Formation Of Surface Blebs Research Articles

Modifications in plasma membrane lipid composition and morphological features of AH-130 hepatoma cells by polyenylphosphatidylcholine in vivo treatment

The plasma membrane lipid composition in AH-130 hepatoma cells was found to change remarkably after polyenylphosphatidylcholine (PPC) treatment. Plasma membranes from cells grown in rats treated for 7 days i.v. with 20mg/kg/day PPC, when compared to those of control cells, did not show significantly different amounts of cholesterol or phospholipids relative to protein content, but, surprisingly, the individual phospholipid distribution inside the two membrane leaflets changed dramatically. Phosphatidylcholine (PC), the major phospholipid in the external membrane leaflet, increased ~47% (p<0.001). By contrast, phosphatidylethanolamine (PE), the most important component of the inner leaflet, decreased nearly 37% (p<0.001), while sphingomyelin (SM) also decreased ~17%, (p=0.1). Tumor cells collected from control rats at the same time interval and observed by scanning electron microscopy, exhibited a spherical shape with numerous and randomly distributed long microvilli, the same morphological and ultrastructural features displayed by the implanted cells. Conversely, tumor cells from PPC-treated rats no longer showed the roundish cell profile, and microvilli appeared shortened and enlarged, with the formation of surface blebs. Transmission electron microscopy observations confirmed the morphological and ultrastructural cell changes, mainly seen as loss of microvilli and intense cytoplasmic vacuolization. Taken together, these results indicate that the new phospholipid class distribution in the plasma membrane leaflets, modifying tumor cell viable structures, produced heavy cell damage and in many cases brought about complete cellular disintegration.

Atomic force microscopic study on morphological alterations induced by photodynamic action of Toluidine Blue O in Staphylococcusaureus and Escherichiacoli

Topographical alterations induced by Toluidine Blue O (TBO) mediated photodynamic treatment in Staphylococcus aureus and Escherichia coli was studied using atomic force microscopy (AFM). The AFM images showed distinct differences in the effect of photodynamic treatment on the morphology of S. aureus and E. coli. In S. aureus, photodynamic treatment with TBO resulted in light dose dependent increase in surface bleb formation suggesting breakage in the contact between the cell wall and the membrane with no significant change in the cell dimensions. Photosensitization of E. coli, resulted in surface indentations, significant reduction in the mean cell height, and flattening of bacteria as compared to the bacteria treated with the photosensitizers in the dark. These results indicate damage to the bacterial membrane and reduction of cell volume due to the loss of cytoplasmic materials. Leakage of intracellular contents measured using absorption spectrophotometry was higher and occurred faster in E. coli as compared to S. aureus and correlated with the morphological alterations. The results suggest that with AFM imaging it is possible to distinguish the membranolytic action of TBO in Gram-positive and Gram-negative bacteria.

Active bleb formation is abated in Lytechinus variegatus red spherule coelomocytes after disruption of acto‐myosin contractility

Red spherule coelomocytes are immune cells in the sea urchin Lytechinus variegatus that have been characterized as motile O(2) transport cells. Video microscopy of living red spherule coelomocytes reveals a constitutive, dynamic array of cellular morphologies and movements. Cells continuously send out and retract membrane blebs all over the cell surface as part of their normal cellular physiology. Disruption of microtubules by perfusion with either nocodazole or taxol had no effect on bleb formation or motility. Perfusion with cytochalasin B abated bleb formation and revealed cells that exhibited multiple small spheres attached by short membrane extensions. Attenuation of blebbing and intracellular organelle motility were restored by washing out with cytochalasin B. Treatment with phalloidin also abated bleb formation and revealed a smooth, spherical cellular morphology. The effects of phalloidin were completely reversible after washout. Red spherule coelomocytes treated with blebbistatin rounded up with an irreversible retraction of blebs into surface blebs that were greatly reduced in size, number and motility. Normal cell surface bleb formation and intracellular organelle motility were not restored after washout of the drug. These results indicate that the acto-myosin contractile mechanism contributes to the dynamics of constitutive cell surface membrane blebbing in invertebrate immune cells.

Calyculin A-induced endothelial cell shape changes are independent of [Ca 2+] i elevation and may involve actin polymerization

Changes in endothelial cell (EC) shape result in inter-EC gap formation and subsequently regulate transendothelial passage. In this work, we investigated the effects of protein phosphorylation (induced by inhibition of protein phosphatases) on EC shape changes. Treatment of bovine pulmonary artery endothelial cells (BPAEC) with calyculin A (100 nM, an inhibitor of protein Ser/Thr phosphatases 1 and 2A) resulted in cell retraction, surface bleb formation and cell rounding. Trypan blue and electrophysiological experiments suggested that the plasma membrane of these rounded cells maintained functional integrity. Calyculin A-induced morphological changes were strongly inhibited by staurosporine, but not affected by specific inhibitors of the myosin light chain (MLC) kinase, protein kinases A, C and G, and tyrosine kinases. The calyculin A effects were not mimicked by phorbol myristate acetate, dibutyryl cAMP, 8-bromo-cGMP or ionomycin. Cytochalasin B (an inhibitor of actin polymerization) almost completely abolished such shape changes while colchicine (an inhibitor of microtubule polymerization) had no inhibitory effect at all. Ca 2+ imaging experiments showed that the morphological changes were not associated with any global or local cytosolic Ca 2+ concentration ([Ca 2+] i) elevation. The results suggest that calyculin A unmasked the basal activities of some protein Ser/Thr kinases other than MLC kinase and protein kinases A, C and G; these unknown kinases might cause BPAEC shape changes by a mechanism involving actin polymerization but not [Ca 2+] i elevation.

The hepatotoxicity of rhein involves impairment of mitochondrial functions

Metabolism of rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) in primary cultures of rat hepatocytes caused production of oxygen-derived free radicals by redox cycling; this was shown as an increased rate of superoxide-dismutasesensitive NAD(P)H oxidation and NAD(P)H-cytochrome c reduction. Furthermore, rhein caused a depletion of intracellular reduced glutathione and an immediate, almost 10-fold increase in intracellular free Ca 2+. Exposure to rhein also induced the following: a decrease in the mitochondrial membrane potential, as analyzed by uptake of rhodamine 123 (Rh 123); initiation of lipid peroxidation, measured as accumulation of malondialdehyde and 4-hydroxyalkenals; and cell death (LD 50 = 20 μM). Pretreatment of cell cultures with dithiothreitol (DTT), nifedipin or N′, N′-diphenyl- p-phenylenediamine (DPPD) increased the intracellular free Ca 2+ concentration 5-fold but inhibited rhein-induced cytotoxicity. Moreover, addition of these protecting substances maintained the level of ATP and glutathione (GSH) and prevented accumulation of lipid peroxidation products. Depletion of intracellular glutathione by pretreatment with buthionine sulfoximine (BSO), or inhibition of glutathione reductase with 1,3-bis-2-chloroethyl-1-nitrosourea (BCNU) decreased cell viability (LD 50 = 2.5 μM). On the other hand, increasing GSH by pretreatment with l-2-oxothiazolidine-4-carboxylic acid (OTC) did not provide complete protection. In summary, rhein undergoes redox cycling that gives rise to oxygen metabolites that affect the mitochondrial membranes (recorded as a decreased membrane potential) and alter the plasma membrane (i.e. induced the formation of surface blebs). Mitochondrial malfunction also causes changes in Ca 2+ homeostasis and depletion of ATP, which eventually lead to cell death.

Induction of daunorubicin carbonyl reducing enzymes in carcinoma cells

Metabolism of rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) in primary cultures of rat hepatocytes caused production of oxygen-derived free radicals by redox cycling; this was shown as an increased rate of superoxide-dismutasesensitive NAD(P)H oxidation and NAD(P)H-cytochrome c reduction. Furthermore, rhein caused a depletion of intracellular reduced glutathione and an immediate, almost 10-fold increase in intracellular free Ca2+. Exposure to rhein also induced the following: a decrease in the mitochondrial membrane potential, as analyzed by uptake of rhodamine 123 (Rh 123); initiation of lipid peroxidation, measured as accumulation of malondialdehyde and 4-hydroxyalkenals; and cell death (LD50 = 20 μM). Pretreatment of cell cultures with dithiothreitol (DTT), nifedipin or N′,N′-diphenyl-p-phenylenediamine (DPPD) increased the intracellular free Ca2+ concentration 5-fold but inhibited rhein-induced cytotoxicity. Moreover, addition of these protecting substances maintained the level of ATP and glutathione (GSH) and prevented accumulation of lipid peroxidation products. Depletion of intracellular glutathione by pretreatment with buthionine sulfoximine (BSO), or inhibition of glutathione reductase with 1,3-bis-2-chloroethyl-1-nitrosourea (BCNU) decreased cell viability (LD50 = 2.5 μM). On the other hand, increasing GSH by pretreatment with l-2-oxothiazolidine-4-carboxylic acid (OTC) did not provide complete protection. In summary, rhein undergoes redox cycling that gives rise to oxygen metabolites that affect the mitochondrial membranes (recorded as a decreased membrane potential) and alter the plasma membrane (i.e. induced the formation of surface blebs). Mitochondrial malfunction also causes changes in Ca2+ homeostasis and depletion of ATP, which eventually lead to cell death.

Fas-Mediated Apoptosis in Primary Cultured Mouse Hepatocytes

The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.

The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei

Five oral isolates each of Candida albicans and Candida krusei were studied for their sensitivity to the fungicidal effect of human lactoferrin. Significant inter- and intraspecies variations were observed and with most isolates the sensitivity of C. krusei to lactoferrin was greater than that of C. albicans. Fungicidal activity of lactoferrin was dose-dependent and observable only with the irondashfree form of the molecule (apo-lactoferrin). Irondashsaturated lactoferrin was ineffective against all isolates. Supernatant protein assays and scanning electron microscopy indicated cell surface alterations—leakage of proteins and formation of surface blebs—only in those Candida isolates that were sensitive to apo-lactoferrin. As lactoferrin is a common, nondashimmune, mucosal defence protein, its varying mode of action against C. albicans and C. krusei may be related to their different oral carriage rates.

Two different pathways for necrotic cell death induced by free radicals.

Plasma membrane modifications have been widely recognized as crucial factors in cell injury and death. One of these modifications, surface blebbing, has been considered as an injury-marker associated with a series of biochemical and physiological modifications. Our study focused on the different effects of free radical-induced cell damage by quinone menadione (2-methyl-1,4-naphthoquinone) and by hyperthermic shock (45 degrees C) on the erythroleukemic cell line K562. Different techniques including immunofluorescence, freeze-fracturing, and electron paramagnetic resonance spectroscopy were employed. Menadione induced the formation of surface blebs, accompanied by a rearrangement of the microfilament system and changes in the distribution of plasma membrane proteins. In contrast, heat-shocked cells showed neither blebbing nor important cytoskeletal changes. Finally, the electron paramagnetic resonance results showed an increase in membrane order not specifically related to the type of free radical-induced stress. These cell death features appear to suggest the existence of two different types of pathways for necrotic cell death: both treatments induce cell injury and eventual death by modifying plasma membrane integrity and function. However, one involves cytoskeleton-dependent surface blebbing, whereas the other does not.

Cellular Effects of Some Metabolic Oxidation Products Pertinent to 4‐Ethoxyaniline

The toxicity of some metabolic products pertinent to 4-ethoxyaniline in isolated hepatocytes were investigated. The compounds investigated were 4-ethoxynitrosobenzene (1), 4-ethoxy-4'-nitrosodiphenylamine (2), 3,6-bis(4-ethoxy-phenylimino)-4-ethoxy-1,4-cyclohexadienylamine (3), 4-(4-ethoxyphenylimino)-2,3-dimethyl-2,5-cyclohexadiene-1-one (4) and 4-(4-ethoxyphenylimino)-2,6-dimethyl-2,5-cyclohexadiene-1-one (5). Of these, 1, 2 and 3 are oxidation products of 4-ethoxyaniline. Compounds 4 and 5 are dimethyl analogues of previously investigated oxidation product 4-(4-ethoxyphenylimino(-2,5-cyclohexadiene-1-one (NEPBQI). Among the investigated compounds, 1 and 2 were the most toxic towards isolated hepatocytes. In hepatocytes treated with compounds 1, 2 and 4, loss of cell viability was also accompanied by surface bleb formation. All compounds except 3 reacted with GSH resulting in depletion of cellular GSH. No formation of GSSG was observed, however. Thus, the GSH depletion was apparently due to conjugate formation rather than oxidation. No superoxide dismutase inhibitable reduction of acetylated cytochrome c was observed, thus none of the compounds undergoes measurable redox cycling.

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 μ M with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca 2+-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.

On the mode of action ofClostridium difficile toxin A: anin vitro study

C. difficile toxin A causes the rounding up and the formation of surface blebs in normal rat intestinal crypt cells (IEC-6). These phenomena, together with the inhibition of DNA synthesis and the cell lysis, could be the mechanisms leading the toxin-treated cells to death.

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: Alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphtoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs, and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.

Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant.

To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086-1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton-permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.

On the role of mitochondria in cell injury caused by vanadate-induced Ca 2+ overload

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca 2+ in the intracellular compartments. Vanadate- induced Ca 2+ accumulation was related to inhibition of the plasma membrane Ca 2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca 2+ or the enhanced operation of a putative Na +/Ca 2+ exchanger. After an initial rise in the cytosolic free Ca 2+ concentration, as revealed by phosphorylase activation, Ca 2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca 2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca 2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca 2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca 2+ overload.

Effect of thiol manipulation on chemopotentiation by nitroimidazoles

Highly electron affinic compounds such as the nitroimidazole misonidazole (MISO) have been shown both in vitro and in vivo to be effective potentiators of certain conventional chemotherapeutic agents. Mechanistically, the observation that nitroheterocyclics reduce intra-cellular thiols by enhancing the oxidation of glutathione (GSH), has suggested that thiol depletion by MISO may be a key factor in this enhancement. The present investigations were undertaken to determine whether the use of buthionine sulfoximine (BSO) to affect GSH metabolism may lead to more effective potentiation of chemotherapeutic agents by sensitizers. KHT/iv cells were treated in exponential phase under hypoxic conditions with variable doses of the activated form of cyclophosphamide (4-hydroxy-cyclophosphamide, 40H-CY) administered concominantly with or without MISO (2.5 mM) for an exposure time of 4 hr. Inclusion of the sensitizer in the treatment protocol resulted in a dose modifying factor of ∼2.4. Exposing cells to 1.0 mM BSO for 2 hr prior to treatment reduced intracellular GSH levels to 70–80% of control and increased the efficacy of 40H-CY ∼1.2-fold. If BSO was administered prior to the 40H-CY + MISO combination, severe tumor cell toxicity resulted. For example, when combined with 40H-CY, similar cell kill could be achieved with 5 to 6-fold lower MISO doses in the presence of BSO as in the absence of BSO. Ultrastructural evaluations revealed that in the three agent combination, membrane damage, as reflected by the formation of surface blebs, may play a key role in the mechanism of the observed enhanced cytotoxicity.

Anoxic hepatocyte injury: role of reversible changes in elemental content and distribution.

Examination of anoxic isolated hepatocytes by light and electron microscopy indicated that initial morphologic changes were largely localized to the periphery of the cells. This early phase consisted of surface bleb formation but was not accompanied by alterations in parameters of plasma membrane integrity (leakage of cellular enzymes, exclusion of trypan blue). The time course of changes in structure was temporally related to alterations in the elemental distribution and content of various subcellular compartments. These studies, which employed electron probe X-ray microanalysis, demonstrated that rapid increases in the sodium and chlorine content and decreases in the potassium content of the cytoplasm, mitochondria and nucleus occurred, whereas no change in the calcium content of any subcellular compartment was detected. Concurrently, two cellular functions known to be dependent upon ion homeostasis, sodium-dependent taurocholate uptake and mitochondrial respiratory control, became markedly impaired. Reoxygenation within 30 min resulted in the restoration of both elemental distribution and the latter two functions to baseline. These data are consistent with the hypothesis that some early functional changes may be mediated by altered ion homeostasis. In contrast, additional studies indicated that sodium and water fluxes could be dissociated from the appearance of plasma membrane blebs. Thus, this study provides direct evidence that the structural and functional changes of early anoxic hepatocyte injury cannot be explained by a single mechanistic cascade, but apparently involve multiple mechanisms which may not be directly linked.

Non-uniform fine structural alteration in the sinus node after cessation or reduction of blood flow

The sinus node of the dog was examined, using a large-specimen resin-embedding method for combined light and electron microscopy, after 30-240 min of total in vitro ischaemia and after 15-180 min of reduced blood flow in hearts with 2:1 AV block. Although progressive fine structural alteration was observed in the in vitro series, not all cells developed changes at the same rate. Complete loss of glycogen stores and swelling of organelles could be seen in many after only 30 min, whilst others retained their glycogen and showed minimal alteration even after 90 min. Despite relatively severe alteration throughout the node after 120 or more min, some groups of cells showed more extreme degrees of swelling and surface bleb formation than others. In the partially ischaemic in vivo series focal alteration, consisting chiefly of mitochondrial swelling and/or gross dilatation of sarcoplasmic reticulum vesicles, was detectable after 20 min and increased little in extent or degree during longer periods of reduced blood flow. This non-uniform response of sinus nodal cells to ischaemic injury parallels that of contractile atrial muscle cells.

Protection of rat hepatocytes from tert-butyl hydroperoxide-induced injury by catechol

Metabolism of tert-butyl hydroperoxide (TBHP, 2.0 m m) by glutathione peroxidase within isolated rat hepatocytes caused a rapid oxidation of intracellular reduced glutathione and ultimately NADPH through glutathione reductase. TBHP also caused the formation of surface blebs in the hepatocyte plasma membrane followed by the leakage of cytosolic enzymes, such as lactate dehydrogenase, into the incubation medium. Catechol (0.1 m m) protected hepatocytes from the cytotoxic effects of TBHP but did not prevent the rapid oxidation of glutathione indicating normal metabolism of TBHP through glutathione reductase. In contrast, addition of catechol to the hepatocyte incubations prevented TBHP-induced depletion of intracellular NADPH and increased the total NADP + + NADPH concentration without altering significantly the intracellular NADP + content or the NADPH NADP + NADPH ratio. Catechol did not alter TBHP stimulation of the pentose phosphate pathway. Hepatocytes incubated with sublethal concentrations of TBHP (1.0 m m) did not leak lactate dehydrogenase into the medium but did lose intracellular potassium. In these experiments, TBHP caused a sustained increase in phosphorylase a activity suggesting that TBHP metabolism may be associated with a sustained increase in cytosolic free Ca 2+. In the presence of catechol, phosphorylase a activity was increased by 5 min but returned toward control by 20 min. These data suggest that catechol may be protecting hepatocytes from TBHP-induced injury by preventing a sustained rise in cytosolic free Ca 2+ concentration.

Influence of calcium‐perturbing agents on schistosomes: Comparison of effects of praziquantel and verapamil on worm tegument

AbstractA “slow calcium channel” blocking agent, Verapamil (VPL), induces tegumental disruption in schistosomes. The response of worms to VPL was compared to that caused by a widely used antischistosomal drug, Praziquantel (PZQ), whose mode of action has been related to a rapid influx Ca++.Scanning microscopy of worms exposed to micromolar concentrations of either PZQ or VPL in vitro revealed a striking similarity of tegumental damage. At high dosage (10−5 M × 1 h), both compounds cause extensive surface bleb formation, which can proceed to shredding and denudation of the tegument. Early VPL‐induced changes in male Schistosoma mansoni appear to consist of small intertubercular lesions, as well as head and neck microblebs and bullae. After longer exposure, the bleb formations develop into large lesions, which after fixation for scanning microscopy, appear as flower‐like outgrowths on and around the tubercles. Females show much less surface disruption than males.Physiologically, PZQ typically causes quick contraction of worm pairs into a tight, agonal coil. While VPL also induces spasm and clonic jerking, worms soon lengthen into a gentle coil. Both drugs kill the parasites in vitro.The findings suggest that both PZQ and VPL have a surface target, whose disruption can be noted visually as lipoidal oozing and eventual tegumental blebbing. The inference drawn is that a direct affect on worm internal calcium metabolism, or on calcium channel activities, can lead to pathological changes which are grossly indistinguishable. These two drugs have markedly dissimilar structures, and thus probably different mechanisms of action. Yet, they result in similar final pathological events. These studies raise the possibility that various calcium‐perturbing agents might be useful candidates in a search for alternative or synergistic chemotherapeutic agents against trematodes and cestodes.