations comprised between 0.1 to 2 µM (data not shown). We then investigated whether CsA affected the [Ca2+]i pattern response to intracellular Ca2+ mobilizing agents, such as the tumor promoter sesquiterpene lactone TG. In Ca2+ containing medium, 5 µM TG produced a biphasic response: cytosolic Ca2+ peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca2+]i, which remains above basal levels (lower curve of Figure 1). However, it was found that CsA did not influence the [Ca2+]i peak response to TG, but it significantly affected the following sustained phase. As shown in Figure 1 (upper curve), the addition of 5 µM TG to cells preincubated with 400 nM CsA for 10 min led to a potentiation of plateau phase after the [Ca2+]i peak response; [Ca2+]i was 156 ± 4.84 after 300 s-TG addition in CsA-treated cells versus 130 ± 3.28 nmol in control cells stimulated with equimolar amounts of TG in the absence of CsA (mean ± SEM, n = 6, P < 0.001). This effect became evident when the cells were exposed to CsA concentration of 400 nM, and it was maximal after incubation with 1 µM CsA (data not shown). To closely examine whether the amplification of the sustained phase after CsA application ensued from Ca2+ influx stimulation or inhibition of Ca2+ extrusion mechanisms, we looked at the effects of 400 nM CsA on Mn2+ binding and quenching of fura-2 fluorescence. Mn2+ can traverse Ca2+ channels and its influx rate through Ca2+ channels is proportional to the Ca2+ influx rate (18,23,35). Mn2+ binding to fura-2 rapidly quenches fura-2 fluorescence; on these basis, when extracellular Mn2+ is added to fura-2 loaded cells, the rate of decline in fura-2 fluorescence is an index of the Mn2+ influx rate; as such, it represents a good estimate of the Ca2+ influx rate.



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  		Figure 1. Effects of Cyclosporine A (CsA) on thapsigargin (TG)-induced intracellular calcium ([Ca2+]i) increase. Cells grown on coverslips and kept under identical experimental conditions were loaded with fluorescent dye as described in Materials and Methods. (Lower curve) A typical [Ca2+]i profile induced by 5 µM TG under control conditions. (Upper curve) Application of 400 nM CsA for 10 min resulted in an amplification of the [Ca2+]i sustained phase. Both traces are representative of at least six experiments.

 
As shown in Figure 2, the rate of Mn2+ influx was low in unstimulated cells, meanwhile the stimulation of LLC-PK1 cells with 5 µM TG increased the fura-2 fluorescence quenching rate, thus revealing an increase in Ca2+ influx that was insignificantly affected (approximately 5%) by the pretreatment for 10 min with 400 nM CsA. It follows that the CsA amplification of Ca2+ response to TG might depend on changes in Ca2+ entry rate only to a minimum degree.



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  		Figure 2. Effect of CsA on TG-stimulated Mn2+ influx in LLC-PK1 cells. Cells were prepared and loaded as described in Materials and Methods. Excitation wavelength was set at 360 nm and emission at 510 nm. At 0 sec (arrow), 0.5 mM MnCl2 was added to cells incubated for 2 min with 5 µM TG in the presence or absence of 400 nM CsA, and the resulting change in 360 nm fluorescence was measured at 1-s interval. The fluorescent pattern in control cells is shown in the upper curve. Traces are representative of four paired experiments.

 
All these experiments focused on the possibility that the maintenance of an elevated plateau phase after CsA addition was mainly mediated by inhibition of Ca2+ efflux. Ca2+ export from the cell occurs via stimulation of plasma membrane Ca2+ ATPase or Na/Ca exchanger. This latter operates in reverse mode, depending on the net electrochemical force on the exchanger, and therefore represents a basic mechanism involved in cell sodium and calcium homeostasis in cells where it is expressed. The Na/Ca exchanger expression is controlled and regulated under the influence of different promoters in a tissue-specific fashion (20); e.g., in mammalian heart cells the Na/Ca exchanger represents the main Ca2+ extrusion pathway, while in erythrocytes this role is mainly accomplished by the PMCA. It is therefore expected that the influence of the exchanger on Ca2+ homeostasis depends on cell types. However, the finding that CsA impairs the exchanger activity in concanavalin A-stimulated T lymphocytes (19) suggested the possibility that effects of CsA on Ca2+ efflux in LLC-PK1 cells could be mediated by exchanger imbalance. A set of experiments was then planned to ascertain the role of the exchanger on Ca2+ export in LLC-PK1 cells. As shown in Figure 3, addition of 5 µM TG to LLC-PK1 cells resulted in a sustained elevation in [Ca2+]i under conditions of complete inhibition of [Ca2+]i extrusion via the exchanger produced by the removal of extracellular Na+ (42); the outcome of this protocol clearly discloses a significant contribution of Na/Ca pathway to Ca2+ homeostasis in LLC-PK1. On the other hand, the amplification of Ca2+ response after the incubation with 400 nM CsA for 10 min still persisted under these conditions, suggesting that the effects of CsA on Ca2+ signaling do not involve the exchanger activity.



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  		Figure 3. Effects of isotonic substitution of extracellular Na+ with choline on [Ca2+]i upon stimulation with 5 µM TG. Cells were loaded with fluorescent dye as described in Materials and Methods and then bathed with Na+-free medium. 5 µM TG were added after a 10-min incubation in Na+-free medium. Each bar represents the mean ± SEM of six cell calcium measurements taken at 250 s from TG addition. * significantly different (P < 0.002 or less) from the control group (only TG).

 
On this basis, it is conceivable that the actions of CsA are mediated through an effect on Ca2+ efflux via the plasma membrane Ca2+-ATPase. To address this issue, we examined the effects of CsA on PMCA activity in membranes of LLC-PK1 cells. As shown in Figure 4, the preincubation with CsA at doses between 0.2 and 2 µM for 10 min produced a 9.6 to 69.5% decrease of PMCA activity (46.96 ± 0.26 and 16.3 ± 0.60, respectively, versus 53.5 ± 1.96 in control group, nmol · mg of protein-1 · min-1 mean ± SEM, n = 6, P < 0.02 and 0.001), which was substantially unaffected by longer incubations. It should be emphasized that the incubation with 200 and 2000 nM CsA analog, D-MeVal-11-CS (CsH), was associated with a similar reduction of the PMCA activity; PM Ca2+ pump activity was, on the other hand, increased of approximately 11% after 10 min incubation with TG alone (59.8 ± 0.49).



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  		Figure 4. Effect of CsA and CsH on plasma membrane Ca2+ pump (PMCA) activity of LLC-PK1 membranes activated by 10 µM CaM. 1.5 mg/ml of membranes of LLC-PK1 cells were pre-incubated for 10 min with appropriate amounts of test agents. The samples were subjected to PMCA assay as described in Materials and Methods. Each bar represents the mean ± SEM of six paired experiments done in triplicate. * Significantly different (P < 0.02 or less) from control.

 
The enzymatic activity measured in the presence of exogenous CaM (10 µg/ml) showed a progressive decrease as the CsA concentration increased (Figure 5). We next studied the dependence of the enzyme activity on the phosphorylating substrate concentration under different experimental conditions. As evidenced in Figure 6, the enzyme activity did not show a significant dependence on the ATP concentration in the range 0.5 to 2 mM, irrespective of the presence of CaM or CsA, either alone or on a combined basis. It is then evident that CsA does not compete with the ATP binding sites because the inhibition is not reversed by increasing the ATP concentration. Lastly, the possibility was evaluated that the effects of CsA on PMCA activity were due to an inhibition of the stimulation by CaM. We therefore examined the effect of increasing medium CaM concentration in the presence and absence of the minimum inhibiting CsA concentration (200 nM); as reported in Figure 7, the maximal stimulation of PMCA, obtained at 10 µg/ml CaM under control conditions, shifted to 15 µg/ml in the presence of CsA.



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  		Figure 5. Inhibition of LLC-PK1 PMCA activity by CsA. The reaction mixture, prepared as described in Materials and Methods, was incubated with the appropriate amounts of CsA for 10 min. Experiments were carried out at 37°C in the presence of 10 µg/ml calmodulin. Each point represents the mean ± SEM of four experiments.

 


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  		Figure 6. The dependence of PMCA activity as a function of ATP concentration in the presence of 10 µg/ml CaM ({blacktriangleup}), 200 nM CsA plus 10 µg/ml CaM ({blacksquare}), 200 nM CsA (•), and in the absence of CaM and CsA. ({blacktriangledown}). Each point represents the mean ± SEM of four experiments.

 


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  		Figure 7. The dependence of PMCA activity as a function of CaM concentration in the presence (+) or absence (-) of 200 nM CsA. Each point represents the mean ± SEM of five experiments. * Significantly different (P < 0.02 or less) from the paired control in the absence of CsA.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The bulk of data arising from this study addresses a model of CsA toxicity mainly based on the changes in Ca2+ signaling. The chain of events activated by CsA may be summarized as follows: (1) inhibition of the PMCA activity; (2) a consequent decrease of Ca2+ export rate and, (3) amplification of the [Ca2+]i response to Ca2+-coupled receptor activation.

As shown in Figure 1, CsA did not affect the early Ca2+ response to TG, whereas it modestly stimulated the sustained component of TG-induced Ca2+ response. In intact cells, the tumor promoter TG mimics the most distal steps of [Ca2+]i response to extracellular Ca2+ receptors activation; it inhibits microsomal Ca2+-ATPase, thus increasing [Ca2+]i levels and promoting Ca2+ influx with no activity on the inositol lipids pathway (41). The [Ca2+]i increase brought about by TG displays, in fact, two phases: an early, rapid spike-like inositol-1,4,5-trisphosphate (InsP3) sensitive [Ca2+]i release followed by a secondary, sustained elevation of [Ca2+]i, resulting predominantly from Ca2+ entry across the plasma membrane. According to Putney’s proposal of "capacitative model" (32), this second phase of [Ca2+]i response derives from an increased Ca2+ permeability of plasma membrane induced by depletion of intracellular Ca2+ stores. On this basis, the sustained, late component of calcium response could be considered as the result of a dynamic balance between Ca2+ influx and efflux (41). In keeping with these observations, we found an increased Ca2+ entry rate during the sustained component of [Ca2+]i response to TG, as shown in Figure 2. To address the question as to whether the CsA amplification of the sustained phase to TG exposure arose from an increase of Ca2+ entry or a decrease of Ca2+ export rate, we measured the Ca2+ entry rate by Mn2+ binding and quenching of fura-2 fluorescence. The rationale of these experiments was that if CsA stimulated Ca2+ influx, then a further increase in Ca2+ entry rate was expected. However, a striking result became apparent; as shown in Figure 2, cells stimulated with 5 µM TG in the presence of 400 nM CsA did not display different Mn2+ quenching rate from the those exposed to equimolar amounts of TG in the absence of CsA. It follows that the amplification of the late phase of Ca2+ response after CsA application was not due to stimulation of Ca2+ influx, but rather to a dampening of Ca2+ extrusion mechanisms.

Finally, it could be demonstrated that the inhibition of Ca2+ extrusion rate by CsA was mediated by the inhibition of PMCA. As shown in Figure 4, the addition of CsA to incubation medium resulted in a significant inhibition of PMCA. Was this effect mediated by the same mechanisms leading to immunosuppression in T cells? When inside the T cell CsA, in fact, binds to cyclophilins A, a member of immunophilins, a family of ubiquitous cytosolic proteins provided with isomerase activity; the complex CsA-Cyclophilin A then forms a ternary complex with calcineurin, thereby inhibiting its phosphatase activity and hence the transcriptional activation of genes encoding cytokines through inhibition of nucleus entry of NF-AT (33). Two lines of evidence support the observation that, at least within the limits of the short-term experimental design and the cell line used, the mechanisms leading to immunosuppression in T cells do not account for the abnormal Ca2+ handling in LLC-PK1 cells: (a) CsH, a derivative devoid of immunosuppressive activity (28), is also able to inhibit to a similar extent the PMCA; (b) PMCA inhibition occurs in 10 min, a time that is too short to involve a genomic response. As shown in Figure 6, the incubation of membrane preparations with calmodulin resulted in approximately 2.5-fold increase in PMCA activity. The stimulating effect of calmodulin on the enzyme activity is now well established: an autoinhibitory domain of about 9 kD, located at the C-terminus of the enzyme, is removed upon calmodulin binding, leaving the substrates free access to its active site (9). It is also evident that the main physiologic consequences of CsA on Ca2+ signaling become manifest only upon the apparent affinity of PMCA for CaM; interestingly, this observation adequately explains why the addition of CsA to intact cells did not affect basal [Ca2+]i but it modified Ca2+ levels under conditions of maximal PMCA rate, as it occurs during the secondary, sustained phase following TG stimulation or Ca2+ receptor activation.

In the present study, the CsA inhibited the PMCA at concentration (200 nM) lower than that required to affect [Ca2+]i response to TG in intact cells (400 nM). The reason for such a discrepancy is unclear, but we may speculate that, in consideration of the lipid solubility of CsA, the different experimental settings may influence its partitioning process into the lipid components of cellular membranes (43).

The evidence accruing from characterization of the inhibition suggests that it could be mediated at the calmodulin binding domain level. As shown in Figure 7, the apparent affinity of PMCA for CaM is influenced by the presence of CsA; it is therefore conceivable that the drug affects the CaM binding to the enzyme. The finding that the enzyme inhibition took place in a preparation deprived of cell organelles suggests that, within the limits of the experimental model adopted, the effects of CsA on calmodulin domain of the enzyme are direct; this leaves open the question of whether these effects are mediated by changes of the affinity sites for calmodulin or nonspecific conformational disturbance induced by CsA. On the other hand, it cannot be excluded that more complex mechanisms might be involved in intact cells; e.g., CsA and/or its non-immunosuppressant analogues affect several intracellular proteins, the functions of which have yet to be fully defined (11). In addition, it must be considered that the observed effects of CsA on Ca2+ signaling pertain to an epithelial cell line in which the response could be different from non-polarized cells, such as mesangial or smooth muscular cells, that are also targets of CsA toxicity. However, with these limitations in mind, the effects of CsA on Ca2+ extrusion mechanisms might have a remarkable physiologic significance. The increased Ca2+ efflux rate during the sustained phase of receptor-activated Ca2+ response is mainly accomplished by triggering plasma membrane Ca2+ pump which thus appears to be the major mechanism of Ca2+ export from the non-excitable cells (15). The active extrusion of Ca2+ across the basolateral membrane is related to several diverse functions of structurally polarized epithelial cells. In non-excitable cells, PMCA represents a mechanism for the fine tuning of [Ca2+]i over the long term, because it is the only component that can move Ca2+ between the cell and the semi-infinite Ca2+ reservoir of the extracellular space (31).

If one views the Ca2+-ATPase as a defense system (40) that reestablishes basal [Ca2+]i after a Ca2+ signaling event has occurred, it is then conceivable that a substance such as CsA that slows the recovery of the basal [Ca2+]i may have a significant impact on cellular processes such as cell division and growth, secretion, inflammation, etc.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication June 24, 2002. Accepted for publication February 19, 2003.





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