Biomedical subjects

A Gamberucci

Publications and source records attributed to A Gamberucci.

At least 19 recordsLinked to original sources

PubMed2006

Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status.

The contribution of different proteolytic systems, in particular calpains and effector caspases, in apoptotic cell death is still controversial. In this paper, we show that during cisplatin-induced apoptosis of human metastatic melanoma cells, calpain activation, as measured in intact cells by two different fluorescent substrates, is an early event, taking place well before caspase-3/-7 activation, and progressively increasing during 48 h of treatment. Such activation appears to be independent from any intracellular calcium imbalance; in fact, an increase of cytosolic calcium along with emptying of the reticular stores occur only at very late stages, uniquely in frankly apoptotic, detached cells. Calpain activation proves to be an early and crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples co-treated with the calpain inhibitors, MDL 28170, calpeptin and PD 150606, where a variable but significant reduction of both caspase-3/-7 activity and cell detachment is observed. Consistently, such a protective effect can be at least partially due to the impairment of cisplatin-induced p53 activation, occurring early in committed, preapoptotic cells. Furthermore, in late apoptotic cells, calpain activity is also responsible for the formation of a novel p53 proteolytic fragment (approximately 26 kDa), whose function is so far to be elucidated.

Antineoplastic Agents↗

PubMed2000

Glucose-6-phosphatase in the insulin secreting cell line INS-1.

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form. On the other hand, hardly detectable levels of glucose-6-phosphatase activity and protein were present in the parent glucose insensitive RINm5F cell line. The mRNA of the glucose-6-phosphate transporter was also more abundant in the INS-1 cells. The results support the view that the glucose-6-phosphatase system of the beta-cell is associated with the regulation of insulin secretion.

Animals↗

PubMed1999

Caffeine releases a glucose-primed endoplasmic reticulum Ca2+ pool in the insulin secreting cell line INS-1.

Caffeine mobilized an intracellular Ca2+ pool in intact fura-2-loaded INS-1 cells in suspension exposed to high (16 mM) [glucose], while a minor effect was observed with low (2 mM) [glucose]. Cells were kept in a medium containing diaxozide or no Ca2+ to prevent the influx of extracellular Ca2+. The caffeine-sensitive intracellular Ca2+ pool was within the endoplasmic reticulum since it was depleted by the inhibitor of the reticular Ca2+ pumps thapsigargin and the InsP3-dependent agonist carbachol. No effect of caffeine was observed in the parent glucose-insensitive RINmF5 cells. In microsomes from INS-1 but not RINmF5 cells, the type 2 ryanodine receptor was present as revealed by Western blotting. It was concluded that the endoplasmic reticulum of INS-1 cells possesses caffeine-sensitive type 2 ryanodine receptors Ca2+ channels.

Animals↗

PubMed1998

On the mechanism of action of econazole, the capacitative calcium inflow blocker.

The ability of bovine serum albumin to reverse the inhibitory action of econazole and the unsaturated fatty acid oleate on store-dependent Ca2+ inflow was examined in Ehrlich ascites tumour cells. We report that inhibition of Ca2+ inflow by both compounds is reversed immediately upon addition of bovine serum albumin. It is concluded that the inhibitory action of econazole resembles that of unsaturated fatty acids. The mechanism appears to be one pertaining to nonspecific events at the plasma membrane, possibly involving alterations in plasma membrane fluidity/structure.

Animals↗

PubMed1998

Histones and basic polypeptides activate Ca2+/cation influx in various cell types.

Histone H2A (1-10 microg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr>4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.

Animals↗

PubMed1997

Unsaturated fatty acids mobilize intracellular calcium independent of IP3 generation and VIA insertion at the plasma membrane.

Addition of oleic and arachidonic acids to Ehrlich ascites tumor cells mobilizes Ca2+ from the same intracellular pool as that mobilized by thapsigargin. Such mobilization occurs in the presence of the phospholipase C inhibitor U73122 as well as in cells treated with pertussis toxin. Co-addition of fatty acids and thapsigargin leads to initial rates of Ca2+ mobilization much greater than that induced by either compound alone. The responses induced by the fatty acids are observed also with other lipophiles like sphingosine, bromo-palmitate and the Ca2+ influx inhibitor econazole; all responses are rapidly reversed by addition of bovine serum albumin. Many of the above effects of fatty acids are observed also in Jurkat T lymphocytes and Friend erythroleukemia cells. The experiments provide evidence of lipid-induced plasma membrane perturbations that influence intracellular Ca2+ mobilization independent of the generation of currently known second messengers.

Animals↗

PubMed1997

Inhibition of store-dependent capacitative Ca2+ influx by unsaturated fatty acids.

The effects of the unsaturated fatty acids, arachidonic and oleic acid, on the influx of Ca2+ activated by depletion of intracellular stores with thapsigargin were investigated in various cell types. By using a Ca2+ free/Ca2+ reintroduction protocol, we observed that arachidonic acid (2 to 5 microM) inhibited thapsigargin-induced rises in cytosolic free Ca2+ ([Ca2+]i) in Ehrlich tumor cells, Jurkat T lymphocytes, rat thymocytes, and Friend erythroleukemia and PC12 rat pheochromocytoma cells. This effect was attributed to the inhibition of Ca2+ entry, since arachidonate also inhibited thapsigargin-stimulated unidirectional entry of the Ca2+ surrogates Ba2+ and Mn2+. In Ehrlich cells, the IC50 for arachidonic and oleic acid was 1.2 and 1.8 microM, respectively. The inhibition appeared to depend on the ratio [fatty acid]/[cells] rather than on the absolute fatty acid concentration. Experiments with [3H]-oleic acid revealed that the inhibitory activity was not correlated with cell internalisation and metabolism of the fatty acid. The inhibition was reverted by removal of the fatty acid bound to cell membrane by fatty acid-free albumin treatment. The unsaturated fatty acids had no effect on ATP/ADP cell levels and plasma membrane potential. Pharmacological evidence indicated that cell phosphorylation/dephosphorylation events, and pertussis toxin-sensitive G proteins were not involved. Other amphipathic lipophilic compounds, i.e. 2-bromopalmitic acid, retinoic acid, sphingosine, and dihydrosphingosine, mimicked arachidonic/oleic acid as they inhibited thapsigargin-stimulated Ca2+ influx in an albumin-reversible fashion. These results suggest that physiologically relevant (unsaturated) fatty acids can inhibit capacitative Ca2+ influx possibly because they intercalate into the plasma membrane and directly affect the activity of the channels involved.

Animals↗

PubMed1996

Inhibition of glucuronidation by an acyl-CoA-mediated indirect mechanism.

The mechanism of the inhibition of glucuronidation by long-chain fatty acyl-CoAs was studied in rat liver microsomal membranes and in isolated hepatocytes. Palmitoyl- and oleoyl-CoA did not affect p-nitrophenol UDP-glucuronosyltransferase activity in native microsomes but were inhibitory in permeabilised vesicles. The extent of inhibition was dependent on the effectiveness of permeabilisation and was constant in time in fully permeabilised microsomes. Fatty acyl-CoAs mobilised calcium from calcium-loaded microsomes. Elevation of the intracellular acyl-CoA level by the addition of palmitate or oleate inhibited the glucuronidation of p-nitrophenol in isolated hepatocytes. This effect could be abolished by emptying the intracellular calcium stores. Therefore, it is concluded that fatty acyl-CoAs inhibit glucuronidation indirectly, presumably via calcium mobilisation.

Animals↗

PubMed1996

Biochemical and functional heterogeneity of rat cerebrum microsomal membranes in relation to SERCA Ca(2+)-ATPases and Ca2+ release channels.

Rat cerebrum microsomes were subfractionated on isopycnic linear sucrose (20-42%) density gradients. The Ca2+ loading/release properties and the distribution of intracellular Ca2+ store channels, inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine (Ry) receptor, and SERCA pumps, were monitored in each subfraction by ligand binding and 45Ca2+ loading/release assays. Three different classes of vesicles were identified: (i) heavy density vesicles with high content of Ry receptors and Ca2+ pumps and high thapsigargin (TG)-sensitivity of Ca2+ loading; (ii) intermediate sucrose density vesicles with high content of IP3 receptor, high IP(S)3-sensitivity of Ca2+ loading and low content of Ry receptors; and (iii) light sucrose density vesicles with high content of Ry receptors, low content of IP3 receptors and low content of SERCA pumps highly sensitive to TG. Isolation of molecularly heterogeneous rat cerebrum microsomes and identification of specific Ca2+ loading/release properties support the presence of multiple, potentially active, heterogeneous rapidly exchanging Ca2+ stores in rat cerebrum.

Animals↗

PubMed1995

Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes.

In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition.

Acyl Coenzyme A↗

PubMed1995

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca2+ pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin.

To investigate the presence and the size of different non-mitochondrial Ca2+ pools of Ehrlich ascites tumor cells (EATCs), digitonin-permeabilized cells were allowed to accumulate Ca2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca(2+)-ATPase inhibitor thapsigargin, IP3 and the Ca2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca2+ stores prevented any Ca2+ release by IP3, and, after IP3 addition, little or no Ca2+ was released by thapsigargin. In both instances, a further Ca2+ release was accomplished by A23187. The IP3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondrial stored Ca2+, respectively. In intact EATCs, IP3-dependent agonists and thapsigargin discharged Ca2+ pools almost completely overlapping, and A32187 released a minor residual Ca2+ pool. The IP3-insensitive pool appeared to have a relatively low affinity for Ca2+ (below 600 nM). The high affinity, IP3-sensitive Ca2+ pool was discharged in a 'quantal' manner following step additions of sub maximal [IP3], and the IP3-induced fractional Ca2+ release was more marked at higher concentrations of stored (luminal) Ca2+, The IP3-sensitive Ca2+ pool appeared to be devoid of the Ca(2+)-activated Ca2+ release channel since caffeine did not released any Ca2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.

Animals↗

PubMed1995

Alteration of Na+ homeostasis as a critical step in the development of irreversible hepatocyte injury after adenosine triphosphate depletion.

The exposure of isolated hepatocytes to the redox-cycling quinone menadione caused an early loss of mitochondrial membrane potential, adenosine triphosphate (ATP) depletion, and decreased intracellular pH. These alterations were followed by an increase in intracellular Na+ and, ultimately, cell death. If HCO3- was omitted from the incubation buffer, or the hepatocytes were incubated in an acidic medium (pH 6.5) the accumulation of Na+ was markedly reduced. Inhibition of the Na+/H+ exchanger and of the Na+/HCO3- cotransporter by, respectively, amiloride and 4,4'-di-isothiocyano-2,2'-disulfonic acid stilbene (DIDS) suppressed the initial Na+ influx but did not prevent subsequent Na+ accumulation, because amiloride and DIDS inhibited the Na+/K+ pump. The omission of HCO3- from the extracellular medium or the incubation in acidic conditions also prevented menadione toxicity, without interfering with the loss of mitochondrial membrane potential and with ATP depletion. A similar protection was evident when hepatocytes were incubated with menadione in a medium without Na+. The preservation of adequate levels of ATP by supplementing hepatocytes with fructose allowed the initial Na+ load to be recovered and provided partial protection against menadione toxicity. These effects were suppressed if Na+/K(+)-ATPase was inhibited with ouabain. Taken together, these results indicated that the activation of the Na+/HCO3- cotransporter and of the Na+/H+ exchanger in response to the decrease of intracellular pH stimulated an enhanced influx of Na+. When the activity of the Na+/K+ pump was not able to control Na+ levels because of ATP depletion, such an uncontrolled Na+ influx precipitated irreversible injury and caused hepatocyte death.

Adenosine Triphosphate↗

PubMed1994

Modulation of Ca2+ influx dependent on store depletion by intracellular adenine-guanine nucleotide levels.

The effects of a number of metabolic inhibitors on the influx of Ca2+ activated by stimulation of receptors coupled to inositol 1,4,5-trisphosphate generation or by depletion of intracellular Ca2+ stores with thapsigargin were investigated in four different cell types: Ehrlich ascites tumor cells, Jurkat and HeLa cell lines, and rat hepatocytes. Independently of their chemical structure and site of inhibition, all of these metabolic poisons markedly inhibited Ca2+ influx without significantly affecting Ca2+ release. This inhibition was not due to membrane potential depolarization or to alteration in cytosolic pH but appeared correlated to a drop in the cellular concentration of ATP. The decreases in cellular [ATP] were paralleled by decreases in [GTP] and by increases in [ADP] and [GDP]. The reduction in ATP level necessary to drastically reduce Ca2+ influx was quite small, e.g. a 50% inhibition for a 5% reduction in [ATP], thus within the range of fluctuation presumably occurring under physiological conditions. We suggest that changes in the adenine or guanine nucleotide concentrations may represent an important modulatory mechanism of Ca2+ influx activated by store depletion.

Adenine Nucleotides↗

PubMed1994

Evidence for the intraluminal positioning of p-nitrophenol UDP-glucuronosyltransferase activity in rat liver microsomal vesicles.

Addition of p-nitrophenol and UDP-glucuronic acid to rat hepatic microsomes enhanced the MgATP-stimulated Ca2+ sequestration. This stimulatory effect was more explicit in the presence of the activator of glucuronidation, UDP-N-acetylglucosamine. The stimulation of Ca2+ uptake was dependent on the p-nitrophenol concentration and showed a good correlation with the rate of p-nitrophenol glucuronidation. The stimulation of Ca2+ sequestration was probably due to its coaccumulation with the intraluminar Pi originated during glucuronidation. The increase in extravesicular osmolarity due to the addition of UDP-glucuronic acid to microsomes resuspended in an hyposmotic medium caused a rapid and prolonged shrinking as revealed by light-scattering measurements. This indicates a poor permeability of microsomal membrane to UDP-glucuronic acid. The subsequent addition of the pore-forming compound alamethicin resulted in an immediate swelling of vesicles indicating a rapid entry of UDP-glucuronic acid. Alamethicin also caused an about 15-fold increase in p-nitrophenol UDP-glucuronosyltransferase activity. These results support the hypothesis of the intravesicular compartmentation of the microsomal UDP-glucuronosyltransferase catalytic site.

Adenosine Triphosphate↗

PubMed1994

Alterations of hepatocyte Ca2+ homeostasis by triethylated lead (Et3Pb+): are they correlated with cytotoxicity?

Isolated rat hepatocytes were used to investigate the biochemical mechanisms of toxicity of triethyllead (Et3Pb+), a highly neurotoxic degradation product of the antiknocking petrol additive tetraethyllead. As early as 5 min from the addition of 50 microM Et3Pb+ to hepatocyte suspensions a decrease of mitochondrial membrane potential and of the capacity of mitochondria and microsomes to retain Ca2+ occurred. A dose-dependent release of mitochondrial Ca2+ as well as an inhibition of microsomal Ca(2+)-ATPase activity were also evident when Et3Pb+ (from 2.5 microM up to 50 microM) was added to, respectively, isolated liver mitochondria and microsomes. Further experiments using hepatocytes loaded with the Ca2+ indicator Fura-2AM demonstrate that 1 min from addition of Et3Pb+ the cytosolic free Ca2+ levels increased by about 3-fold. High affinity plasma membrane Ca(2+)-ATPase activity was also significantly inhibited in hepatocytes treated with Et3Pb+, suggesting that an impairement of the mechanisms controlling the efflux of extracellular Ca2+ was concomitantly involved in the rise in cytosolic Ca2+ concentration. The increase in the cytosolic Ca2+ levels caused by Et3Pb+ was followed by a rapid decline of cell viability. However, the addition of EGTA or of the intracellular Ca2+ chelator BAPTA/AM did not affect either the time-course or the extent of cytotoxicity. Conversely, fructose, a glycolytic substrate that was able to support ATP production, prevented hepatocyte death. Thus, the depletion of cellular energy stores rather than the increase in cytosolic Ca2+ appears to be the mechanism by which Et3Pb+ causes irreversible injury in isolated hepatocytes.

Animals↗

PubMed1994

Fatty acyl-CoA esters induce calcium release from terminal cisternae of skeletal muscle.

The effect of palmitoyl-CoA (PCoA) on Ca2+ fluxes in unfractionated SR, longitudinal tubules (LSR) and terminal cisternae (TC) subfractions, obtained from rabbit fast-twitch skeletal muscles, was investigated. After MgATP-dependent Ca2+ preloading, PCoA released Ca2+ from unfractionated SR and TC, but not from LSR. Both the extent and the rate of PCoA-induced Ca2+ release from TC were increased in a dose-dependent manner, the half-maximal effect being attained at [PCoA] of approximately 6 microM. Ruthenium red, a Ca2+ release channel blocker, completely inhibited PCoA-induced Ca2+ release, whereas caffeine, a Ca2+ release channel agonist, depleted TC of Ca2+ and prevented the PCoA action. Scatchard plot analysis of [3H]-ryanodine binding showed that PCoA increased the affinity without affecting Bmax. The action of PCoA was mimicked by a nonhydrolysable analog. The present results indicate that PCoA interacts and opens the Ca2+ release channel (ryanodine receptor) of TC and that the mechanism of action involves binding rather than hydrolysis.

Acyl Coenzyme A↗

PubMed1993

CoA and fatty acyl-CoA derivatives mobilize calcium from a liver reticular pool.

The effect of CoA and fatty acyl-CoA esters on Ca2+ fluxes has been studied in isolated liver microsomes and in digitonin-permeabilized hepatocytes. When microsomes were loaded with increasing concentrations of Ca2+ (6-29 nmol/mg of protein), the extent to which CoA and palmitoyl-CoA released Ca2+ increased. At 23 nmol of Ca2+/mg of protein, half-maximal [CoA] and [palmitoyl-CoA] were 35 and 50 microM respectively. Under conditions of minimal Ca2+ loading, net release of Ca2+ was absent, but Ca2+ translocation from a CoA-sensitive to a CoA-insensitive pool took place. The effect of CoA required the presence of fatty acids, probably to form fatty acyl esters. In permeabilized hepatocytes, the pool(s) mobilized by CoA (or by palmitoyl-CoA) appeared to be different from that mobilized by Ins(1,4,5)P3.

Acyl Coenzyme A↗

PubMed1993

Evidence that stimulation of plasma-membrane Ca2+ inflow is an early action of glucagon and dibutyryl cyclic AMP in rat hepatocytes.

The ability of glucagon (1 nM) and of dibutyryl cyclic AMP (50 microM) to increase cytosolic free Ca2+ concentration ([Ca2+]i) in Fura-loaded rat hepatocytes was examined in a system wherein Ca2+ inflow was induced by the re-admission of excess Ca2+ to a nominally Ca(2+)-free medium. An increase in [Ca2+]i did not occur in the absence of either agonist, but did so after co-addition of either agonist with Ca2+. Increasing the time between addition of dibutyryl cyclic AMP (or of glucagon) and Ca2+ led to increases in [Ca2+]i; half-maximal and maximal increases were observed at 0 s (i.e. at co-addition) and 5-7 s respectively. Dibutyryl cyclic AMP and Ca2+ each exhibited a concentration-dependence when their respective concentrations were changed for a fixed time interval between additions. Half-maximal and maximal effects were obtained with 30 microM and 50 microM dibutyryl cyclic AMP and with 0.5 mM and approx. 1 mM Ca2+ respectively. The data demonstrate an early action of glucagon and dibutyryl cyclic AMP on [Ca2+]i. It is argued that the agonist-induced rise in [Ca2+]i results from an increase in plasma-membrane Ca2+ inflow, an effect that appears to occur much earlier than that on mobilization of internal stores of Ca2+.

Animals↗