Biomedical subjects

P Dell'Antone

Publications and source records attributed to P Dell'Antone.

At least 19 recordsLinked to original sources

PubMed2006

Inactivation of H+-vacuolar ATPase by the energy blocker 3-bromopyruvate, a new antitumour agent.

3-bromopyruvate (3-Br PA), a strong alkylating agent, was found to cause a dramatic disruption of pH gradients in acidic compartments of Ehrlich ascites tumour cells (EATCs), as well as of rat thymocytes, at concentrations similar to those reported to cause ATP depletion in hepatocellular carcinoma cells. However, in the condition of complete disruption of pH gradients, ATP depletion was not, in either cell type, as serious as pH gradient dissipation. Moreover, the 3-Br PA effect on acidic compartments preceded severe cell ATP depletion, indicating that the former was not merely linked to energy deprivation elicited by 3-Br PA. Experiments conducted on isolated lysosomes supported this view in that the drug inactivated H(+)-vacuolar ATPase, the enzyme that makes certain compartments in the cell acidic. Inactivation probably involved alkylation of the enzyme on a thiol group, essential for H(+)-ATPase activity for dithiothreitol secured complete protection from 3-Br PA inactivation. The findings are discussed with regards to a possible involvement of lysosome destabilization in 3-Br PA induced cell death.

Animals↗

PubMed2000

The interactions of trialkyltin compounds with lysosomes from rat liver.

Interactions of two trialkyltin compounds with the lysosomes from a rat liver have been studied. It is shown that these compounds induce a fast alkalinisation in the matrix of energised lysosomes. The fast alkalinisation rate is similar to the one obtained with uncouplers of the oxidative phosphorylation. An identical effect has been obtained with lysosomes energised in a chloride-free medium. This supports the hypothesis that trialkyltin compounds behave not only as Cl-/OH- exchangers, but also as proton carriers in biological membranes. This result could explain the toxicity and in particular the neurotoxicity of trialkyltin compounds.

Animals↗

PubMed1997

The antineoplastic drug lonidamine interferes with the acidification mechanism of cell organelles.

The effect of the anticancer drug lonidamine (LND) on the pH of intracellular organelles was studied in isolated rat thymocytes by fluorimetric analysis of the (bafilomycin-nigericin sensitive) uptake of the acridine orange dye (AO) into acidic compartments. LND brought about a marked reduction (> 60%) in the above pH gradients with a half maximal decrease at a concentration of 0.25 mM. LND also caused a decrease, although to a lesser extent, in the ATP levels. In isolated rat liver lysosomes, 0.6 mM LND was found to inhibit ATP-driven organelle acidification by about 80%; minor inhibition of ATPase activity was observed in the same conditions. In addition, LND was able to promote proton efflux from isolated lysosomes. On the basis of our results it is suggested that the effect of LND on intracellular proton gradients may be due partially to the decrease in ATP levels, and mostly to a drug-induced increase in the ion (proton) permeability of the membranes.

Adenosine Triphosphatases↗

PubMed1996

Mitochondrial bioenergetics as affected by cationic detergents.

This paper examines the accumulation and toxicity mechanism of a cationic detergent, cetyltrimethylammonium (bromide) (CTAB), in energized rat liver mitochondria. The results suggest that: (1) the CTAB ion is accumulated in the mitochondrial matrix by a membrane potential-driven uptake mechanism; (2) accumulation may lead to a toxic effect, since it gives rise to collapse of the mitochondrial membrane potential (delta phi) which is correlated with ATP synthesis and which regulates Ca++ uptake; (3) collapse of delta phi may be due to enhanced permeability of the mitochondrial membrane to the ions (detergent effect); and (4) delta phi collapse and Ca++ and K+ release were also observed in another cationic detergent, NTAB, but not in the presence of anionic detergents.

Adenosine Triphosphate↗

PubMed1995

Anticholinesterasic drugs: tacrine but not physostigmine, accumulates in acidic compartments of the cells.

Tacrine (THA) and physostigmine (PHS) have been used in Alzheimer's disease therapy for their anticholinesterasic activity. Here we show that THA is taken up in rat lysosomes in an energy-dependent manner, and that it is also accumulated in acidic compartments of rat thymocytes and neuroblastoma cells. A concentration of THA less than 1 mM dissipated the pH gradient (delta pH) in all the above mentioned in vitro systems. On the contrary higher concentrations of PHS (1-2 mM) were ineffective. The accumulation of THA in acidic organelles of the cell may be relevant for the pharmacological action of THA in Alzheimer's treatment.

Acridine Orange↗

PubMed1994

Metabolic pathways in Ehrlich ascites tumor cells recovering from a low bioenergetic status.

Ehrlich ascites tumor cells were found to be in a low bioenergetic status, as evaluated by acridine orange uptake and ATP content, when resuspended in a glucose medium shortly after removal from the animal. Dye uptake as well as ATP content then increased for about 2 h at room temperature. This effect was only slightly inhibited by oligomycin. Cells resuspended in a glucose-free medium initially showed high dye uptake and ATP level, which were stable over time: in this case oligomycin caused a drop in both dye uptake and ATP level. The above findings, which are indicative of a marked Crabtree effect in Ehrlich ascites tumor cells, means that it is unlikely that limiting ADP and Pi play an important role in the glucose-induced inhibition of oxidative phosphorylation in this system.

Acridine Orange↗

PubMed1994

A new in vitro toxicity test based on the response to toxic substances in solutions of mitochondria from beef heart.

A simple, rapid and inexpensive in vitro method which may be used as a biological sensor for the valuation of toxicity of chemical compounds in water is proposed. The method utilizes the response of the respiratory chain of beef heart mitochondria to a wide range of toxic substances. By means of simple voltametric measurements, the method not only gives an indication of toxicity in water but, by using different substrates of the respiratory chain and exploiting some features of the toxic substances, it also allows selective detection of some toxic substance or groups of substances. Since the method reproduces the standard "rainbow trout" test, it may also be proposed as a screening test to precede whole bioorganisms bioassays or as a method to assess the presence of certain toxic substances in water.

Adenosine Triphosphate↗

PubMed1989

Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes.

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.

Acridine Orange↗

PubMed1988

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes.

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.

Adenosine Triphosphate↗

PubMed1987

Evidence for a delta pH-driven Ca2+ uptake in EGTA-treated bovine spermatozoa.

Calcium efflux from ejaculated bovine spermatozoa occurred upon incubation in Ca2+/EGTA buffers with Ca2+ ion concentrations ranging from 0.1 microM to 1 nM. Both total cellular calcium and cytosol free Ca2+ concentrations, the latter measured with Quin 2, were inversely correlated with the Ca2+ activity of the medium. An influx of radioactive 45Ca2+ parallel to a net efflux of calcium took place in spermatozoa incubated in 45Ca2+/EGTA buffers with 45Ca2+ activity of 0.01 microM or 0.1 microM. The uptake of the radioactive isotope was higher in spermatozoa incubated at pH 7.8 than that found at pH 6.8, increased in the presence of acetate or amiloride but decreased when ammonium chloride or monensin was added to the incubation mixture. Addition of acetate produced a decrease of the cytoplasmic pH, determined with the indicator carboxyfluorescein, whereas addition of NH4Cl or monensin caused a pH increase. Addition of either nigericin or monensin to spermatozoa suspended in a choline medium containing low concentrations of Na+, K+ and Ca2+ produced a cytosolic acidification, the subsequent addition of Ca2+ caused a cytosolic alkalinization parallel to an increase of the cytosolic free Ca2+. Addition of CaCl2 to EGTA-pretreated spermatozoa resuspended in a poorly buffered medium induced an evident decrease of extracellular pH suggesting a cellular proton extrusion. Both monensin and nigericin caused an increase of the calcium transport in spermatozoa suspended in a choline medium containing a physiological concentration of 1.5 mM CaCl2. Taken together the present results indicate that, under the experimental conditions used, a delta pH-driven Ca2+ uptake occurs in ejaculated bovine spermatozoa and suggest that Ca2+ is taken up in exchange with H+.

Acetates↗

PubMed1984

Electrogenicity of the lysosomal proton pump.

Using acridine orange as a pH gradient probe, the effects of valinomycin and FCCP on the pH gradient across lysosomal membranes in an ATP-free medium as well as on the rate of the inward ATP-driven proton translocation were investigated. Both lysosome-enriched and highly purified lysosomal preparations from rat liver were used with identical results. Ionophore effects were found to be different depending upon whether passive ion fluxes or ATP-driven H+ translocation were involved and supported the existence of a membrane potential in the latter case. Anions stimulated the rate of ATP-driven proton translocation and stimulation increased with increasing anion lipophilicity. These results strongly support the electrogenic nature of the lysosomal proton pump.

Adenosine Triphosphate↗

PubMed1982