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The Journal of Immunology, 1998, 161: 6122-6127.
Copyright © 1998 by The American Association of Immunologists

Cyclosporin A Inhibits Inositol 1,4,5-Trisphosphate Binding to Its Receptors and Release of Calcium from Intracellular Stores in Peritoneal Macrophages1

Uma K. Misra, Govind Gawdi and Salvatore V. Pizzo2

Department of Pathology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have studied the effects of the immunosuppressive drug cyclosporin A (CsA) on the generation of inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ levels elicited upon ligation of murine macrophage receptors for {alpha}2-macroglobulin, bradykinin, epidermal growth factor, and platelet-derived growth factor. Preincubation of cells with CsA (500 ng/ml), either alone or with the various ligands, did not inhibit the synthesis of IP3. However, we observed 70–80% inhibition of the binding of [3H]IP3 to IP3 receptors on macrophage membranes isolated from CsA-treated macrophages. Preincubation of macrophages with CsA abolished IP3-mediated release of Ca2+ from intracellular stores and Ca2+ entry from the extracellular medium observed when macrophage receptors were stimulated with ligands in the absence of CsA. Preincubation of macrophages with CsA also significantly inhibited DNA synthesis induced by ligands for all four receptors studied. Thus in macrophages, as in T cells, CsA blocks receptor-activated signal transmission pathways characterized by an initial increase in intracellular Ca2+ concentration. This inhibition appears to result from a drug effect on IP3 receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The structurally different immunosuppressant agents cyclosporin A (CsA)3 and FK506 are front line drugs used to prevent graft rejection following organ transplantation (see Refs. 1–3 for review). These drugs block T cell function by preventing transcriptional activation of genes that encode the T cell growth factor IL-2 and other immunologically important T cell-derived lymphokines following antigenic stimulation (1, 2, 3). Triggering of the T cell Ag receptor as well as ligation of many other receptors, including the {alpha}2-macroglobulin signaling receptor ({alpha}2MSR) (4, 5), bradykinin receptor (BKR) (6, 7), epidermal growth factor receptor (EGFR) (8, 9), and platelet-derived growth factor receptor (PDGFR) (10, 11), results in activation of tyrosine kinase as well as rapid hydrolysis of membrane phosphatidyl inositol 4,5-bisphosphate. This activation generates inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. The latter is important because of its role in activating protein kinase C (12). IP3 binds to intracellular IP3 receptors (IP3R), changes their conformation, and opens the Ca2+ channel causing an increase in intracellular Ca2+ [Ca2+]i (13). Receptor-mediated Ca2+ release from internal stores is often followed by Ca2+ influx across the plasma membrane (14, 15). The mechanism of this capacitative Ca2+ entry is not fully understood but is thought to involve a novel diffusible activator, a G protein, tyrosine kinase, cGMP, and direct coupling between proteins of the endoplasmic reticulum and the plasma membrane (14, 15). Intracellular Ca2+ release is predominantly mediated by the ryanodine receptor (RyR) and IP3R (13, 16, 17). These Ca2+ release channels display extensive amino acid homology and functional similarities. Prolonged elevation of [Ca2+]i is linked to IL-2 gene expression and to DNA synthesis in activated T cells (16, 17).

In T cells, CsA and FK506 inhibit signal transmission pathways from the cell surface to the nucleus that are characterized by an initial increase in [Ca2+]i (1, 2, 3). CsA and FK506 bind to their cognate intracellular receptor immunophilins, cyclophilin and FKBP12, respectively (1, 2, 3). The complexes of CsA-cyclophilin and FK506-FKBP12 bind to calcineurin, a Ca2+/calmodulin-dependent serine/threonine phosphatase, which plays a critical role in signaling pathways necessary for T cell activation. This complex results in inhibition of its phosphatase activity, impairing the translocation of nuclear transcription factors from the cytosol to the nucleus and the formation of a functional transcription complex, which in part regulates the expression of cytokine genes (1, 2, 3, 18). The binding site for the NF-AT transcription complex is present within the enhancer sequences of several genes and is required for their regulation by the antigen receptor or by agents that both mobilize Ca2+ and activate protein kinase C (1, 2, 3, 18). CsA and FK506 also block the production of TNF-{alpha} by B cells (19). In mast cells, CsA and FK506 block degranulation as well as transcriptional activation of IL-3 and IL-5 and genes involved in leukotriene synthesis (20, 21, 22). In monocyte/macrophages, CsA inhibits LPS-induced expression of tissue factor both at the transcriptional and functional levels, as well as translocation of NF-{kappa}B (23).

Colocalization of FKBP12 with calcineurin in brain (1, 2, 3, 16), association of FKBP12 with the Ca2+ release channels RyR and IP3R, suggests that immunophilins play a direct role in signal transduction (24, 25, 26, 27, 28). Cyclophilin and FKBP12 possess cis/trans-peptidylprolyl isomerase activity that is inhibited by binding of CsA and FK506, respectively (1, 2, 3, 29, 30). Cyclophilins aid in protein folding, and they increase folding rates of human carbonic anhydrase, ribonuclease Ti, and collagen in vitro (31, 32, 33). Cyclophilins may also act as chaperones (34), chemotactic agents (35), and stress response proteins (36). CsA binds to the p55 Gag protein of the human immunodeficiency virus (37). In the yeast two hybrid system, FKBP12 has been found in association with the TGF-ßR (38).

We have studied the effects of CsA in murine peritoneal macrophages with respect to the second messengers elicited upon ligation of {alpha}2MSR with the {alpha}2M cloned and expressed receptor binding fragment (RBF), of BKR with bradykinin, of EGFR with EGF, and of PDGFR with PDGF Ab. We report here that in macrophages, CsA treatment before the addition of agonists does not affect IP3 generation, but it inhibits the binding of [3H]IP3 to IP3R. CsA also inhibits IP3-mediated release of Ca2+ from intracellular Ca2+ stores and entry of Ca2+ from the medium. Finally, CsA inhibits RBF-, EGF-, and PDGF-induced DNA synthesis in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Endotoxin-free RBF was cloned, expressed, and purified as described previously (4, 5). Culture media were purchased from Life Technologies (Grand Island, NY). Fatty acid free BSA, bradykinin, EGF, CsA, and eiconozole were purchased from Sigma (St. Louis, MO). PDGF AB was procured from R&D Systems (Minneapolis, MN). Fura 2/AM and BAPTA/AM were obtained from Molecular Probes (Eugene, OR). Myo-[2-3H]inositol (specific activity, 10–20 Ci/mmol) and [3H]thymidine (specific activity, 70 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were the highest purity grade commercially available.

Isolation of peritoneal macrophages

Pathogen-free C57B1/6 mice (6 wk old) were obtained from Charles River Laboratories (Raleigh, NC). Thioglycollate-elicited macrophages were routinely obtained by peritoneal lavage with HBSS containing 10 mM HEPES (pH 7.2) and 3.5 mM NaHCO3 (HHBSS) (4, 5). The cells were washed once with HHBSS, suspended in RPMI 1640 medium containing 2 mM glutamine, 12.5 U/ml penicillin, 6.25 µg/ml streptomycin, and 5% FCS, and plated at cell density of 5 x 105/cm2 for Ca2+ measurements, or 2–4 x 106/well in 6-well plates for other studies. The macrophages were incubated for 2 h at 37°C in a humidified CO2 (5%) incubator. The nonadherent cells were removed by washing with HHBSS as above, and the adherent cells were incubated in RPMI 1640 medium under the specified conditions.

Measurement of IP3 and [Ca2+]i

Changes in IP3 and [Ca2+]i resulting from exposure of thioglycollate-elicited murine peritoneal macrophages to RBF (50 pM) bradykinin (50 nM), EGF (200 ng/ml), and PDGF Ab (50 ng/ml) in the absence and presence of CsA were measured as described previously (4, 5). [Ca2+]i was studied by digital imaging microscopy. The effect of RBF, bradykinin, EGF, and PDGF Ab on IP3 synthesis was determined by employing myo-[2-3H] inositol as the substrate (4, 5). Ligand concentrations were identical to those used to study [Ca2+]i.

[3H]IP3 binding to IP3R

Binding of [3H]IP3 to IP3R in microsomal preparations from thioglycollate-elicited macrophages was studied according to the methods used for brain microsomal preparation as described previously (39).

Macrophages (10–12 x 106 cells) were incubated in a CO2 (5%) incubator for 16–18 h at 37°C in RPMI 1640 medium containing 1 mM glutamine, 12.5 U/ml penicillin, 6.5 µg/ml streptomycin, and 5% FBS. Monolayers were washed three times with cold HHBSS and treated with either buffer or CsA (500 ng/ml) for 16 min at 37°C. To the cells was added a volume of buffer containing 50 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, and 10 µM leupeptin (pH 8.3). The cells were scraped off the tissue culture wells into tubes maintained at 4°C. The cell suspension was then passed through a sterile 27.5-gauge needle 40–45 times on ice and the lysate centrifuged at 289,000 x g for 30 min at 4°C. The pellet was suspended in another volume of the Tris-HCl buffer described above, and the protein content was measured (40). The binding of [3H]IP3 (New England Nuclear (Boston, MA); specific activity, 21 Ci/mmol) to membranes from buffer-treated or CsA-treated cells (~50 µg protein in a volume of 100 µl) was studied in 1.5 ml Eppendorf tubes at 4°C for 10 min under the desired conditions. After incubation, the tubes were centrifuged in an Eppendorf microcentrifuge for 4 min at 4°C and the pellet washed four times with centrifugation in the Tris-HCl buffer described above. The pellet was dissolved in a volume of 1 N NaOH, and radioactivity was determined by liquid scintillation counting. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled IP3, which was then subtracted from total binding to give specific binding.

Measurements of DNA synthesis

DNA synthesis in macrophages was essentially measured as described previously (41). Briefly, thioglycollate-elicited macrophages (4 x 106 cells) were incubated in RPMI 1640 medium containing 1 mM glutamine, 12.5 U/ml penicillin, 6.5 µg/ml streptomycin, and 0.1% fatty acid free BSA for 2 h at 37°C in a humidified CO2 (5%) incubator. The monolayers were washed three times with ice-cold HHBSS and a volume of RPMI medium added. To each well was added [3H]thymidine (2 µCi/well) followed by the addition of the various ligands. The cells were incubated for 20 h as above, and the incubations were terminated by aspirating the medium. A volume of cold TCA (5%) was added to each well and the plate left on ice for 30 min. TCA was removed and cells washed once more with TCA (5%) followed by three washings with cold HHBSS. The cells were lysed in 1 N NaOH, and radioactivity was determined by liquid scintillation counting. When the effects of CsA on DNA synthesis was studied, it was added 15 min before the addition of ligands and it was present during the incubation. In experiments where internal Ca2+ levels were manipulated by thapsigargin (100 nM for 20 min at 37°C, which depletes both IP3-sensitive and IP3-insensitive intracellular Ca2+ stores), BAPTA/AM (10 µM for 30 min at 37°C, a chelator of intracellular Ca2+) or eiconozole (10 µM for 30 min at 37°C, which prevents entry of Ca2+ through plasma membrane), these agents were added before CsA (500 ng/ml) and RBF (50 pM) in that order and were present during the incubation. Other details for measuring DNA synthesis under these conditions were the same as described above. For protein measurements, cells were incubated identically, but untreated, were washed and dissolved in 0.1 N NaOH, and protein was estimated according to Bradford (40).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CsA and generation of IP3

Treatment of macrophages with CsA alone showed very little effect on the generation of IP3 (Fig. 1GoA). CsA also did not inhibit the generation of IP3 in macrophages stimulated with RBF, bradykinin, EGF, and PDGF (Fig. 1Go). These results show that CsA does not affect receptor-stimulated second messenger events at the cell surface. Receptor-activated increase in IP3 rapidly declines due to its further metabolism either by a phosphatase or kinase or both (42). In cells, however, to which CsA was added before RBF, bradykinin, EGF, and PDGF, respectively, the levels of IP3 remained significantly elevated compared with CsA untreated cells. These results suggest that CsA impairs the metabolism of IP3. The difference in the elevation caused by bradykinin in cells treated with CsA as compared with untreated cells is less significant. The reason for this difference is unclear. From the mechanistic standpoint RBF, like EGF and PDGF, functions like a growth factor (4, 5), which is not the case with bradykinin. Thus RBF, EGF, and PDGF may activate different signaling mechanisms downstream from IP3 generation.



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  		FIGURE 1. CsA and IP3 generation in macrophages. Details of measurements of IP3 in [3H]myoinositol-labeled macrophages (2 x 106 cells/well) stimulated with various agonists preincubated with or without CsA (500 ng/ml) for 15 min at 37°C have been described previously (4, 5). Values are mean ± SEM from two independent experiments performed in duplicate and are expressed as % change over basal value at zero time. A, Changes in IP3 levels induced by RBF (50 pM) (•), CsA ({triangleup}), and CsA + RBF ({circ}). B, Bradykinin (50 nM)-induced IP3 generation (•), CsA({triangleup}), CsA + bradykinin ({circ}). C, Changes in IP3 levels induced by EGF (200 ng/ml) (•), CsA ({triangleup}), CsA + EGF ({circ}). D, PDGF (50 ng/ml)-induced changes in IP3 levels (•), CsA ({triangleup}), and CsA + PDGF ({circ}).

 
CsA and intracellular Ca2+ levels

The effect of CsA on increased [Ca2+]i levels observed in macrophages after stimulation with RBF, bradykinin, EGF, and PDGF is shown in Fig. 2Go. CsA itself showed very little effect on [Ca2+]i levels (Fig. 2GoA). RBF transiently increased [Ca2+]i levels by about 4-fold within 5–20 sec after stimulation over the basal value in 85–90% cells studied (150–160 cells in four independent experiments; Fig. 2GoA). Depletion of IP3-sensitive intracellular Ca2+ stores triggers capacitative Ca2+ entry across the plasma membrane, which sustains elevated [Ca2+]i levels (13, 14). We studied this Ca2+ entry under our experimental conditions by adding 1 mM Ca2+ to the medium (Fig. 2GoA). Consistent with our previous studies (43), addition of Ca2+ 5 min after RBF stimulation further increased [Ca2+]i levels by 2- to 3-fold in 85–90% of the cells (Fig. 2GoA). Incubation of macrophages with CsA for 15 min at 37°C before the addition of RBF completely inhibited both IP3-mediated release of Ca2+ from intracellular Ca2+ stores and capacitative Ca2+ entry in 80–85% of cells studied (120–130 cells in four independent experiments; Fig. 2GoA).



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  		FIGURE 2. CsA and intracellular Ca2+ levels. Details of measurements of Ca2+ in Fura-2/AM loaded macrophages have been described previously (4, 5). Change shown in [Ca2+]i under the experimental conditions is representative of four independent experiments for each agonist with or without preincubation with CsA (500 ng/ml for 15 min), in low Ca2+ (75 µM) HBSS containing 10 mM HEPES, pH 7.2 and 3.5 mM NaHCO3, using about 110–160 cell in each case. A, Changes in [Ca2+]i induced by RBF (50 pM) (•), CsA + RBF ({circ}), or CsA (500 ng/ml) alone ({triangleup}). B, Changes in [Ca2+]i induced by bradykinin (50 nM) (•), and CsA + bradykinin ({circ}). C, Changes in [Ca2+]i induced by EGF (200 ng/ml) (•), and CsA + EGF ({circ}). D, Changes in [Ca2+]i induced by PDGF (50 ng/ml) (•), and CsA + PDGF ({circ}). Arrows indicate the time of additions.

 
Ligation of BKR on macrophages with bradykinin transiently increased [Ca2+]i levels by about 3-fold over the basal value within 5–20 sec after stimulation in 75–80% of the cells (100–120 cells in four individual experiments). Addition of 1 mM Ca2+, 4 min after bradykinin, caused a further sustained increase in [Ca2+]i levels in 80–85% cells studied (Fig. 2GoB). Incubation of macrophages with CsA, drastically inhibited both the IP3-mediated increase in [Ca2+]i as well as Ca2+ entry in 75–80% of the cells (100–110 cells in four experiments; Fig. 2GoB). We also studied the effects of CsA on IP3 induction mediated by EGF and PDGF as well as capacitative entry of [ Ca2+]i. Both these growth factors increased IP3-mediated and capacitative entry-mediated increase in [Ca2+]i in 80–85% of the cells (120–130 cells in four separate experiments in each case; Fig. 2Go, C and D). Incubation of macrophages with CsA before the addition of EGF or PDGF drastically inhibited both the IP3-mediated and capacitative entry-mediated increase in [Ca2+]i in 70–80% of the cells (100–120 cells in four independent experiments; Fig. 2Go, C and D).

CsA and [3H]IP3 binding to IP3R

The data presented above indicate that CsA does not prevent the generation of IP3 in macrophages on ligation of {alpha}2MSR, BKR, EGFR, and PDGFR (Fig. 1Go); however, it does nearly abolish IP3-mediated transient release of Ca2+ from intracellular Ca2+ stores (Fig. 2Go). One possible explanation for these observations is that CsA may prevent the binding of IP3 to IP3R and the opening of Ca2+ channels. Therefore, we next studied the binding of [3H]IP3 to IP3R in membrane preparations from macrophages treated with either buffer or CsA. We have shown previously that the binding of [3H]IP3 in macrophage macrosomal preparations is concentration-dependent, and [3H]IP3 binds to a single class of IP3 binding sites with a Kd of 4.3 ± 0.6 pM (39). The binding of [3H]IP3 to IP3R in membranes prepared from CsA-treated cells was reduced by 70–80% compared with buffer-treated controls (Fig. 3Go).



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  		FIGURE 3. CsA and binding of [3H]IP3 to IP3R in macrophage microsomal preparations. Details for measuring binding are described in Materials and Methods. Values are the mean ± SEM from three experiments performed in quadruplicate and are expressed as fmol bound/mg protein. Data are shown for [3H]IP3 binding to buffer-treated cells (•) and to CsA-treated cells ({circ}).

 
CsA and agonist-induced DNA synthesis

In view of the results described above and the known regulatory role of elevated [Ca2+]i in gene expression, we studied the effects of CsA on DNA synthesis (Fig. 4Go). Stimulation of macrophages with RBF, EGF, and PDGF caused a 2- to 3-fold increase in DNA synthesis (Fig. 4Go). Incubation of macrophages with CsA before the addition of the respective ligands inhibited DNA synthesis by 50–90%, depending on the ligand (Fig. 4Go).



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  		FIGURE 4. CsA and DNA synthesis. Details for measuring DNA synthesis in macrophages preincubated with buffer or CsA (500 ng/ml/15 min) and then stimulated with different ligands are described in Materials and Methods. Valus are the mean ± SEM from two experiments performed in quadruplicate and are expressed as fmol DNA synthesis/mg cell protein. A, The symbols are buffer (lane 1), RBF (lane 2), and CsA + RBF (lane 3). B, The symbols are buffer (lane 1), EGF (lane 2), and CsA + EGF (lane 3). C, The symbols are buffer (lane 1), PDGF (lane 2), and CsA + PDGF (lane 3).

 
Modulations of [Ca2+]i levels and DNA synthesis

The effects of modulating [Ca2+]i levels in macrophages was studied in three ways: 1) with thapsigargin a depletor of Ca2+ from intracellular IP3-sensitive and IP3-insensitive Ca2+ stores; 2) BAPTA/AM, a chelator of intracellular Ca2+; and 3) eiconozole, an inhibitor of Ca2+ entry from the medium (44). In each study, these agents were added to the cells before addition of CsA followed by RBF and incubation for 20 h at 37°C (Fig. 5Go). Thapsigargin by itself slightly increased macrophage DNA synthesis; however, when thapsigargin was added 20 min before CsA and RBF to cells, it resulted in a further reduction of DNA synthesis as compared with the decrease observed in cells treated with CsA with RBF (Fig. 5Go). Likewise, modulation of [Ca2+]i levels with BAPTA/AM or eiconozole before adding CsA and RBF further reduced DNA synthesis observed in cells treated with CsA plus RBF (Fig. 5Go).



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  		FIGURE 5. Modulations of [Ca2+]i and effect of CsA on RBF-induced DNA synthesis. Details of measuring DNA synthesis under the experimental conditions were the same as described in Materials and Methods. Values are mean ± SEM from two independent experiments performed in quadruplicate and are expressed as fmol DNA synthesis/mg cell protein. The symbols are buffer (lane 1), RBF (lane 2), CsA + RBF (lane 3), thapsigargin (lane 4), thapsigargin + CsA + RBF (lane 5), BAPTA/AM + CsA + RBF (lane 6), and eiconozole + CsA + RBF (lane 7).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary findings of this study with peritoneal macrophages are: 1) CsA at nontoxic doses does not inhibit IP3 generation consequent to ligation of {alpha}2MSR, BKR, EGFR, and PDGFR; 2) CsA inhibits the binding of [3HP]IP3 to IP3R; 3) CsA inhibits both the initial IP3-mediated release of Ca2+ from internal stores as well as capacitative Ca2+ entry; 4) CsA inhibits DNA synthesis observed in macrophages when these cells are stimulated with RBF, EGF, and PDGF, respectively; and 5) modulation of [Ca2+]i levels with thapsigargin, BAPTA/AM, or eiconozole further reduces DNA synthesis observed in cells treated with CsA before stimulation with RBF. These results show that signal transduction events generated upon cell surface receptor ligation are impaired by CsA in macrophages as well as in T-cells (2). They are the first observations suggesting that the action of CsA may involve a direct effect on IP3R.

There is a growing body of literature suggesting that capacitative Ca2+ entry into cells, as well as intracellular mobilization of Ca2+, affects many cellular functions requiring protein and DNA synthesis (14). Ca2+ mobilization has been implicated in lymphocyte and fibroblast mitogenesis, posttranslational processing and trafficking of newly synthesized lysosomal and membrane proteins, translocation of transcription factors from the cytoplasm to the nucleus, expression of cytokine genes, modulation of specific cell cycle events, and reinitiation of DNA synthesis (14). Addition of thapsigargin and di- t-butylhydroquinone to Swiss 3T3 cells at lower concentrations causes reinitiation of DNA synthesis in synergy with either phorbol 12,13-dibutyrate or bombesin (14, 18, 44, 45, 46, 47, 48, 49). At higher concentrations, these agents inhibit DNA and protein synthesis in smooth muscle cells, where cell growth has been linked to intracellular Ca2+ pool contents (44, 45, 46, 47, 48). Elevated [Ca2+]i pools have been reported to modulate DNA synthesis in rheumatoid synovial fibroblasts and murine macrophages on stimulation with RBF (41). In Jurkat T lymphocytes, which demonstrate defective capacitative Ca2+ entry, maintenance of elevated [Ca2+]i levels during early T cell activation is required for IL-2 gene induction (50). In several T cell lines and T lymphocytes, interruption of Ca2+ signaling by chelating extracellular Ca2+ in the absence of protein synthesis results in the export of nuclear transcription factors to the cytoplasm within 5 min (18).

IP3R mediates intracellular Ca2+ release elicited by hormones and neurotransmitters that bind to cell surface receptors to activated phosphatidylinositol-specific phospholipase C and generate IP3 (13, 42). While most of IP3R is associated with the endoplasmic reticulum, some may occur on plasma membranes and mediate Ca2+ entry (50, 51). Intracellular Ca2+ release is predominantly mediated by RyR and IP3R Ca2+ release channels (16, 24, 25, 26, 27, 28). FKBP12 is physiologically associated with the intracellular Ca2+ release channels, RyR and IP3R (28). Both of these channels are tetramers of four identical subunits each of 80 kDa and possess 40% homology in the transmembrane region (13, 52, 53). In preparations of IP3R, FKBP12 binds to it very tightly and FK506 and rapamycin dissociate this complex, making it more leaky and reduce Ca2+ accumulation (24). Recent studies demonstrate that calcineurin is physiologicly associated in a physical and functional complex with IP3R and FKBP12 (27). This association is disrupted by FK506 and rapamycin but not by CsA, suggesting that calcineurin is bound on IP3R at a site different from FKBP12 (27). In a IP3R complex, calcineurin retains its catalytic activity toward known substrates such as mitogen-activating protein-2 and it regulates the phosphorylation status of IP3R induced by Ca2+-activated kinases and phosphatases (27). Inhibition of calcineurin by CsA is a consequence of altering the phosphorylation status of IP3R and it will affect Ca2+ conductance. It would also inhibit calcineurin activity toward other cellular substrates such as NF-AT and thus alter Ca2+ fluxes and the resultant physiologic responses (27). The cis/trans-peptidylprolyl isomerase activity of immunophilins has been associated with the folding of proteins (31, 32, 33). Therefore, it is conceivable that this activity is involved in maintaining the proper conformation of the ion channels, RyR and IP3R. Inhibition of the prolyl isomerase activity of immunophilins by the binding of CsA might then alter the conformation of IP3R in a manner unfavorable to the binding of IP3, resulting in Ca2+ release channels which remain closed, thus preventing Ca2+ entry into the cytoplasm. The absolute magnitude of IP3 production consequent to receptor activation in CsA-treated and untreated macrophages was nearly comparable; however, there was a significant difference in that IP3 levels were much more sustained in CsA-treated cells. This potentiated and sustained increase in IP3 levels in CsA-treated cells compared with untreated cells may be consequent to the inhibition of inositol trisphosphate breakdown by phosphatases in CsA-treated cells. CsA is an inhibitor of calcineurin, a Ca2+-dependent phosphatase, thereby preventing the removal of phosphate groups from its substrates and thus impairing their biologic functions (1, 2, 3, 13, 16, 17, 18, 20, 24, 25, 26, 27).


   Footnotes
 
1 This work was supported by Grant HL-24066 from the National Heart, Lung, and Blood Institute. Back

2 Address correspondence and reprint requests to Dr. Salvatore V. Pizzo, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710. E-mail address: Back

3 Abbreviations used in this paper: CsA, cyclosporin; {alpha}2MSR, the {alpha}2-macroglobulin signaling receptor, BKR, bradykinin receptor; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; [Ca2+]i, intracellular Ca2+ concentration; RyR, ryanodine receptor; RBF, receptor binding fragment; BAPTA/AM, 1,2-(bis[2-aminophenoxy]ethane-N,N,N'N'-tetraacetic acetoxymethylester; Fura-2/AM, 1-[2-(5-carbo-xyoxazol-2-yl)-6-aminobenzofuran-5-oxyl-2]-2'amino-5-methyl-phenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxy methylester. Back

Received for publication May 5, 1998. Accepted for publication August 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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