甲状旁腺素对大鼠心肌细胞内游离钙和细胞凋亡的影响

目的　研究甲状旁腺素 (PTH)对心肌细胞内游离钙以及细胞肥大和凋亡的影响。方法　利用培养的新生大鼠心肌细胞 ,以Fluo 3/AM负载 ,通过激光共聚焦显微镜(LSCM)测定细胞内游离钙浓度 ([Ca2 + ] i) ;以细胞面积和细胞蛋白含量作为心肌细胞肥大指标 ;采用电镜和流式细胞术观察细胞凋亡的变化。结果　PTH1～ 34 0 0 1和 0 1 μmol·L- 1 刺激 7d后 ,心肌细胞内钙荧光强度以及心肌细胞面积和蛋白含量、细胞凋亡率较对照组显著增加。而 0 1 μmol·L- 1 PTH1～ 34 刺激的同时分别加入 1、1 0 μmol·L- 1 硝苯地平 ,上述指标改善 ,但未能达正常。结论　PTH1～ 34 可显著增加心肌细胞 [Ca2 + ] i,诱导细胞肥大和凋亡 ,并呈浓度依赖性 ,电压依赖性钙通道开放引起的细胞外钙内流增加为其机制之一     

Inhibition of calcium uptake and catecholamine release by 8-(n,n-diethylamino)- octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) In cultured bovine adrenal chromaffin cells

Effects of intracellular calcium antagonists, 8-(f,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and 1-(5-(p-nitrophenyl)-furfurylidene-amino) hydantoin sodium hydrate (dantrolene sodium), on catecholamine release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. TMB-8 inhibited carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner with a similar potency. On the contrary, dantrolene sodium did not show obvious inhibitory effects of catecholamine release and ⁴⁵Ca²⁺ uptake. Although TMB-8 inhibited the high K⁺-evoked catecholamine release and ⁴⁵Ca²⁺ uptake, the potency of the drug was approximately 100-fold less than when used to inhibit the carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake. The inhibitory effect of TMB-8 on the carbamylcholine-evoked catecholamine release was not overcome by an increase in an extracellular calcium concentration, and was not due to competitive antagonism at the nicotinic receptor site. Moreover, TMB-8 inhibited the carbamylcholine-stimulated ⁴⁵Ca²⁺ efflux, but dantrolene sodium failed to affect it. These results suggest that TMB-8, a well-known intracellular calcium antagonist, prevents the cellular calcium uptake in cultured adrenal chromaffin cells, and thus prevents catecholamine release.     

Indomethacin-induced free radical-mediated changes in the intestinal brush border membranes

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause small intestinal damage but the pathogenesis of this toxicity is not well established. Our earlier work has shown that villus enterocytes are most susceptible to the effects of indomethacin, a commonly used NSAID. This study looked at the acute effect of indomethacin on brush border membranes (BBM), which are present mainly in the villus cells and are in immediate contact with the contents of the small intestinal lumen. Evidence of oxidative stress was found in the mucosa of the small intestine of rats dosed with indomethacin, as indicated by increased activity of xanthine oxidase with corresponding decrease in the levels of several free radical scavenging enzymes. These changes were associated with an increase in peroxidation parameters in the BBM and a fall in the level of alpha-tocopherol. These BBM also exhibited impairment in glucose transport. Significant changes were seen in the lipid composition of these membranes, with upregulation of an 85kDa isoform of phospholipase A(2). Pretreatment of animals with allopurinol, arginine or zinc protected against these effects of indomethacin. Thus this study suggests that in an acute model of indomethacin dosing there is impairment in structure and function of the BBM in enterocytes, with the effects possibly mediated by free radicals and phospholipases.     

Inhibition of lymphocyte function by 9-deazaadenosine

9-Deazaadenosine (c⁹Ado), a novel C-nucleoside, has been found to inhibit lymphocytemediated cytolysis (LMC) in a time-dependent manner. c⁹Ado inhibited LMC by 50% at concentrations of 10 and 0.07 μM after drug-pretreatment periods of 3 and 22 hr, respectively, although a 1-hr pretreatment of cytolytic lymphocytes with 100 μM c⁹Ado had no effect upon this lymphocyte function. c⁹Ado was metabolized rapidly and extensively to 9-deazaadenosine 5'-triphosphate (c⁹ATP) both by mouse cytolytic lymphocytes and by human erythrocytes. Adenosine kinase purified from rabbit liver phosphorylated c⁹Ado with a K_m of 200 μM and a V_max of 8% that for adenosine. The metabolic buildup of c⁹ATP in lymphocytes was accompanied by a large, time-dependent decrease in cellular ATP and by smaller percentage decreases in CTP, UTP and GTP. Among other biochemical effects examined, c⁹Ado was found to cause a decrease in lymphocyte cAMP content and appeared to be neither an inhibitor nor a substrate for S-adenosylhomocysteine hydrolase. Consistent with this latter result, l-homocysteine thiolactone had no effect on the inhibition of LMC by c⁹Ado. Neither the inhibition of LMC by c⁹Ado nor the metabolic formation of c⁹ATP in lymphocytes was affected by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), indicating that c⁹Ado is not a substrate for adenosine deaminase. 5-Iodotubercidin, a non-competitive inhibitor (K₁₅ = 9 nM, K_ii = 20 nM) of adenosine kinase, prevented the above effects of c⁹Ado on lymphocyte function, c⁹ATP formation, and ATP levels. Either complete preservation (with coformycin) or partial replenishment (with adenosine plus EHNA) of ATP levels in c⁹Ado-treated lymphocytes resulted in partial restoration of cytolytic function to cells containing large amounts of c⁹ATP. These results suggest that c⁹Ado is inhibitory to LMC both because it causes a decrease in the absolute concentration of ATP within the cytolytic lymphocytes and because it permits the establishment within these cells of an unfavorable c⁹ATP: ATP ratio which impedes the utilization of ATP in a reaction essential to the execution of this lymphocyte function.     

Effects of cysteine derivatives of styrene on the transport ofp-aminohippurate ion in renal plasma membrane vesicles

The effects of cysteine conjugates of styrene, e.g. S-1/2-(phenyl-hydroxyethyl) cysteine (PEC) and its N-acetyl derivative (NAPEC) on the transport ofp-aminohippurate (PAH) ion in plasma membranes were studied in vitro using isolated rat renal brush-border membrane (BBM) and basolateral membrane (BLM) vesicles. The uptake of PAH was significantly inhibited by both PEC and NAPEC in both the membrane vesicles, as verified by decrease of the membrane/medium concentration ratio of PAH as the concentration of either PEC or NAPEC in the medium increased. These results show that both PEC and NAPEC are capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. The inhibition of PAH uptake in BBM vesicles due to 10 mM PEC or NAPEC was found to be nearly competitive, almost similar to probenecid, whereas in BLM vesicles such inhibition was found to be partially noncompetitive, as verified by the double reciprocal plots. Both PEC and NAPEC showed dose-dependent inhibition of the specific activity of the marker enzyme in each membrane, e. g. gamma-glutamyl transferase in BBM and Na⁺-K⁺-ATPase in BLM vesicles. However, no such inhibition was noticed with probenecid. The in vitro pretreatment with probenecid prevented the inhibition of gamma-glutamyl transferase activity in BBM due to PEC or NAPEC, but such was not the case for the Na⁺-K⁺-ATPase activity in BLM. In conclusion, the data suggest that the transport of cysteine or N-acetylcysteine conjugates of styrene by renal proximal tubular cells across both the membrane vesicles accompanied by the inhibition of the membrane-specific enzymes may lead to cellular dysfunction and consequently to the initial development of their nephrotoxicity.Portions of this work were presented at the 29th Annual Meeting of the Society of Toxicology, Miami Beach, Florida 1990 and at the 33rd Annual Meeting of Canadian Federation of Biological Societies, Halifax, Nova Scotia, 1990     

Lack of intestinal transport of [3H]-demethylphalloin: Comparative studies with phallotoxins and bile acids on isolated small intestinal cells and ileal brush border membrane vesicles

Summary Several earlier studies suggested that the uptake of phallotoxins by liver cells is a carrier mediated process using a transport system normally handling bile acids (see Frimmer 1982). In this study we have shown whether ileal cells, well known to transport bile acids too, are able to take up phallotoxins. Isolated epithelial cells prepared from guinea pig ileum accumulated [¹⁴C]-cholate, whereas [³H]-demethylphalloin ([³H]-DMP) was not taken up. The same observation was made with isolated jejunal cells but the uptake of [¹⁴C]-cholate was much slower. [³H]-DMP, however, was partly bound to intestinal cells. This process was not inhibited by cholate, iodipamide, oligomycin and carbonylcyano-chlorophenylhydrazone (CCCP), compounds known to decrease the uptake of phallotoxins into liver cells. Substituting Na⁺ for choline⁺ and also Cl^– for SCN^– did not influence the binding of [³H]-DMP. Frozen intestinal cells from the guinea pig bound two times more [³H]-DMP after thawing compared with intact cells. Supplementary uptake experiments on isolated brush border membrane vesicles from rat ileum revealed that phalloidin does not inhibit taurocholate uptake and that taurocholate does not interfer with [³H]-DMP binding.The results suggest that [³H]-demethylphalloin is not recognized by the bile acid carrier of the guinea pig and the rat ileum. It is concluded that the transport system for bile acids present in ileal cells is different from that of liver cells.Abbreviations TC taurocholic acid - DMP demethylphalloin - CCCP carbonylcyano-chlorophenylhydrazone - EGTA ethylene-glycol-bis-(2-aminoethylether)-N,N-tetraacetic acid - FCCP carbonylcyano-p-trifluoromethoxyphenylhydrazine This work was supported by the Deutsche Forschungsgemeinschaft     

Inhibition by phenothiazine derivatives of the adenylate cyclase of amphibian oocytes

The adenylate cyclase activity of membranes of Xenopus laevis oocytes and follicle cells was affected by the presence of 2-chloro-10-(3-aminopropyl)phenothiazine (CAPP) and two other antipsychotic drugs, fluphenazine and penfluridol. CAPP, at concentrations of 10 and 100 microM, had opposite effects on the activation of the oocyte adenylate cyclase by effectors that act through the G/F regulatory subunit. Under these conditions, the drug stimulated the activation by fluoride and drastically inhibited the activation by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and by cholera toxin and GTP. The activity of the catalytic subunit measured in the presence of either Mn2+ or forskolin was not affected by 100 microM CAPP. however, concentrations of this drug above 100 microM inhibited the adenylate cyclase activated by fluoride or by forskolin and also inhibited the activity of a calmodulin-independent cyclic nucleotide phosphodiesterase present in the same oocyte membrane preparation. Oocyte adenylate cyclase has been shown previously to be inhibited by the hormone progesterone. The inhibitory effect of CAPP is additive to that measured with the hormone, indicating that these compounds act through different mechanisms. CAPP did not modify the concentration of Gpp(NH)p required to yield half-maximal activation and, although the drug inhibited more strongly at lower concentrations of Gpp(NH)p, saturating amounts of the guanine nucleotide did not reverse completely the inhibition caused by CAPP. The effects of these antipsychotic drugs on oocyte adenylate cyclase did not require the presence of free Ca2+ and were not altered by the addition of exogenous calmodulin and calcium.     

Effect of cyclooxygenase inhibitors and protease inhibitors on phorbol-induced stimulation of oxygen consumption and superoxide production by rat pulmonary macrophages

Oxygen consumption and superoxide anion production by pulmonary macrophages are both increased by phorbol myristate acetate (PMA) but the two processes have been separated using protease inhibitors and cyclooxygenase inhibitors. Pretreatment with the protease inhibitors (L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as with the cyclooxygenase inhibitors acetylsalicylic acid (ASA) and ibuprofen (IBU), inhibited the stimulation of superoxide production and oxygen consumption by phorbol myristate acetate. However, whereas the order of potency for inhibition of stimulation of superoxide production was TPCK greater than TLCK greater than IBU greater than ASA, the order of potency for inhibition of stimulation of oxygen consumption was ASA greater than IBU greater than TPCK = TLCK. Although all four agents were effective inhibitors of PMA-stimulated superoxide production and oxygen consumption when added before PMA, in contrast to the cyclooxygenase inhibitors. TPCK was unable to inhibit oxygen consumption by more than 70-80% regardless of the concentration used, although superoxide generation could be inhibited completely. When added after PMA, ASA did not suppress either oxygen consumption or superoxide production and ibuprofen was only one-half as effective as an inhibitor. TPCK and TLCK, when added after PMA, accelerated the return to basal rates of both oxygen consumption and superoxide production. None of the four agents had any effect on basal superoxide production or oxygen consumption at the concentrations used. The data support the interpretation that both prostaglandin biosynthesis and protease activity may be associated with the activation of the superoxide-generating system of pulmonary macrophages. The consumption of molecular oxygen following stimulation of the cells with phorbol myristate acetate is not due solely to the generation of superoxide, however, since each process is inhibited with different potency by the same group of inhibitors. There appears to be a component of oxygen consumption which results from the activation of cyclooxygenase and, unlike superoxide production, cannot be completely inhibited by treatment with protease inhibitors.     

Impact of elevated intact parathyroid hormone on mortality and renal disease progression in patients with chronic kidney disease stages 3 and 4

Abstract

Objective:

To estimate the impact of elevated intact parathyroid hormone levels on time to death and renal replacement therapy in patients with chronic kidney disease stages 3 and 4.     

Histamine synthesis in intact and disrupted rat mast cells

Histamine production by purified intact rat peritoneal mast cells, as measured by formation of [β-³H]histamine from [β-³H]l-histidine or by release of ¹⁴CO₂ from ¹⁴C-carboxyl-labeled histidine, was ten to thirty times greater than that of disrupted cells or soluble extracts of these cells. Loss of activity was evident whether cells were disrupted by sonification, freezing and thawing, or lysis, both in the absence and presence of inhibitors of proteolytic enzymes and agents known to preserve enzyme activity. Studies with decarboxylase inhibitors indicated that a specific histidine decarboxylase was responsible for histamine formation in both the intact cells and cell extracts. In the presence of subsaturating concentrations of histidine, various histidine analogs and glutamine inhibited histidine uptake and histamine formation in intact mast cells but did not inhibit synthesis in cell extracts. These data indicate that, at physiological concentrations of histidine, blockade of histidine transport (through system N) may limit histamine synthesis in the intact cell and that measurement of histidine decarboxylase activity in tissue homogenates or cell extracts may not reflect actual histidine decarboxylase activity in vivo.     

Synthesis and structural identification of fluorine‐18 labeled parathyroid hormone

Parathyroid hormone (PTH) is an 84 amino acid peptide hormone that plays a key role in bone and mineral metabolism. The biological actions of PTH are mediated via the N‐terminal PTH(1–34) fragment, serving as the PTH receptor‐binding sequence, and which is therefore used clinically to treat conditions of low bone mass such as osteoporosis. In this study, PTH(1–34) was conjugated with non‐radioactive (stable F isotope) N‐succinimidyl 4‐fluorobenzoate (SFB) leading to three isomeric mono‐fluorobenzoated (FBz) PTH followed by Liquid chromatography‐Tandem mass spectrometry (LC‐MS/MS) assisted structural identification. Corresponding [¹⁸F]SFB‐labeled PTH derivatives were prepared respectively and the Lys¹³ site‐specific labeled [¹⁸F]FBz PTH was isolated by HPLC with radiochemical purity >99% and specific activity of 2.78 GBq/µmol, suitable for future application with in vivo pharmacokinetic/pharmacodynamic studies of PTH, using preclinical Positron Emission Tomography Computed Tomography (PET/CT) imaging.     

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol,and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.     

甲状旁腺激素、1，25（OH）2D3对鼠心肌细胞c—myc基因表达的影响

目的　研究不同浓度水平的甲状旁腺激素 (PTH)和 1,2 5 (OH) 2 D3 对新生大鼠心肌细胞c mycmRNA表达的影响。 方法　采用新生大鼠心肌细胞原代培养和逆转录多聚酶链反应 (RT PCR)技术 ,比较正常对照组、不同浓度PTH组和 1,2 5 (OH) 2 D3 组心肌细胞c mycmRNA表达程度。结果　 (1)高浓度PTH组 (10 -8M )心肌细胞c mycmRNA表达较正常对照组和低浓度PTH组(10 -10 M)明显增高 (分别为 1 84± 0 38、0 11± 0 16和 0 10± 0 14,P <0 0 1) ,而后两组之间没有差异 (P >0 0 5 ) ;(2 )两种浓度 1,2 5 (OH) 2 D3 组心肌细胞均未见c mycmRNA表达。 结论　高浓度PTH明显刺激心肌细胞c mycmRNA表达 ,1,2 5 (OH) 2 D3 能抑制c mycmRNA表达     

2-Mercapto-1-(beta-4-pyridethyl) benzimidazole inhibition of basal and aldosterone-stimulated sodium transport but prolongation of the transient theophylline-induced stimulation in the toad bladder

2-Mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB) was originally introduced as a reversible inhibitor of RNA synthesis, but subsequent findings made this suggestion doubtful. We examined the effect of MPB on active sodium transport, measured as short-circuit current (scc), across the isolated urinary bladder of the toad (Bufo marinus). The drug caused a rapid, dose-dependent inhibition of baseline scc; 25 micrograms/ml MPB reduced it by 70%. Sensitivity to MPB was the same in the presence and absence of metabolizable substrate. The transport stimulation by aldosterone (7 X 10(-8)M) was abolished entirely when MPB was introduced 30 min before the hormone. In bladders incubated with MPB with or without aldosterone, removal of both agents resulted in a rise in scc, which was more rapid in the aldosterone-pretreated hemibladders; a significant difference was observed after 30 min. This suggests that MPB inhibited transport at a site distal to messenger RNA accumulation. The effect of 3 hr of pretreatment with MPB on the response of the bladders to antidiuretic hormone (ADH, 20 mU) and cyclic AMP (cAMP, 10 mM) was then examined. The absolute increment in scc due to these agents was the same as in the absence of MPB, though the baseline was much reduced by the drug. After challenging MPB-pretreated bladders with theophylline (22.5 mM), sodium transport rose continuously for 90 min, in contrast to the small, short-lived rise in the absence of MPB. It is proposed that, in the toad bladder, MPB may: (1) inhibit cAMP-dependent protein kinase, as found by us in other tissues; and (2) counteract the accumulation of a transport inhibitor, possibly calcium or cyclic GMP, in tissues treated with endogenous or exogenous cAMP.     

Inhibition of lymphocyte-mediated cytolysis by 2-fluoroadenosine--evidence for two discrete mechanisms of drug action.

2-Fluoroadenosine (F-Ado) is a potent, irreversible inhibitor of lymphocyte-mediated cytolysis (LMC) in vitro: the irreversibility of this inhibition has been attributed to the metabolism of F-Ado to 2-fluoroadenosine t′-triphosphate (F-ATP) and 2-fluoroadenosine 3′, 5′-monophosphate (F-cAMP) within the cytotoxic lymphocytes [T. P. Zimmerman, J. L. Rideout, G. Wolberg, G.S. Duncan and G. B. Elion, J. biol. Chem.251, 6757 (1976)]. The present study was undertaken to define better the biochemical events intrinsic to the inhibition of LMC by F-Ado. Several purine ribonucleosides, which are themselves non-inhibitory towar LMC, have been found to inhibit the metabolism of F-Ado to F-ATP and F-cAMP by the cytotoxic lymphocytes. The reduction in F-cAMP formation caused by these ribonucleosides was counterbalanced by their augmentation of the elevation of lymphocytic cyclic AMP (cAMP) caused by F-Ado. While interference with the metabolism of F-Ado had little or no effect on the immediate inhibitory activity of F-Ado toward LMC, prevention of the cellular formation of F-ATP and F-cAMP did allow most of the inhibitory activity of F-Ado to be reversed after washing the lymphocytes free of exogenous F-Ado. The relative efficacy of these ribonucleosides in allowing reversibility of the inhibitory activity of F-Ado toward LMC followed the same order as did their efficacy in preventing the metabolism of F-Ado by the cytotoxic lymphocytes: 8-aza-adenosine > inosine > guanosine. Cytotoxic lymphocytes which had been preloaded with nucleotides of F-Ado (via prior incubation with F-Ado and subsequent washout of residual extracellular drug) exhibited increased inhibition of their cytolytic activity upon subsequent incubation with an inhibitor (Ro 20-1724) of cAMP phosphodiesterase. Under these latter experimental conditions, Ro 20-1724 caused a 2- to 3-fold elevation of F-cAMP in the cytotoxic lymphocytes but did not raise cAMP above control levels. These results suggest that F-Ado can inhibit LMC by either of two distinct mechanisms: (1) an extracellular mechanism, wherein F-Ado binds reversibly to an adenosine receptor present on the plasma membrane of the cytotoxic lymphocytes and reversibly activates a functionally associated adenylate cyclase, thereby causing an elevation of cellular cAMP; and (2) an intracellular mechanism, wherein F-Ado is metabolized irreversibly (during the 1- to 2-hr experimental period) by the cytotoxic lymphocytes to F-cAMP which, by reason of its ability to activate cAMP-dependent protein kinase, mimics the effect of elevated cellular levels of cAMP.     

Lipid peroxidation by bovine heart submitochondrial particles stimulated by 1,1′-dimethyl-4,4′-bipyridylium dichloride (paraquat)

Paraquat enhanced the NADH-dependent lipid peroxidation of bovine heart submitochondrial particles in the presence of ADE-Fe³⁺ chelate. The enhancement at physiological pH was about 3-fold. The pH optimum of the lipid peroxidation was shifted from pH 6.5 by paraquat. The submitochondrial particles catalyzed the reduction of paraquat when incubated anaerobically with NADH, whereas they did not reduce paraquat with succinate. The reduction was inhibited by phydroxymercuribenzoate or amytal, but it was not inhibited by rotenone, antimycin A or cyanide. The respiratory-chain inhibitors similarly affected the NADH-dependent O₂ consumption stimulated by paraquat, indicating that the NADH-dehydrogenase is involved in the reduction of paraquat at a region between the mercurial-sensitive site and the rotenone-sensitive site. The NADH-dependent reduction of ADP-Fe³⁺ chelate, a key step in lipid peroxidation, was stimulated by paraquat about 5-fold at physiological pH. The stimulation could mainly be ascribed to the direct electron transfer from a paraquat radical to the chelate and partially to the electron transfer from O₂^? produced by the reoxidation of the paraquat radical. ADP-Fe²⁺ produced lipid hydroperoxide in liposomes and decomposed cumene hydroperoxide. These reactions, the initiation reaction and the propagation reaction of peroxidation, were stimulated by paraquat. These results suggest that paraquat enhanced lipid peroxidation by stimulating (1) the reduction of ADP-Fe³⁺ chelate, and (2) the ADP-Fe²⁺-dependent initiation and propagation reactions of the peroxidation.