Biomedical subjects

A Gamberucci

Publications and source records attributed to A Gamberucci.

25 records · Page 2Linked to original sources

PubMed1993

Physiological concentrations of inorganic phosphate affect MgATP-dependent Ca2+ storage and inositol trisphosphate-induced Ca2+ efflux in microsomal vesicles from non-hepatic cells.

1. MgATP-dependent 45Ca2+ uptake by microsomes obtained from various non-hepatic tissues, namely rat brain, rat solid Morris hepatoma 3924A and human platelets, was measured in the presence of P(i) at low, cytosol-like, concentrations. 2. Increasing P(i) concentrations (0.5-3 mM) caused a progressive enlargement of the 45Ca(2+)-storage capacity of all the microsomal fractions. 3. As a result of P(i) stimulation of Ca2+ uptake, 45Ca2+ and [32P]P(i) were co-accumulated by the three microsomal fractions. 4. The time course for 45Ca2+ and [32P]P(i) accumulation in brain microsomes revealed a biphasic 45Ca2+ uptake: a rapid phase was followed by a second, slower, phase, which depended on the presence of P(i). During the P(i)-dependent phase, the uptake of 45Ca2+ was paralleled by the uptake of [32P]Pi. 5. The passive efflux of Ca2+ was paralleled by the efflux of P(i) and vice versa. In fact, the inhibition of active Ca2+ uptake by excess EGTA, or lowering the P(i) concentration of the incubation system by dilution, caused the release of 45Ca2+ and [32P]P(i) from 45Ca2+ or [32P]P(i) pre-loaded brain microsomes. The Ca2+ ionophore A23187 also released 45Ca2+ and [32P]P(i). 6. Ca2+ efflux by A23187 was rapid (t 1/2 approx. 2 s) and independent of the extent of intravesicular Ca2+ loading, which indicates that Ca2+ and P(i) do not form intravesicular insoluble complexes. 7. The progressive increase in Ca2+ accumulation, depending on P(i) stimulation, resulted in a proportional increase in the amount of Ca2+ releasable by InsP3 in the three non-hepatic microsomal fractions and in digitonin-permeabilized platelets. 8. Concomitantly to Ca2+, microsomal P(i) was also released by InsP3.

Adenosine Triphosphate↗

PubMed1992

Permeability of rat liver microsomal membrane to glucose 6-phosphate.

Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.

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

PubMed1991

Liver glucose-6-phosphatase activity is not modulated by physiological intracellular Ca2+ concentrations.

1. In the presence of MgATP and increasing amounts of added Ca2+, isolated liver microsomal vesicles accumulate approx. 10 nmol of Ca2+/mg of protein and buffer ambient free Ca2+ at increasing concentrations (0.22-10.9 microM). Under these experimental conditions, microsomal glucose-6-phosphatase activity is unaffected by the concentration of extravesicular free Ca2+. 2. Different levels of intravesicular Ca2+ were obtained by treating microsomes with the Ca2+ ionophore A23187 and by stimulating active microsomal Ca2+ accumulation with Pi (3 mM). In both instances, microsomal glucose-6-phosphatase activity is unaffected by the level of intravesicular Ca2+.

Adenosine Triphosphate↗

PubMed1991

Ca(2+)-dependent and independent mitochondrial damage in hepatocellular injury.

The alterations of mitochondrial membrane potential during the development of irreversible cell damage were investigated by measuring rhodamine-123 uptake and distribution in primary cultures as well as in suspensions of rat hepatocytes exposed to different toxic agents. Direct and indirect mechanisms of mitochondrial damage have been identified and a role for Ca2+ in the development of this type of injury by selected compounds was assessed by using extracellular as well as intracellular Ca2+ chelators. In addition, mitochondrial uncoupling by carbonylcyanide-m-chloro-phenylhydrazone (CCCP) resulted in a marked depletion of cellular ATP that was followed by an increase in cytosolic Ca2+ concentration, immediately preceding cell death. These results support the existence of a close relationship linking, in a sort of reverberating circuit, the occurrence of mitochondrial dysfunction and the alterations in cellular Ca2+ homeostasis during hepatocyte injury.

Adenosine Triphosphate↗

PubMed1991

Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.

Adenosine Triphosphate↗

PubMed1990

MgATP-dependent accumulation of calcium ions and inorganic phosphate in a liver reticular pool.

1. MgATP-dependent Ca2+ uptake by rat liver microsomal preparations and permeabilized hepatocytes was measured in the presence or absence of Pi. 2. Monitoring of free Ca2+ in incubation systems with a Ca2+ electrode in the presence of Pi (2-7 mM) revealed a biphasic Ca2+ uptake, with the onset of a second, Pi-dependent, Ca2+ accumulation. 3. Increasing Pi concentrations (up to 10 mM) caused a progressive enlargement of 45Ca2(+)-loading capacity of microsomal fractions. 4. As a result of Pi stimulation of active Ca2+ uptake, [32P]Pi and 45Ca2+ were co-accumulated. 5. Experiments with permeabilized hepatocytes revealed that the amount of Ca2+ releasable by myo-inositol 1,4,5-trisphosphate is unaffected by Pi.

Adenosine Triphosphate↗

PubMedDate not supplied

Low levels of glucose-6-phosphate hydrolysis in the sarcoplasmic reticulum of skeletal muscle: involvement of glucose-6-phosphatase.

Glucose-6-phosphate hydrolysis was measured in a fraction obtained from rabbit fast-twitch skeletal muscle and corresponding to total sarcoplasmic reticulum, as well as in three subfractions containing longitudinal tubules, terminal cisternae or both structures. In all cases the levels of hydrolysis measured both in native and disrupted membranes were approximately 60-100 times lower than the microsomal glucose-6-phosphatase activity of the corresponding livers. In contrast to liver microsomes, most (up to 80%) of the glucose-6-phosphate hydrolysing activity in muscle sarcoplasmic reticulum membranes was not inactivated by pH 5.0 pre-incubation indicating that it was not catalysed by the specific glucose-6-phosphatase enzyme. Osmotically induced changes in light-scattering intensity of sarcoplasmic reticulum vesicles revealed that, in contrast to liver microsomes, sarcoplasmic reticulum vesicles were not selectively permeable to glucose-6-phosphate as mannose-6-phosphate was also permeable and in addition they were poorly permeable to glucose. Immunoblot experiments using antibodies raised against the glucose-6-phosphatase enzyme, and liver endoplasmic reticulum glucose and Pi translocases, failed to detect the presence of these protein components in sarcoplasmic reticulum membranes. Southern blot analysis of reverse transcriptase-polymerase chain reaction products from rat muscle revealed that glucose-6-phosphatase mRNA is present in muscle. Quantification of Northern blot analysis of liver and muscle mRNA indicated that muscle contains less than 2% of the amount of glucose-6-phosphate mRNA found in corresponding livers. We conclude that very low levels of specific glucose-6-phosphatase (e.g. as in liver; E.C. 3.1.3.9) are present in muscle sarcoplasmic reticulum and that the muscle and liver glucose-6-phosphatase systems have several different properties.

Animals↗