Enhanced uptake of spermidine and methylglyoxal-bis(guanylhydrazone) by rat liver mitochondria following outer membrane lysis

Isolated rat liver mitochondria rapidly bound the 14C-labeled organic cations spermidine, a physiologically important polyamine, and methylglyoxal-bis(guanylhydrazone) (MGBG), an anticancer drug. This rapid, Mg2+-sensitive, respiration-independent binding is assumed to involve adsorption to anionic surface groups. A slower progressive uptake of the organic cations exhibited respiration dependence, indicating that it involves transport across the inner mitochondrial membrane into the matrix compartment. Addition of digitonin, to lyse the outer mitochondrial membrane, caused an increase in the mitochondrial content of the organic cations and enhanced the rate of progressive, respiration-dependent cation uptake. The data are consistent with the interpretation that the outer mitochondrial membrane limits access of the organic cations, spermidine and MGBG, to the inner mitochondrial membrane. This conclusion is supported also by published data indicating that outer membrane lysis enhances inhibitory effects of the organic cations on mitochondrial respiration. The uptake of spermidine by mitochondria was inhibited by MGBG.     

The effect of methylglyoxal-bis(guanylhydrazone) on mitochondrial Ca(2+) fluxes

Methylglyoxal-bis(guanylhydrazone) (MGBG) induces a dose-dependent inhibition of the electrophoretic Ca(2+) uptake by rat liver mitochondria (RLM) without affecting the electrical membrane potential. MGBG is also able to inhibit the electroneutral Ca(2+) release from mitochondria. These effects result in a progressive increase of Ca(2+) level in suspending medium indicating that Ca(2+) uptake is inhibited at higher extent than Ca(2+) efflux. Spermine instead, induces a lowering of external Ca(2+) concentration. This action is reversed by MGBG which again raises the external Ca(2+) concentration then in the absence of spermine, though at a lower extent. The mechanism of MGBG effects and their implications on energy metabolism are discussed.     

Cellular pharmacodynamics of the cytotoxic guanidino-containing drug CHS 828. Comparison with methylglyoxal-bis(guanylhydrazone)

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828) is a new guanidino-containing compound with antitumoral activity both in vitro and in vivo. Its activity profile differs from those of standard cytotoxic drugs but the mechanism of action is not yet fully understood. CHS 828 is presently in early phase I and II clinical trials. In the present study, the pharmacodynamic effects at the cellular level of CHS 828 was compared to another compound containing two guanidino groups, methylglyoxal-bis(guanylhydrazone) (MGBG). MGBG is known to inhibit the synthesis of polyamines, which are important in, e.g., proliferation and macromolecular synthesis. The concentration-response relationship of CHS 828 closely resembled that of MGBG and the drugs were similar with respect to inhibition of DNA and protein synthesis. On the other hand, CHS 828 induced a significant increase in cellular metabolism while MGBG did not. The cytotoxic effect of MGBG was reversed by the addition of exogenous polyamines, while that of CHS 828 was unaffected. Unlike MGBG, there was also no effect of CHS 828 on the levels of decarboxylating enzymes in the polyamine biosynthesis. In conclusion, CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG. Further studies will be required to define the exact mechanism of action of CHS 828.     

The effects of methylglyoxal-bis(guanylhydrazone) on spermine binding and transport in liver mitochondria

This study evaluated the effect of the anticancer drug methylglyoxal-bis(guanylhydrazone) (MGBG) on the binding of the polyamine spermine to the mitochondrial membrane and its transport into the inner compartment of this organelle. Spermine binding was studied by applying a new thermodynamic treatment of ligand-receptor interactions (Di Noto et al., Macromol Theory Simul 5: 165-181, 1996). Results showed that MGBG inhibited the binding of spermine to the site competent for the first step in polyamine transport; the interaction of spermine with this site, termed S1, also mediates the inhibitory effect of the polyamine on the mitochondrial permeability transition (Dalla Via et al., Biochim Biophys Acta 1284: 247-252, 1996). In the presence of 1 mM MGBG, the binding capacity and affinity of this site were reduced by about 2.6-fold; on the contrary, the binding capacity of the S2 site, which is most likely responsible for the internalization of cytoplasmic proteins (see Dalla Via et al., reference cited above), increased by about 1.3-fold, and its binding affinity remained unaffected. MGBG also inhibited the initial rate of spermine transport in a dose-dependent manner by establishing apparently sigmoidal kinetics. Consequently, the total extent of spermine accumulation inside mitochondria was inhibited. This inhibition in transport seems to reflect a conformational change at the level of the channel protein constituting the polyamine transport system, rather than competitive inhibition at the inner active site of the channel, thereby excluding the possibility that the polyamine and drug use the same transport pathway. Furthermore, it is suggested that, in the presence of MGBG, the S2 site is able to participate in residual spermine transport. MGBG also strongly inhibits deltapH-dependent spermine efflux, resulting in a complete block in the bidirectional flux of the polyamine and its sequestration inside the matrix space. The effects of MGBG on spermine accumulation are consistent with in vivo disruption of the regulator of energy metabolism and replication of the mitochondrial genome.     

Immunosuppressive effects of methylglyoxal-bis(guanylhydrazone) on mouse bone marrow and spleen cells and their antagonism by spermidine

Changes in immunological function and in the activity of S-adenosylmethionine decarboxylase were studied in bone marrow and spleen cells from mice immunized with sheep red blood cells and given multiple doses of methylglyoxal-bis(guanylhydrazone) (CH₃-G) or CH₃-G in combination with spermidine. Inhibition by CH₃-G was found in the complement-dependent and complement-independent immune responses of cells from both tissue sources; however, this inhibition was greater and more persistent in bone marrow than in spleen cells. Co-administration of spermidine with CH₃-G completely prevented this inhibition in the spleen at all the time points studied, whereas in the bone marrow prevention appeared later and was lower in magnitude. The increase in the activity of S-adenosylmethione decarboxylase, a key enzyme in spermidine biosynthesis, was greater in bone marrow than in spleen after the administration of CH₃-G. Co-administration of spermidine with CH₃-G consistently reduced this increase in enzyme activity in spleen but not in bone marrow. In addition to prevention, appropriate scheduling of spermidine subsequent to CH₃-G resulted in reversal of both the immunosuppression and the increase in S-adenosylmethionine decarbooxylase activity produced by CH₃-G in spleen cells.     

Protective action of methylglyoxal bis (guanylhydrazone) on the mitochondrial membrane

At low concentrations (0.5-1.0 mM) methylglyoxal bis (guanylhydrazone) (MGBG) exhibited a clearcut protection of rat liver mitochondria against the deenergizing action of either Ca2+, or oxidizing agents (butylhydroperoxide and oxaloacetate). Such a protection resulted from the prevention of transmembrane potential decay, discharge of accumulated Ca2+, release of mitochondrial Mg2+, adenine nucleotides and pyridine nucleotides and mitochondrial swelling. At high concentrations (5-10 mM) MGBG induced functional alterations of mitochondria (decrease of transmembrane potential, lower capability to accumulate and to retain Ca2+) which can be reversed by resuspension of mitochondria in a MGBG free medium. These reversible mitochondrial alterations by high MGBG concentrations are interpreted as a consequence of an aggregation and coprecipitation of suspended mitochondria.     

Selective effects of methylglyoxal-bis-(guanylhydrazone) on the development of antibody-forming cells in mice

The effects of methylglyoxal-bis(guanylhydrazone) on selected cellular immune functions and on S-adenosylmethionine decarboxylase activity were studied in spleen cells from mice given sublethal doses of this drug. The development of antibody-forming cells was markedly inhibited after administration of methylglyoxal-bis(guanylhydrazone) whereas the functions of cytotoxic effector cells and phagocytes were not affected. The activity of S-adenosylmethionine decarboxylase was found to be greatly increased in spleen cells from mice given the drug, and this increase was 4- to 5-fold greater in nylon wool-adherent cell populations as compared to that in non-adherent cell populations. The uptake of methylglyoxal-bis(guanylhydrazone) was 1.5- to 2-fold greater in nylon wool-adherent cells than in non-adherent cells. These studies suggest that methylglyoxal-bis(guanylhydrazone) selectively affects antibody-forming cells among mouse spleen cell populations.     

Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM)

Summary The inhibition by N-ethylmaleimide (NEM) of uptake₁ and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) ³H-noradrenaline uptake into intact PC12 cells; (2) ³H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) ³H-desipramine binding to isolated PC12 plasma membrane vesicles.In plasma membrane vesicles, NEM inhibited ³H-desipramine binding and ³H-noradrenaline uptake with similar potency (the IC₅₀'s were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting ³H-noradrenaline uptake (IC₅₀ = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na^{+/K +}-ATPase and not to a direct interaction with the noradrenaline carrier.The inactivation by NEM of ³H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake₁ (amezinium, 1 mmol/l) protected desipramine binding from inactivation.These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB176) Send offprint requests to E. Schömig at the above address     

Mitochondrial effects of the guanidino group-containing cytostatic drugs, m-iodobenzylguanidine and methylglyoxal bis (guanylhydrazone)

The involvement of mitochondrial damage in the antiproliferative effects of m-iodobenzylguanidine [MIBG] and methylglyoxal bis (guanylhydrazone) [methylGAG] was studied in human neuroblastoma SK-N-SH, mouse neuroblastoma N1E115 and mouse lymphosarcoma S49 cells. Proliferation of SK-N-SH cells was insensitive to MIBG (100 microM gave 15% inhibition), but sensitive to methylGAG (IC50 = 50 microM). MIBG and methylGAG were approximately equitoxic to N1E115 cells (IC50 of 92 and 87 microM, respectively). S49 cells were most sensitive to both MIBG (IC50 = 11 microM) and methylGAG (IC50 = 5 microM). In isolated sonicated mitochondria, MIBG inhibited respiration a complex I of the respiratory chain (EC50 = 0.5 mM), whereas methylGAG was much less effective (EC50 greater than 15 mM). In intact cells, MIBG at 31 microM impaired mitochondrial respiration and stimulated the glycolytic flux. In contrast, equimolar concentrations of methylGAG had no effect on oxygen consumption, ATP content, glucose consumption and lactate production. MethylGAG significantly increased putrescine levels in N1E115 and S49 cells within 12 hr via inhibition of S-adenosylmethionine decarboxylase. No such effects were seen in SK-N-SH cells for up to 48 hr. Equimolar concentrations of MIBG had no effect on the putrescine levels in the various cell lines, suggesting that MIBG did not inhibit S-adenosylmethionine decarboxylase. It is concluded that the antiproliferative mechanisms of the guanidino compounds are essentially different. MIBG inhibited mitochondrial respiration at complex I with concomitant stimulation of the glycolytic flux but was essentially without effect on polyamine levels. On the other hand, cytotoxicity of methylGAG was not associated with mitochondrial dysfunction.     

Inhibition by methylglyoxal bis(guanylhydrazone) of drug oxidation reactions catalyzed by mouse liver microsomes in vivo and in vitro

The activity of coumarin 7-hydroxylase (coumarin 7-hydroxylation) was inhibited in B6 mouse liver after a single injection of methylglyoxal bis(guanylhydrazone (MGBG). The decrease in the activity in vivo was greatest (70%) one day after the drug injection and the hydroxylase activity in microsomal fraction prepared from livers of MGBG-treated B6 mice was still 25% decreased 5 days after the drug. The amount of cytochrome P-450 also was decreased in MGBG-treated livers with the same time-dependency as the inhibition of coumarin 7-hydroxylation. MGBG and its close derivative 1,1'-[methylethanediylidene)dinitrilo)bis(3-aminoguanidine) (MBAG) inhibited the activity in vitro of coumarin 7-hydroxylase, benzo(a)pyrene hydroxylase and 7-ethoxy 0-de-ethylase when microsomes were prepared from livers of untreated B6 mice. In every case MBAG was a better inhibitor than MGBG in vitro. The in vitro inhibition of MGBG of several drug metabolizing enzymes was not reversed when microsomes were preincubated with 1 mM putrescine, spermidine or spermine. These results suggest that the anti-cancer drug, MGBG, has a severe effect(s) on the drug metabolizing system at concentrations reached during the treatment of patients with MGBG.     

肺动脉瓣狭窄(PS)采用肺动脉轴位(Pulmonary artery)投射(Project)的影像技术探讨

目的使用心血管造影机对7例肺动脉瓣狭窄(PS)患者进行经皮肺动脉瓣球囊扩张成形术(PBPV)采用肺动脉轴位投射体位的影像技术探讨。方法回顾性分析2011年至2013年我院共7例肺动脉瓣狭窄患者(5例单纯性肺动脉狭窄、1例合并房间隔缺损加房间隔封堵术、1例合并卵圆孔未闭)在心血管造影机下行经皮肺动脉瓣球囊扩张成形术的影像技术配合。结果 7例患者通过严格的术前、术中、术后X线造影,手术成功,无1例复发,通过术后1个月、3个月、6个月、1年的随访,无临床并发症。结论根据不同年龄的患者、病变部位、性质采用肺动脉轴位体位投影,既为术者提供最直观、最便捷的影像,也给术者提供给了狭窄瓣膜的精准值,方便术者对球囊的选择,有效缩短手术时间,大幅度降低手术并发症。     

小檗碱通过抑制脂肪酸摄取降低小鼠原代肝细胞脂质沉积

目的观察小檗碱对小鼠原代肝细胞脂质沉积的影响及其是否依赖于磷酸腺苷活化蛋白激酶(adenosine 5'-monophosphate-activated protein kinase,AMPK)通路,探讨小檗碱治疗非酒精性脂肪肝的作用机制。方法用不同浓度白蛋白偶联的油酸(OA)孵育小鼠原代肝细胞,确定OA诱导脂肪变的具体浓度,同时以二甲双胍为阳性对照,给予小檗碱处理24 h,乳酸脱氢酶(LDH)试剂盒检测确定小檗碱的安全剂量;油红染色定性判断细胞内脂质水平;酶法定量检测细胞内甘油三酯含量;Western blot法检测AMPK以及乙酰辅酶A羧化酶(ACC)磷酸化水平。AMPK的特异性阻断剂Compound C(CC)孵育原代肝细胞1 h后,再用小檗碱处理细胞24 h,油红染色及甘油三酯检测判断AMPK被抑制时小檗碱对脂质沉积的作用。生化分析仪检测OA组、小檗碱组、鱼藤酮(线粒体复合物I抑制剂)组细胞上清液中脂肪酸的含量,观察细胞对脂肪酸的摄取情况与复合物I抑制之间的关系。结果小檗碱在无细胞毒性的剂量下可明显降低小鼠原代肝细胞中油酸所致脂质沉积。同时,小檗碱可使AMPK及ACC的磷酸化水平增加。此外,在加入CC即AMPK活性被抑制的情况下,小檗碱降低肝细胞脂质沉积的作用不受影响。小檗碱和鱼藤酮分别使肝细胞的油酸摄取减少约31.2%和23.6%。结论小檗碱通过抑制电子传递链线粒体复合物I抑制脂肪酸摄取,进而降低小鼠原代肝细胞脂质沉积,这一作用并不依赖于AMPK通路。