Cyclosporin A Inhibits the Activation of Membrane-Bound Guanylate Cyclase GC-A of Atrial Natriuretic Factor via NAD(P)H Oxidase

Abstract

Cyclosporin A (CsA) is a common immunosuppressant wildly used in patients with organ transplant and autoimmune diseases; however, it can cause several adverse effects, such as nephrotoxicity and hypertension. The detailed mechanisms have not been completely understood. Atrial natriuretic factor (ANF) and its receptor (mGC-A) have been shown to play a crucial role in the regulation of blood pressure. Here, we investigated the effects of CsA on the activation of mGC-A in ANF-treated LLC-PK1 cells. In our study, ANF-induced mGC-A activities and superoxide generation in LLC-PK1 cells were measured by guanosine 3′,5′-cyclic monophosphate (cGMP) radioimmunoassay and lucigenin-dependent chemiluminescence, respectively. We found that CsA can reduce about 60% of mGC-A activities in ANF-treated LLC-PK1 cells. CsA is known to induce superoxide. Addition of superoxide generators menadione and diamide mimicked the effects of CsA, whereas DPI (a reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor) and Tiron (a superoxide quencher) blocked the suppressive effects of CsA on ANF-induced mGC-A activities. We previously showed that the catalytic domain of GC-A (GC-c) expresses guanylate cyclase activities. Addition of menadione, diamide, or peroxynitrite or transfection of Nox-4 NAD(P)H oxidase abolished GC-c activities. In conclusion, CsA inhibits ANF-stimulated mGC-A activities through superoxide and/or peroxynitrite generated by an NAD(P)H oxidase by interacting with the catalytic domain of mGC-A.

Introduction

Cyclosporin A (CsA) is an immunosuppressive agent that has been wildly used to improve the outcome of organ transplantation and certain autoimmune diseases. Nevertheless, chronic CsA treatment can cause nephrotoxicity and hypertension.^1,2) The molecular mechanisms leading to the development of these side effects are still unclear. Several pathways were demonstrated to account for the effects of CsA on hypertension including endothelin-1,^3,4) thromboxane,^4,5) free radical generation,^6–10) the renin-angiotensin system,¹¹⁾ the renal kallikrein-kinin system,^12,13) and the renal sensory nerve ending.¹⁴⁾

There are two forms of guanylate cyclases, soluble form (sGC) and membrane-bound receptor form (mGC). Nitric oxide (NO) and atrial natriuretic factor (ANF) can activate sGC and GC-A (one subtype of mGC), respectively. We have shown that CsA attenuates the activation of sGC through superoxide produced by reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in bradykinin-treated porcine proximal tubular epithelial cell line (LLC-PK1 cells).¹⁰⁾ Several transgenic and gene targeting studies also demonstrated that the blood pressure can be regulated by ANF/mGC-A system.^15–18) Furthermore, Kook et al reported that CsA treatment impairs mGC-A activation by inhibiting mGC-A activities in the glomerulus.¹⁹⁾ These findings suggested that CsA can affect the ANF/mGC-A system besides the NO/sGC pathway; nevertheless, the underlying mechanisms remain unknown. Here, we provide evidence that CsA activated NAD(P)H oxidase and produced superoxide/peroxynitrite in ANF-treated LLC-PK1 cells, and that the generated superoxide/peroxynitrite interacts with the catalytic domain of mGC-A and suppresses its activation.

Experimental

Materials

RPMI-1640 and Dulbecco’s modified Eagle’s medium (DMEM) medium were purchased from Gibco (Gaithersburg, MD, U.S.A.). Protein assay reagent was purchased from Bio-Rad (Hercules, CA, U.S.A.). Enhanced chemiluminescence kit was purchased from Amersham (Arlington Heights, IL, U.S.A.). Immobilon®-P polyvinylidene fluoride (PVDF) membrane and GenePORTER transfection reagent were purchased from Fisher Scientific (Hanover, IL, U.S.A.) and Genlantis (San Diego, CA, U.S.A.), respectively.

Measurement of Guanosine 3′,5′-Cyclic Monophosphate (cGMP)

LLC-PK1 and transfected cells were cultured in 6-well plates. After a wash step, these cells were incubated with DMEM medium containing 0.5 mM isobutylmethylxanthine at 37 °C for 10 min. Cells were treated with various concentrations of ANF and incubated for another 10 min. The levels of intracellular cGMP were measured by radioimmunoassay.^10,20)

Expression of the Catalytic Domain of GC-A (GC-c) and Nox-4 in COS-7 Cells

mGC-A contains an ANF-binding domain, a kinase-like domain and a catalytic domain. We previous showed that the catalytic domain plus three amino acids from the kinase-like domain of GC-A (242 amino acids) are capable of performing the catalytic function of guanylate cyclase.²¹⁾ The cDNA corresponding to this region (bp 2673 to 3401) of GC-A (GC-c) was inserted into pcDNA3.1.^21,22) COS-7 cells were transfected with plasmids (pcDNA3.1) containing GC-c, and/or NAD(P)H oxidase Nox-4 plasmids by the GenePORTER transfection reagent.^21–24) In control group, cells were transfected with pcDNA3.1 plasmid.

NAD(P)H Oxidase Assay

In addition, these cells were also treated with 10 µM NAD(P)H oxidase inhibitor (DPI), 30 µM ketoconazole, 10 µM oxypurinol, 10 mM Tiron for 24 h at 37 °C.¹⁰⁾ COS-7 cells and LLC-PK1 cells were treated with or without CsA (1 µM) at 37 °C for 24 h. The activities of NAD(P)H oxidase in whole cell lysates were determined by lucigenin-dependent chemiluminescence.¹⁰⁾ Chemiluminescent photoemission was analyzed using Lumat LB 9501 Luminometer in relative light units (RLU).^10,25)

Statistical Analysis

Data analysis was performed using the Student’s t-test. Error bars represent the standard deviations from the mean of four experimental replicates. A p-value less than 0.05 is considered statistically significant.

Results

Effects of Cyclosporin A (CsA) on mGC-A Activities in ANF-Treated LLC-PK1 Cells

We investigated whether CsA can affect mGC-A activation in LLC-PK1 cells. CsA-treated cells were incubated with various concentrations of ANF. The level of cGMP was used as an index of mGC-A activities. Figure 1(a) shows that CsA can decrease the production of cGMP by about 60% in ANF-treated LLC-PK1 cells.

Fig. 1. Effects of Cyclosporin A (CsA) on Atrial Natriuretic Factor (ANF)-Stimulated Guanylate Cyclase (GC-A) Activity in LLC-PK1 Cells

LLC-PK1 cells were incubated with 1 µM CsA for 14 h in the CO₂ incubator. (a) After incubation, cells were exposed to 0.5 mM isobutylmethylxanthine at 37 °C for 10 min and challenged with various concentrations of ANF for an additional 10 min. (b) After CsA treatment, cells were exposed to 0.5 mM isobutylmethylxanthine at 37 °C for 10 min, then incubated with 10 µM NAD(P)H oxidase inhibitor (DPI), 10 mM Tiron, 10 µM oxypurinol (oxy) or 30 µM ketoconazole (Ket) for 10 min and challenged with 0.1 µM ANF for additional 10 min. The reaction was terminated with 10% trichloroacetic acid. The generated cGMP was measured with the radioimmunoassay. The error bar represents the deviation from the mean of the four replicates. * p < 0.001 vs. control (a), and * p < 0.01, ANF/CsA vs. ANF/CsA/DPI and ANF/CsA vs. ANF/CsA/Tiron (b).

Effects of NAD(P)H Oxidase, CYP and Xanthine Oxidase Inhibitors on CsA-Mediated Suppression of mGC-A Activities in ANF-Treated LLC-PK1 Cells

NAD(P)H oxidase has been suggested to play a crucial role in cardiovascular disease.^26–28) We have shown that CsA stimulates the activation of NAD(P)H oxidase in LLC-PK1 cells.¹⁰⁾ To investigate whether NAD(P)H oxidase is involved in the suppressive effects of CsA on the activation of mGC-A, we examined the effects of DPI and superoxide quencher (Tiron) in ANF-treated LLC-PK1 cells. We found that DPI and Tiron had little effects on basal cGMP levels (data not shown); nevertheless, these two agents can restore the production of cGMP in LLC-PK1 cells treated with CsA and ANF (Fig. 1b). On the other hand, superoxide can be generated by the CYP and xanthine oxidase pathways.^29,30) We found that ketoconazole (CYP inhibitor, 30 µM) and oxypurinol (xanthine oxidase inhibitor, 10 µM) did not affect the inhibitory effects of CsA on mGC-A activities in ANF-treated LLC-PK1 cells (Fig. 1b). These results suggest that superoxide produced by NAD(P)H oxidase is involved in the suppressive effects of CsA on ANF-induced mGC-A activities.

Effects of Menadione and Diamide on ANF-Induced mGC-A Activities in LLC-PK1 Cells

Menadione and diamide can stimulate the activation of NAD(P)H oxidase and production of superoxide in several cell types.^31–34) To further examine whether superoxide produced by NAD(P)H oxidase mediates the suppressive effects of CsA on mGC-A activities, we measured the effects of menadione and diamide on ANF-treated LLC-PK1 cells. As shown in Fig. 2, these two agents can decrease the production of cGMP in ANF-treated LLC-PK1 cells. Thus, menadione and diamide mimic the inhibitory effects of CsA on ANF-induced mGC-A activities.

Fig. 2. Inhibition of Atrial Natriuretic Factor (ANF)-Stimulated Guanylate Cyclase (GC-A) Activity by Menadione and Diamide in LLC-PK1 Cells

LLC-PK1 cells were exposed to 0.5 mM isobutylmethylxanthine at 37 °C for 10 min, then incubated with various concentrations of menadione (a) or diamide (b) for 10 min and challenged with various concentrations of ANF for additional 10 min. The reaction was terminated with 10% trichloroacetic acid. The generated cGMP was measured with the radioimmunoassay. Both menadione and diamide inhibited ANF-stimulated GC-A activity. The error bar represents the deviation from the mean of the four replicates. * p < 0.001, menadione vs. control; * p < 0.01, diamide vs. control.

Inhibition of the Activities of the Catalytic Domain of mGC-A by Nox-4, Diamide, Menadione and Peroxynitrite in COS-7 Cells

mGC-A has a catalytic domain in the intracellular region. We previously showed that the catalytic domain of mGC-A (GC-c) expresses guanylate cyclase activities.^21,22) Renal NAD(P)H oxidase, Nox-4, has been shown to produce superoxide when expressed in NIH 3T3 cells.²³⁾ To determine whether superoxide produced by Nox-4 interacts directly with GC-c, we transfected catalytic domain with or without Nox-4 plasmids into COS-7 cells. The endogenous guanylate cyclase activities in COS-7 cells are very weak. In control group, cells were transfected with pcDNA3.1. One and half days after transfection, we measured the amounts of cGMP and the activities of NAD(P)H oxidase in these transfected cells. We found that expression of Nox-4 can increase the NAD(P)H oxidase activities (Fig. 3a). Furthermore, Expression of GC-c dramatically promoted the production of cGMP; however, co-expression of GC-c and Nox-4 markedly decreased GC-c activities (Fig. 3b).

Fig. 3. Inhibition of Guanylate Cyclase GC-c by Nox-4 in Transfected COS-7 Cells

COS-7 cells were transfected with GC-c with and without Nox-4 plasmids or control vectors by employing the GenePORTER transfection reagent. (a) Two days after transfection, the amount of superoxide release in control and transfected cells was determined by lucigenin-dependent chemiluminescence. (b) Two days after transfection, both transfected and control cells were exposed to 0.5 mM isobutylmethylxanthine at 37 °C for 10 min. The reaction was terminated with 10% trichloroacetic acid. Generated cGMP was measured by radioimmunoassay. The error bar represents the deviation from the mean of the four replicates. * p < 0.01 (a) and p < 0.001 (b), +Nox-4 vs. control.

To examine whether diamide and menadione mimic the effects of Nox-4, we examined the effects of these two agents on the activities of GC-c in transfected cells. As shown in Fig. 4a, menadione and diamide substantially inhibited the GC-c activities (Fig. 4a). These results indicate that the effects of CsA on mGC-A in LLC-PK1 cells is likely mediated by superoxide generated by NAD(P)H oxidase, and that superoxide interacts with the catalytic domain of mGC-A and suppresses its activities.

Fig. 4. Inhibition of the Activity of Guanylate Cyclase GC-c by Diamide, Menadione, and Peroxynitrite in Transfected COS-7 Cells

COS-7 cells were transfected with GC-c plasmids by employing the GenePORTER transfection reagent. Two days after transfection, cells were exposed to 0.5 mM isobutylmethylxanthine at 37 °C for 10 min, and various concentrations of menadione or diamide (a), or 10 µM peroxynitrite or degraded peroxynitrite (b) for another 10 min. The reaction was terminated with 10% trichloroacetic acid. Generated cGMP was measured by radioimmunoassay. The error bar represents the deviation from the mean of the four replicates.

Superoxide is known to interact with NO and produces peroxynitrite. In previous study, we showed that CsA can promote the production of peroxynitrite.¹⁰⁾ To examine whether peroxynitrite mimics the effects of Nox-4, diamide and menadione, we investigated the effects of peroxynitrite on GC-c activities in transfected COS-7 cells. Figure 4(b) shows that peroxynitrite can inhibit GC-c activities in transfected cells. These results indicate that superoxide and peroxynitrite interact with the catalytic domain of mGC-A and inhibit its activities.

Discussion

CsA is widely used in organ transplantation to reduce the rate of graft rejection.^35,36) However, chronic CsA treatment leads to side effects such as nephrotoxicity and hypertension.^35–37) The mechanisms underlying these side effects are not fully understood. We previously found that CsA impairs the bradykinin-stimulated activation of soluble guanylate cyclase (sGC) by quenching NO through superoxide produced by NAD(P)H oxidase in LLC-PK1 cells.¹⁰⁾ Here, we investigated the possible involvement of membrane-bound guanylate cyclase (mGC-A), another guanylate cyclase isoform, in the side effects of CsA in LLC-PK1 cells. The results indicate that superoxide and peroxynitrite generated by CsA treatment can suppress ANF-induced mGC-A activation through interacting with the catalytic domain of mGC-A.

CsA is known to induce oxidative stress that accounts for most of the CsA-induced toxicity.^6–9) The reactive oxygen species generated by CsA are superoxide anions which can be subsequently converted to hydrogen peroxide through the action of superoxide dismutase. Besides NAD(P)H oxidase, CYP has been suggested to be an enzyme activated by CsA to produce superoxide.³⁸⁾ However, in a recent study, we have shown that superoxide produced by CsA in LLC-PK1 cells is NADPH-dependent and not blocked by inhibitors for CYP and xanthine oxidase.¹⁰⁾ Krauskopf et al. also reported that free radical generated by CsA stimulation was not mediated by CYP.⁸⁾ Therefore, the superoxide producing system stimulated by CsA in LLC-PK1 cells is NAD(P)H oxidase. In the current study, we showed that DPI, a specific NAD(P)H oxidase inhibitor, blocked the suppressive effects of CsA on mGC-A activation in ANF-treated LLC-PK1 cells. On the other hand, inhibitors for CYP and xanthine oxidase could not reverse the suppressive effects of CsA on ANF-induced mGC-A activation. These results suggest that NAD(P)H oxidase mediates the inhibitory effects of CsA on mGC-A activation induced by ANF stimulation. Menadione has been shown to produce superoxide through an NAD(P)H oxidase in a variety of cell types such as platelets, neutrophils, B- and T-lymphoid cells and vascular smooth muscle cells.^30–32) Our study showed that addition of menadione mimicked the inhibitory effects of CsA on the mGC-A activation in ANF-treated LLC-PK1 cells. Consistently, addition of Tiron, a superoxide quencher, attenuated the suppressive effects of CsA on ANF-induced mGC-A activities. These results suggest that superoxide produced by NAD(P)H oxidase is involved in the inhibition of mGC-A activities by CsA in ANF-treated LLC-PK1 cells.

mGC-A has a catalytic domain and a kinase-like domain in the intracellular region. In previous studies, we showed that the catalytic domain of mGC-A (GC-c) is capable of expressing guanylate cyclase activity.^21,22) To determine whether NAD(P)H oxidase interacts with GC-c, we examined the effects of menadione and diamide on the activities of GC-c. The results showed that menadione and diamide substantially inhibited the activities of GC-c. There are five NAD(P)H oxidase isoforms.^26,27,38) Nox-1 and Nox-2 are expressed most in colon and phagocytes, respectively. Nox-3 is found mainly in embryonic kidney and Nox-5 in lymphoid organs and testis. Nox-4 is predominantly expressed in the adult and embryonic kidney. To examine whether Nox-4 can affect GC-c activities, we co-transfected GC-c with Nox-4 into COS-7 cells, and found that expression of Nox-4 inhibited GC-c activities, indicating that superoxide generated by Nox-4 interacts with the GC-c and inhibits its enzyme activation. Kook et al. reported that CsA treatment impairs mGC-A activation in the glomerulus by inhibiting mGC-A activities.¹⁹⁾ However, the underlying mechanisms were not known. Our data suggest that CsA treatment stimulates NAD(P)H oxidase and generates superoxide which in turn interacts with the GC-c leading to the inhibition of mGC-A activities.

The mechanism by which superoxide inhibits the catalytic activities of mGC-A remains to be explored. Superoxide is known to form peroxynitrite with NO. It has been demonstrated that CsA can promote the production of peroxynitrite.^8,10) In this study, we showed that peroxynitrite inhibited the activities of GC-c, suggesting that superoxide generated by Nox-4 may be converted into peroxynitrite that interacts with the GC-c leading to mGC-A inactivation. Peroxynitrite is a very strong oxidant that can nitrate tyrosine residues. This type of modification has been identified in patients with Alzheimer’s disease, Parkinson’s disease, chronic lung injury and acute and chronic kidney rejection.^39–43) Recently, we have shown that CsA increases the levels of nitrotyrosine on several proteins in LLC-PK1 cells, and that this increase can be reversed by the addition of the NAD(P)H oxidase inhibitor DPI and superoxide scavenger Tiron.¹⁰⁾ Amino acid analysis reveals that mGC-A contains 9 tyrosine residues in its catalytic domain. It is possible that the superoxide produced by CsA interacts with NO to form peroxynitrite which in turn nitrates tyrosine residues on the catalytic domain of mGC-A leading to a conformational change on mGC-A and an inhibition of the enzyme activation. Consistent with this speculation, we have found that mutation of Tyr 818 or Tyr 1017 into a Phe residue on the catalytic domain abolishes the enzyme activities of GC-c (unpublished results). Further experiments are required to confirm whether peroxynitrite nitrates tyrosine residues on the GC-c leading to its inactivation.

Conclusion

We have found that CsA suppresses the activation of mGC-A by ANF. We have shown that peroxynitrite and superoxide generated by menadione or diamide mimic the effect of CsA, and that the NAD(P)H oxidase inhibitor DPI and superoxide quencher Tiron block the suppressive effects of CsA on ANF-stimulated mGC-A activities. These results suggest that superoxide and/or peroxynitrite mediate the effects of CsA on the inhibition of ANF-stimulated mGC-A activities. Furthermore, the addition of peroxynitrite, menadione, or diamide as well as expression of the renal NAD(P)H oxidase (Nox-4) inhibits the activities of GC-c, indicating that superoxide and/or peroxynitrite generated by CsA interact with the catalytic domain of mGC-A. Thus, CsA suppresses mGC-A activities in ANF-treated LLC-PK1 cells likely through superoxide and/or peroxynitrite generated by Nox-4. Since the ANF/GC-A signaling pathway has been shown to play a curtail role in the regulation of blood pressure,^15–18) the impaired mGC-A activation by ANF may contribute at least partly to the hypertensive effects of CsA in organ transplant patients. Therefore, it is possible that restoring this ANF signaling pathway and the bradykinin/NO/soluble guanylate cyclase pathway¹⁰⁾ with antioxidants, NAD(P)H oxidase inhibitor, or superoxide/peroxynitrite scavenger may provide an opportunity for intervention.

Acknowledgments

This work was supported by grants from the Changhua Christian Hospital Research Foundation (107-2314-B-371-013, 108-2314-B-029-001, 109-CCH-IRP-028, and 110-CCH-MST-126) and the Taiwan Ministry of Science and Technology (MOST 110-2628-B-371-001 and MOST 111-2628-B-371-001).

Conflict of Interest

The authors declare no conflict of interest.

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