Chung-Ho Chang

Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone

NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant‐derived naphthoquinone, has been shown to exert anticarcinogenic and anti‐atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox‐4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox‐4 activity in HEK293 and LN229 cells using lucigenin‐dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox‐4 in a time‐ and dose‐dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH‐ and NADH‐dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox‐cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox‐4 transfected COS‐7 cells. These results indicated that plumbagin directly interacted with Nox‐4 and inhibited its activity.

Cyclosporin A Disrupts Bradykinin Signaling Through Superoxide

Abstract—Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 &mgr;mol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O2− was not significantly reduced by 10 &mgr;mol/L oxypurinol or 30 &mgr;mol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 &mgr;mol/L) as well as the O2− scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 &mgr;mol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O2− scavenger Tiron (10 mmol/L), concomitant with 1 &mgr;mol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O2−. Incubation with 100 &mgr;mol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O2− and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO−).

Role of Phospholipase A2 Isozymes in Agonist-Mediated Signaling in Proximal Tubular Epithelium

Angiotensin II in proximal tubule epithelium is known to stimulate the release of arachidonic acid after stimulation of phospholipase A2 (PLA2) independent of phospholipase C-mediated signaling. Furthermore, an angiotensin II type 2 receptor subtype has been linked to this signaling cascade. We investigated the regulation and differential stimulation of PLA2s by comparing the PLA2 activities associated with the membranes and cytosol of rabbit renal proximal tubular epithelial cells after stimulation with angiotensin II, epidermal growth factor, and bradykinin. Both fractions demonstrated PLA2 activity that was dithiothreitol insensitive, required micromolar concentrations of Ca2+ for optimal activity, and was inhibited in a dose-dependent manner by an antiserum to a cytosolic PLA2 with a molecular mass of 85 kD. However, membrane-associated PLA2 did not demonstrate significant substrate specificity, whereas 1-steroyl-2-[14C]arachidonylphosphatidyl choline was the preferred substrate for cPLA2. An antiserum generated against mastoparan, a known PLA2 activator, inhibited membrane- but not cytosol-associated PLA2 activity. Membrane fractions showed a broad pH range (7.5 to 8.5) for optimal PLA2 activity, whereas cytosol was maximum at pH 9.5. Angiotensin II stimulated membrane-associated PLA2 activity by 88%, whereas bradykinin and epidermal growth factor inhibited activity by 54% and 41%, respectively. The three agonists stimulated cPLA2. Moreover, angiotensin II-induced activation of membrane-associated PLA2 preceded the activation of cPLA2. These results demonstrate differential localization and regulation of proximal tubular epithelial PLA2 isozymes, which may determine the pattern of subsequent arachidonic acid metabolism by the cytochrome P450 system.

Quercetin, a phytoestrogen and dietary flavonoid, activates different membrane‐bound guanylate cyclase isoforms in LLC‐PK1 and PC12 cells

Accumulated evidence suggests that quercetin, a dietary flavonoid, has beneficial effects in protection against cardiovascular diseases and in the inhibition of tumour growth. We have recently shown that antioxidants such as 17β‐estradiol, resveratrol, dithiothreitol and vitamin C activate membrane‐bound guanylate cyclase GC‐A, a receptor for atrial natriuretic factor (ANF). Since quercetin is a phytoestrogen and potent antioxidant, it is possible that it may activate GC‐A or other guanylate cyclase isoforms. We examined whether quercetin activates GC‐A or GC‐B (the receptor for C‐type natriuretic peptide, CNP) in PC12 and porcine kidney proximal tubular LLC‐PK1 cells. The results showed that quercetin activated a guanylate cyclase isoform in both cell types. Quercetin inhibited CNP‐stimulated GC‐B activity, but had little effect on ANF‐stimulated GC‐A activity in PC12 cells, suggesting that quercetin mainly activates GC‐B in PC12 cells. In contrast, CNP had no effect on guanylate cyclase activity in LLC‐PK1 cells, indicating that GC‐B is not expressed in LLC‐PK1 cells. Furthermore, quercetin had a small effect on ANF‐stimulated GC‐A activity and had no effect on soluble guanylate cyclase activity in LLC‐PK1 cells, suggesting that quercetin does not activate GC‐A, GC‐B or soluble guanylate cyclase in LLC‐PK1 cells. However, quercetin did stimulate membrane‐bound guanylate cyclase activity in LLC‐PK1 cell membranes. These results indicate that quercetin activates the GC‐B isoform in PC12 cells, but activates an unknown membrane‐bound guanylate cyclase isoform in LLC‐PK1 cells.

17β-Estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells

Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17beta-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17beta-estradiol decreases cGMP levels in PC12 cells. 17beta-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17beta-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17beta-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17beta-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17beta-estradiol inhibits soluble guanylate cyclase activity through SHP-1.

Cyclophilin A Functions as an Endogenous Inhibitor for Membrane-Bound Guanylate Cyclase-A

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A, is a cis-trans–peptidyl-prolyl isomerase (PPIase). It accelerates the cis-trans isomerization of prolyl-peptide bonds. CypA binds and regulates the activity of a variety of proteins. Atrial natriuretic factor (ANF) and its receptor membrane-bound guanylate cyclase-A (GC-A) are involved in the regulation of blood pressure. We examined whether CypA affects the activation of GC-A by ANF. The results showed that CypA associated with GC-A. Interestingly, binding of ANF to GC-A released CypA. Transfection of CypA inhibited ANF-stimulated GC-A activity, indicating that CypA functions as an endogenous inhibitor for GC-A activation. CypA also inhibits the activity of guanylate cyclase-C (GC-c), the catalytic domain of GC-A, indicating that CypA interacts with the catalytic domain of GC-A. In contrast, transfection of CypA R55A, a CypA mutant expressing low PPIase activity, did not significantly attenuate the activity of GC-c and the activation of GC-A. Inhibition of PPIase activity of CypA with cyclosporin A also blocks the inhibitory effect of CypA on GC-c activity. These results demonstrate that PPIase activity is required for CypA to inhibit GC-c activity and GC-A activation by ANF. Furthermore, mutation of Pro 822, 902, or 958 in GC-c abolished its activity. Therefore, it is likely that CypA binds to GC-A and catalyzes the cis-trans isomerization of Pro 822, 902, or 958, which keeps GC-A in the inactive state, and that binding of ANF to GC-A alters the conformation of the catalytic domain that releases CypA from GC-A leading to enzyme activation.

Mutational Inactivation of the Catalytic Domain of Guanylate Cyclase-A Receptor

Guanylate cyclase-A, the receptor for atrial natriuretic factor, contains a protein kinase-like domain and a catalytic domain in the intracellular region. To investigate the active site (the catalytic cavity) of guanylate cyclase-A, we amplified the catalytic domain plus three amino acids from the kinase-like domain of guanylate cyclase-A (GC-c) with polymerase chain reaction (PCR) and expressed it in Escherichia coli. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Results of cDNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutated protein. The mutated GC-c bound to GTP-agarose as well as the wild-type protein, indicating that the binding capability of mutated GC-c to GTP is not significantly affected by the Arg substitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weight complex. Since mutation of Leu 817 to Arg abolishes the catalytic activity, Leu 817 is likely located near the active site of guanylate cyclase-A. These results demonstrate that the carboxyl fragment of guanylate cyclase-A is an ideal system for studying the active site of guanylate cyclase-A.

Structural requirements of ATP for activation of basal and atrial natriuretic factor-stimulated guanylate cyclase in rat lung membranes

ATP has been reported to increase basal and atrial natriuretic factor (ANF)-stimulated guanylate cyclase activity. The structural features of ATP involved in the activation of guanylate cyclase were examined by employing a variety of ATP analogs with modification either at the phosphate chain or at the ribose moiety. Among the natural adenine nucleotides, ATP and ADP were able to increase both basal and ANF-stimulated guanylate cyclase activities in rat lung membranes. AMP had no effect. ATP was more effective than AMPPCP (the non-hydrolyzable analog of ATP), and ADP was more effective than ADP beta S and AMPCP (the hydrolysis-resistant analogs of ADP) to increase basal and ANF-stimulated guanylate cyclase activities. Removal of the oxygen atom from the ribose moiety of ATP or ADP significantly reduced their potency. Thus, the length of the phosphate chain and the hydroxyl groups at the ribose moiety are both determinants for nucleotide mediated guanylate cyclase activation.

The bradykinin/soluble guanylate cyclase signaling pathway is impaired in androgen-independent prostate cancer cells

The activation of soluble guanylate cyclase by bradykinin and sodium nitroprusside (SNP), a direct activator of soluble guanylate cyclase, was evaluated in androgen-sensitive LNCaP and androgen-independent PC3 and DU145 prostate cancer cells. Bradykinin and SNP activated soluble guanylate cyclase in LNCaP cells, but not in PC3 and DU145 cells. Western blot analysis revealed that the bradykinin B2 receptor, Gqalpha, phospholipase Cgamma and endothelial nitric oxide synthase were expressed in LNCaP, PC3 and DU145 cells. However, both Western blotting and reverse transcriptase--polymerase chain reaction indicated that soluble guanylate cyclase was only expressed in LNCaP cells. These results demonstrate that the impaired bradykinin-soluble guanylate cyclase pathway in PC3 and DU145 cells is likely due to lack of expression of soluble guanylate cyclase.

Antioxidants, vitamin C and dithiothreitol, activate membrane-bound guanylate cyclase in PC12 cells

Antioxidants and antioxidant enzymes are known to protect against cell death induced by reactive oxygen species. However, apart from directly quenching free radicals, little is known about the effect of antioxidants on hormone‐activated second messenger systems. We previously found that antioxidants such as 17‐β estradiol and resveratrol activate membrane‐bound guanylate cyclase GC‐A, the receptor for atrial natriuretic factor (ANF), in PC12 cells. It is possible that other antioxidants may also activate membrane‐bound guanylate cyclase GC‐A. The aim of this study was to determine if dithiothreitol (DTT), vitamin C, and vitamin E activate membrane‐bound guanylate cyclase GC‐A in PC12 cells. The results showed that both DTT and vitamin C increased cGMP levels in PC12 cells, whereas vitamin E had no effect. DTT and vitamin C inhibited membrane‐bound guanylate cyclase activity stimulated by ANF in PC12 cells. In contrast, DTT and vitamin C had no effect on soluble guanylate cyclase activity stimulated by substance P. Furthermore, NO synthase inhibitors L‐NAME and aminoguanidine did not affect DTT‐ and vitamin C‐stimulated guanylate cyclase activity. The results indicate that DTT and vitamin C, but not vitamin E, activate membrane‐bound guanylate cyclase GC‐A in PC12 cells.