Biomedical subjects

C F Bernardes

Publications and source records attributed to C F Bernardes.

12 recordsLinked to original sources

PubMed2001

Suramin inhibits respiration and induces membrane permeability transition in isolated rat liver mitochondria.

Suramin, a polysulfonated naphthylamine, caused a dose dependent inhibition of carbonyl cyanide p-(tri-fluoromethoxy)phenylhydrazone-stimulated respiration supported either by succinate or a cocktail of alphaketoglutarate, malate and isocitrate in isolated rat liver mitochondria. The half-maximum effect was obtained at 40 and 140 microM suramin for NADH- or FADH(2)-linked substrates, respectively. The respiration supported by N,N,N'N'-tetramethyl-p-phenylenediamine oxidation was unaffected by suramin (<or=500 microM). The latter respiratory substrate was used in the studies at examining the effects of suramin on the permeability properties of the inner mitochondrial membrane. These experiments provided evidence that suramin causes Ca(2+)-dependent mitochondrial swelling sensitive to the mitochondrial permeability transition (MPT) inhibitors cyclosporin A, ADP and Mg(2+). In addition, suramin decreased the content of reduced mitochondrial membrane protein thiols suggesting that thiol oxidation is the mechanism underlying suramin-induced MPT.

Adenosine Diphosphate↗

PubMed2000

Effects of 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis.

Cell viability requires the perfect functioning of the processes controlling ATP and Ca(2+) homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with a disruption of ATP and Ca(2+) homeostasis. This study shows that 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits Trypanosoma cruzi epimastigote cell growth. This thiol-reagent thiocyanate derivative was able to inhibit two ecto-enzymes present in this parasite. The ecto-ATPase and ecto-phosphatase activities were inhibited in a dose-dependent manner (K(i)=47.7 and 472.5 microM, respectively), but the 5'nucleotidase and 3'nucleotidase activities were not. DIDS uptake was approached by fluorescence microscopy. Pulse-chase experiments revealed the DIDS accumulation in compartments, presumably endocytic, in the posterior region of epimastigotes. In addition, we show that the T. cruzi mitochondria studied in permeabilized cells are able to accumulate and retain medium Ca(2+) in the absence of DIDS. However, in the presence of increasing concentrations of DIDS (50-200 microM), Ca(2+) transport was inhibited in a dose-dependent manner. DIDS also caused a disruption of the mitochondrial membrane potential, in the same concentration range, thus explaining its effect on Ca(2+) uptake. The presence of EGTA prevented the elimination of the mitochondrial membrane potential (DeltaPsi), supporting previous data suggesting that the binding of Ca(2+) to the mitochondrial membrane exposes buried thiols to react with DIDS. This thiocyanate derivative was also able to inhibit Ca(2+) uptake by the endoplasmic reticulum in a dose-dependent manner. Taken together, the data presented here provide further insights into the mechanisms underlying the antiproliferative actions of DIDS in T. cruzi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Bezold-Jarisch reflex in myocardial infarcted rats.

The Bezold-Jarisch reflex (BJR), produced by the administration of 5-hydroxytryptamine (5-HT, 4-16 micrograms/kg, iv), was evaluated in awake rats bearing short- (1 day) or long-term (30 days) myocardial infarction. Heart chronotropic response produced by acetylcholine was further assessed by Langendorff's isolated heart perfusion technique. Compared to the sham-operated group, infarcted rats showed either hypotension and tachycardia or bradycardia following short- or long-term myocardial infarction, respectively. Whereas the long-term myocardial infarction attenuated 5-HT-induced hypotension and bradycardia by about -25 and -80%, respectively, no significant response changes were observed in short-term infarcted rats. Impairment of BJR correlated significantly (P < 0.01) with the extent of myocardial necrosis in the 30-days infarcted group. Chronotropic responsiveness of the heart to acetylcholine in infarcted rats did not differ from the sham-operated group. Transmural antero-medio-lateral infarcted areas spanned over nearly 37% (1-day group) and 35% (30-days group) of the left ventricular circumference. These results indicate that cardioinhibitory and vasodepressor reflex responses to 5-HT are significantly impaired in chronic myocardial infarction associated with (1) marked hypertrophy of left atrium and/or of non-infarcted left ventricle, which are the main origin of vagal chemosensitive C-fibers, (2) morphological damage of this innervation due to the necrotic injury of the left ventricle, (3), possible attenuation in the vagal afferents located in the lungs and/or (4) enhancement of the chemical sensitivity of cardiac sympathetic afferents.

Animals↗

PubMed1995

[Evaluation of the vagal efferent pathway in rats in the acute and chronic phases of myocardial infarction].

PURPOSE: To investigate the sensitivity of the muscarinic receptors to acetylcholine (Ach) and to vagal stimulation in rats during the acute and the chronic phases of myocardial infarction (MI). METHODS: Male albino rats were submitted to ligature of the descending anterior branches of the left coronary artery to produce MI. Control rats (Con) were submitted to a sham surgery. The animals were studied 1-3 days (acute phase) or 30 days (chronic phase) after surgery. Under anesthesia (ketamine+xylazine) the right vagus nerve was isolated at the neck and stimulated with suprathreshold pulses (2ms, 1-64Hz). Atrial and ventricular rates were measured in the ECG recording. Dose-response curve to Ach (5-80 micrograms) was studied in the isolated hearts perfused according to the Langendorftechnique. Atrial and ventricular rates were evaluated through the surface electrogram recording. The left ventricular pressure was measured with an intraventricular balloon. RESULTS: Basal heart rate in the anesthetized animals was similar in Con and MI rats. The vagal stimulation produced a frequency dependent reduction of the heart rate. This reduction was less intense in the MI groups to stimulation rates of 32 and 64Hz. It was not observed any difference in the sensitivity of sinus and AV nodes to exogenous Ach in infarcted hearts. The reduction of the systolic pressure obtained after Ach administration to the hearts paced artificially (3.3Hz) was similar in MI and Con hearts. CONCLUSION: MI hearts were less sensitive to vagal stimulation than Con hearts. Since the in vitro effects of Ach remained unchanged after infarction, these results suggest an impairment of the cardiac neuroeffector vagal synapse. This may contribute to a less efficient control of the heart rate by the parasympathetic pathway in infarcted individuals.

Acetylcholine↗

PubMed1994

Ca(2+)-dependent permeabilization of the inner mitochondrial membrane by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955-19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and Ca2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca2+ release. The presence of DTT prevented but did not reverse the elimination of delta psi induced by DIDS. EGTA prevented delta psi elimination and decreased the amount of protein aggregates, suggesting that the binding of Ca2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed delta psi by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca(2+)-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

Thapsigargin causes Ca2+ release and collapse of the membrane potential of Trypanosoma brucei mitochondria in situ and of isolated rat liver mitochondria.

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.

Animals↗

PubMed1993

ATP and Ca2+ homeostasis in Trypanosoma cruzi.

Cell viability requires the perfect functioning of the processes controlling ATP and Ca2+ homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with disruption of ATP and Ca2+ homeostasis. Therefore, the study of the mechanisms by which different T. cruzi stages regulate the intracellular Ca2+ distribution and the ATP supply to maintain cell viability could provide new insights into the physiology of these parasites. One important objective of these studies is the identification of possible metabolic differences between host and parasite that could be exploited for the rational design of new and more effective trypanocidal drugs.

Adenosine Triphosphate↗

PubMed1991

Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.

Adenosine Diphosphate↗

PubMed1991

Regulation of intracellular calcium homeostasis in Trypanosoma cruzi. Effects of calmidazolium and trifluoperazine.

Trypanosoma cruzi epimastigotes maintained an intracellular free calcium concentration of about 0.15 microM, as measured with the fluorescent indicator Fura-2. The maintenance of low [Ca2+]i is energy-dependent since it is disrupted by KCN and FCCP. When the cells were permeabilized with digitonin, the steady-state free Ca2+ concentration in the absence of ATP was about 0.7 microM. The additional presence of ATP resulted in a steady-state level close to 0.1-0.2 microM which compares favorably with the concentration detected in intact cells. Intracellular Ca2+ uptake at high levels of free Ca2+ (greater than 1 microM) was due to energy-dependent mitochondrial uptake as indicated by its FCCP-sensitivity. However, as the free Ca2+ concentration was lowered from 1 microM, essentially all uptake was due to the ATP-dependent Ca2+ sequestration by the endoplasmic reticulum as indicated by its stimulation by ATP, and its inhibition by sodium vanadate. High concentrations of the calmodulin antagonist trifluoperazine, inhibited both the Ca2+ uptake by the endoplasmic reticulum and by the mitochondria, while calmidazolium released Ca2+ from both compartments. In addition, trifluoperazine and calmidazolium inhibited respiration and collapsed the mitochondrial membrane potential of T. cruzi, thus indicating non-specific effects unrelated to calmodulin.

Animals↗

PubMed1986

t-Butylhydroperoxide-induced Ca2+ efflux from liver mitochondria in the presence of physiological concentrations of Mg2+ and ATP.

Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM ATP, 4 mM Mg2+ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca2+, a small decrease in delta psi and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca2+ cycling was prevented by ruthenium red, the changes in delta psi, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca2+ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides.

Acetoacetates↗

PubMed1984

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A.

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.

Animals↗

PubMedDate not supplied

Inhibition of succinic dehydrogenase and F0F1-ATP synthase by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

This study shows that incubation of rat liver mitochondria in the presence of the thiol/ amino reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) is followed by inhibition of both succinate supported respiration and oxidative phosphorylation. Half-maximal inhibition of succinic dehydrogenase activity and succinate oxidation by mitochondria was attained at 55.3 and 60.8 microM DIDS, respectively. DIDS did inhibit the net ATP synthesis and ATP<=>[32P]Pi exchange reaction catalyzed by submitochondrial particles in a dose-dependent manner (Ki = 31.7 microM and Ki = 32.7 microM), respectively. The hydrolytic activities of uncoupled heart submitochondrial particles and purified F1-ATPase were also inhibited 50% by 31.9 and 20.9 microM DIDS, respectively.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗