(1/122) Interleukin-1 stimulates Jun N-terminal/stress-activated protein kinase by an arachidonate-dependent mechanism in mesangial cells.
BACKGROUND: We have studied interleukin-1 (IL-1)-stimulated signals and gene expression in mesangial cells (MCs) to identify molecular mechanisms of MC activation, a process characteristic of glomerular inflammation. The JNK1 pathway has been implicated in cell fate decisions, and IL-1 stimulates the Jun N-terminal/stress-activated protein kinases (JNK1/SAPK). However, early postreceptor mechanisms by which IL-1 activates these enzymes remain unclear. Free arachidonic acid (AA) activates several protein kinases, and because IL-1 rapidly stimulates phospholipase A2 (PLA2) activity release AA, IL-1-induced activation of JNK1/SAPK may be mediated by AA release. METHODS: MCs were grown from collagenase-treated glomeruli, and JNK/SAPK activity in MC lysates was determined using an immunocomplex kinase assay. RESULT: Treatment of MCs with IL-1 alpha induced a time-dependent increase in JNK1/SAPK kinase activity, assessed by phosphorylation of the activating transcription factor-2 (ATF-2). Using similar incubation conditions, IL-1 also increased [3H]AA release from MCs. Pretreatment of MCs with aristolochic acid, a PLA2 inhibitor, concordantly reduced IL-1-regulated [3H]AA release and JNK1/SAPK activity, suggesting that cytosolic AA in part mediates IL-1-induced JNK1/SAPK activation. Addition of AA stimulated JNK1/SAPK activity in a time- and concentration-dependent manner. This effect was AA specific, as only AA and its precursor linoleic acid stimulated JNK1/SAPK activity. Other fatty acids failed to activate JNK1/SAPK. Pretreatment of MCs with specific inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase had no effect on either IL-1- or AA-induced JNK1/SAPK activation. Furthermore, stimulation of MCs with the exogenous cyclooxygenase-, lipoxygenase-, phosphodiesterase-, and epoxygenase-derived arachidonate metabolites, in contrast to AA itself, did not activate JNK1/SAPK. CONCLUSION: We conclude that IL-1-stimulated AA release, in part, mediates stimulation of JNK1/SAPK activity and that AA activates JNK1/SAPK by a mechanism that does not require enzymatic oxygenation. JNK1 signaling pathway components may provide molecular switches that mediate structural rearrangements and biochemical processes characteristic of MC activation and could provide a novel target(s) for therapeutic intervention. (+info)
(2/122) Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules.
We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoa). cGMP values ( approximately 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/ HCO3- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 microM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+/-]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity. (+info)
(3/122) Formation of reactive oxygen species by the contracting diaphragm is PLA(2) dependent.
Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. M. Francik, P. A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The intracellular pathways involved in this process are, however, unknown. The purpose of the present study was to test the hypothesis that contraction-related formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A(2) (PLA(2)). Studies were performed by using an in vitro hemidiaphragm preparation submerged in an organ bath, and formation of ROS in muscles was assessed by using a recently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations and then determined whether contraction-related ROS generation could be altered by administration of various PLA(2) inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA(2); arachidonyltrifluoromethyl ketone (AACOCF(3)), an inhibitor of 85-kDa PLA(2); and haloenol lactone suicide substrate (HELSS), an inhibitor of calcium-independent PLA(2). We found 1) little ROS formation [2.0 +/- 0.8 (SE) ng/mg] in noncontracting control diaphragms, 2) a high level of ROS (20.0 +/- 2.0 ng/mg) in electrically stimulated contracting diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s(-1)), 3) near-complete suppression of ROS generation in manoalide (3.0 +/- 0.5 ng/mg, P < 0. 001)- and aristolochic acid-treated contracting diaphragms (4.0 +/- 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF(3) or HELSS on ROS formation in contracting diaphragm. During in vitro studies examining fluorescent measurement of ROS formation in response to a hypoxanthine/xanthine oxidase superoxide-generating solution, manoalide, aristolochic acid, AACOCF(3), and HELSS had no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of inhibitors of the 14-kDa isoform of PLA(2) and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction. (+info)
(4/122) W-7 induces [Ca(2+)](i) increases in Madin-Darby canine kidney (MDCK) cells.
The effect of W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride] on Ca(2+) signaling in Madin-Darby canine kidney cells was investigated. W-7 (0.1-1 mM) induced a [Ca(2+)](i) increase, which comprised an initial increase and a plateau. Ca(2+) removal inhibited the Ca(2+) signals by 80%, suggesting that W-7 activated external Ca(2+) influx and internal Ca(2+) release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (2 microM) and the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished the internal Ca(2+) release induced by 0.5 mM W-7; conversely, pretreatment with W-7 prevented thapsigargin and carbonylcyanide m-chlorophenylhydrazone from releasing internal Ca(2+). W-7 (0.2 mM) induced Mn(2+) quench of fura-2 fluorescence, which was inhibited by La(3+) (0.1 mM) by 80%. La(3+) (0.1 mM) partly inhibited 0.2 mM W-7-induced [Ca(2+)](i) increase. Addition of 5 mM Ca(2+) induced a significant [Ca(2+)](i) increase after pretreating with 0.2 to 1 mM W-7 in Ca(2+)-free medium for 5 min, suggesting that W-7 induced capacitative Ca(2+) entry. W-7 (0.5 mM) potentiated the capacitative Ca(2+) entry induced by 1 microM thapsigargin by 15%. Pretreatment with aristolochic acid (40 microM) to inhibit phospholipase A(2) reduced 0.5 mM W-7-induced internal Ca(2+) release and external Ca(2+) influx by 25 and 80%, respectively. Inhibition of phospholipase C with U73122 (2 microM) or inhibition of phospholipase D with propranolol (0.1 mM) had no effect on the internal Ca(2+) release induced by 0.5 mM W-7. It remains unclear whether W-7 induced [Ca(2+)](i) increases via inhibition of calmodulin. Three other calmodulin inhibitors (phenoxybenzamine, trifluoperazine, and fluphenazine-N-chloroethane) did not alter resting [Ca(2+)](i). (+info)
(5/122) Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene.
The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA. (+info)
(6/122) Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2.
Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists. (+info)
(7/122) Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus.
The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium. (+info)
(8/122) Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi)
BACKGROUND: Chinese-herb nephropathy is a progressive form of renal fibrosis that develops in some patients who take weight-reducing pills containing Chinese herbs. Because of a manufacturing error, one of the herbs in these pills (Stephania tetrandra) was inadvertently replaced by Aristolochia fangchi, which is nephrotoxic and carcinogenic. METHODS: The diagnosis of a neoplastic lesion in the native urinary tract of a renal-transplant recipient who had Chinese-herb nephropathy prompted us to propose regular cystoscopic examinations and the prophylactic removal of the native kidneys and ureters in all our patients with end-stage Chinese-herb nephropathy who were being treated with either transplantation or dialysis. Surgical specimens were examined histologically and analyzed for the presence of DNA adducts formed by aristolochic acid. All prescriptions written for Chinese-herb weight-reducing compounds during the period of exposure (1990 to 1992) in these patients were obtained, and the cumulative doses were calculated. RESULTS: Among 39 patients who agreed to undergo prophylactic surgery, there were 18 cases of urothelial carcinoma (prevalence, 46 percent; 95 percent confidence interval, 29 to 62 percent): 17 cases of carcinoma of the ureter, renal pelvis, or both and 1 papillary bladder tumor. Nineteen of the remaining patients had mild-to-moderate urothelial dysplasia, and two had normal urothelium. All tissue samples analyzed contained aristolochic acid-related DNA adducts. The cumulative dose of aristolochia was a significant risk factor for urothelial carcinoma, with total doses of more than 200 g associated with a higher risk of urothelial carcinoma. CONCLUSIONS: The prevalence of urothelial carcinoma among patients with end-stage Chinese-herb nephropathy (caused by aristolochia species) is a high. (+info)