Biomedical subjects

B van de Water

Publications and source records attributed to B van de Water.

24 records · Page 2Linked to original sources

PubMed1994

Mitochondrial K+ as modulator of Ca(2+)-dependent cytotoxicity in hepatocytes. Novel application of the K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) to assess free mitochondrial K+ concentrations.

In isolated rat hepatocytes a sustained high increase in intracellular free Ca2+ ([Ca2+]i), induced by extracellular ATP, is associated with mitochondrial dysfunction and cell death. The Ca(2+)-induced effects are Pi-dependent and less severe when the intracellular K+ content is low. In this study, the involvement of mitochondrial K+ processing in Ca(2+)-induced loss of mitochondrial membrane potential (MMP) and viability was investigated. The recently introduced K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) has been used in combination with video-microscopy to assess intramitochondrial free K+ concentration ([K+]mito) in rat liver mitochondria in situ. After rapid permeabilization of the plasma membrane to remove cytosolic PBFI, the remaining PBFI was localized in mitochondria, and a 'resting' [K+]mito of approx. 15 mM could be measured. Increased [K+]mito levels were measured after induction of a prolonged increase in [Ca2+]i by ATP. Much lower [K+]mito, more comparable with control levels, were observed when intracellular K+ was depleted by omission of extracellular K+. In permeabilized cells the Ca(2+)-induced, Pi-dependent, dissipation of the MMP was markedly delayed in the absence of K+. These observations suggest involvement of [K+]mito as modulating agent in Ca(2+)-induced cytotoxicity in hepatocytes.

Animals↗

PubMed1994

Activation of rat Kupffer cells to tumoricidal cells by the immunomodulator muramyl tripeptide-phosphatidylethanolamine incorporated into the novel drug carrier lactosylated low density lipoprotein.

Lactosylated low density lipoprotein (lac-LDL) is a potential carrier for the site-specific delivery of lipophilic drugs to liver macrophages (Kupffer cells). In the present study we evaluated the application of lac-LDL as a carrier to target the immunomodulator muramyl tripeptide-phosphatidylethanolamine (MTP-PE) to rat Kupffer cells, to specifically activate these cells to tumor-killing cells. The drug carrier 125I-labeled lac-LDL interacted with a galactose-specific recognition system on isolated rat Kupffer cells. The in vitro association of 125I-lac-LDL at 37 degrees was maximal after 20 min, whereas degradation of 125I-lac-LDL was observed after a lag period of 10 min. Cultured rat Kupffer cells were activated after incubation with MTP-PE incorporated into lac-LDL. Lac-LDL-MTP-PE induced a 2-fold increase in the amount of newly synthesized proteins secreted by Kupffer cells. Lac-LDL-MTP-PE induced a concentration-dependent increase in the cytostatic and cytolytic activities of Kupffer cells towards tumor cells (B16F10 melanoma cells) in vitro. Treatment of rats with lac-LDL-MTP-PE also resulted in dose-dependent activation of Kupffer cells to tumoricidal cells, whereas the drug carrier alone had only a minor effect on this activity of Kupffer cells. The present data show that lac-LDL is an effective carrier for the delivery of the lipophilic immunomodulator MTP-PE to rat Kupffer cells. The specific activation of Kupffer cells to tumoricidal cells by lac-LDL-MTP-PE may be beneficial for the treatment of liver metastases.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1993

The relationship between intracellular Ca2+ and the mitochondrial membrane potential in isolated proximal tubular cells from rat kidney exposed to the nephrotoxin 1,2-dichlorovinyl-cysteine.

The effects of 1,2-dichlorovinyl-cysteine (DCVC) on the intracellular free calcium concentration ([Ca2+]i) and the mitochondrial membrane potential (delta phi) were investigated in freshly isolated rat kidney proximal tubular cells (PTC). Prior to cell death, DCVC induced a rise in [Ca2+]i and a decrease in the delta phi. Omission of extracellular calcium still resulted in a DCVC-induced increase of [Ca2+]i, indicating that calcium was released from intracellular stores. The beta-lyase inhibitor amino-oxyacetic acid completely protected against mitochondrial damage and cell death, indicating that the DCVC effects are dependent on beta-lyase metabolism. Incubation of the PTC with DCVC together with the intracellular-calcium complexing agents EDTA/acetoxy-methyl (AM), EGTA/AM or Quin-2/AM delayed (but did not prevent) the decrease of the delta phi and cell death, which indicates a relationship between [Ca2+]i and the decrease of delta phi. In individual cells four different responses induced by DCVC were observed; an increase of [Ca2+]i without an effect on delta phi, a decrease of delta phi and an increase of [Ca2+]i occurring simultaneously; an increase of [Ca2+]i preceded by a decrease of delta phi and a decrease of delta phi without any increase of [Ca2+]i. This indicates that DCVC-induced effects on [Ca2+]i and delta phi can appear independently. The data show that mitochondrial damage is potentiated by an elevation of [Ca2+]i, thereby creating a situation which rapidly leads to cell death.

Aminoquinolines↗

PubMed1993

Calcium-induced cytotoxicity in hepatocytes after exposure to extracellular ATP is dependent on inorganic phosphate. Effects on mitochondrial calcium.

In isolated mitochondria extensive uptake of Ca2+ in the presence of an "inducing agent," e.g. inorganic phosphate (Pi), causes permeabilization of the mitochondrial inner membrane and a collapse of the mitochondrial membrane potential. In this study we tested whether the effect of phosphate occurs in intact hepatocytes. Rat hepatocytes were incubated with ATP to induce a sustained increase in intracellular Ca2+ ([Ca2+]i), dissipation of the mitochondrial membrane potential, and cell death (Zoeteweij, J. P., van de Water, B., de Bont, H. J. G. M., Mulder, G. J., and Nagelkerke, J. F. (1992) Biochem. J. 288, 207-213). Omission of Pi from the incubation medium delayed the loss of viability. The nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) had similar effects on [Ca2+]i and viability, but now omission of extracellular Pi completely protected against cytotoxicity. Exposure to ATP or ATP gamma S induced a large cellular uptake of Pi. With the use of video-microscopy a significant increase in mitochondrial free calcium was observed before the onset of cell death. Accumulation of mitochondrial calcium was reduced when extracellular Pi was omitted. These results suggest that, after induction of high [Ca2+]i by ATP in hepatocytes, 1) mitochondria accumulate calcium which is associated with cell toxicity and 2) intracellular Pi increases which stimulates mitochondrial calcium uptake. These observations support a calcium-dependent mitochondrial dysfunction, induced by phosphate, as a valid model for ATP-induced cytotoxicity in hepatocytes.

Adenosine Triphosphate↗

PubMed1992

Involvement of intracellular Ca2+ and K+ in dissipation of the mitochondrial membrane potential and cell death induced by extracellular ATP in hepatocytes.

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2+ ([Ca2+]i) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2+]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]i on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80%. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2+ levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.

Adenosine Triphosphate↗

PubMed1991

Role of microtubuli in secretion of very-low-density lipoprotein in isolated rat hepatocytes: early effects of thiol reagents.

The secretion of very-low-density lipoprotein from hepatocytes proceeds through the microtubules. In this study, the role of glutathione in the maintenance of intact microtubules and the secretion of very-low-density lipoprotein has been investigated. When rat hepatocytes were incubated with reagents that deplete glutathione (e.g., diethylmaleate, alpha-bromoisovalerylurea or allyl alcohol) or reacted directly with protein thiols (disulfiram), the secretion of very-low-density lipoprotein by the cells was inhibited and the microtubules were severely damaged as visualized by immunofluorescence staining. Both events occurred within 30 min; long before, an effect on the energy status of the cells became evident. The observed inhibition of the secretion therefore seems due to an effect of the toxicants on the microtubules. For alpha-bromoisovalerylurea, diethylmaleic acid and allyl alcohol, it may be related to glutathione depletion; preincubation of the hepatocytes with N-acetyl-L-cysteine reduced the decrease of glutathione by alpha-bromoisovalerylurea and allyl alcohol (but not of diethylmaleic acid) and almost completely prevented the inhibition of very-low-density lipoprotein secretion and microtubule damage. Depletion of glutathione may result in modification of a small group of essential free protein thiols. Disulfiram did not deplete glutathione, and N-acetyl-L-cysteine could not prevent the effects of disulfiram on microtubules. The binding to protein thiols of radiolabeled disulfiram, which binds to microtubules in vitro, was determined. At 0.2 mmol/L disulfiram, only 3% of total cellular protein thiols were conjugated, but secretion of very-low-density lipoprotein was already inhibited by 25%, and microtubules were severely affected. We propose that modification of a small fraction of cellular protein thiols results in the loss of microtubular ultrastructure and thereby leads to inhibition of very-low-density lipoprotein secretion.

Animals↗