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R Fulceri

Publications and source records attributed to R Fulceri.

At least 37 records · Page 2Linked to original sources
PubMed1996

Permeability of liver microsomal membranes to glucose.

The permeability of rat liver microsomes to glucose has been studied by using (14)C-labelled D-glucose and a light-scattering technique. 1) The microsomal intravesicular apparent isotope space for D-glucose (1mM; after 5 min incubation at 22 degrees C) was 2.34 microl/mg protein, i.e., approximately 72% of the apparent water space. 2) Efflux of [(14)C]D-glucose from microsomal vesicles pre-loaded as in 1) and measured by rapid Millipore filtration after dilution (100 fold) in a glucose-free medium revealed that 15 sec after dilution only 15% of intravesicular glucose was still retained by microsomes. 3) Osmotic behaviour of microsomes upon addition of D-glucose measured by a light-scattering technique revealed a glucose influx, saturable at [D-glucose] > 100 mM, and (partially) inhibited by pentamidine and cytochalasin B. Ascorbic acid, L-glucose and other monosaccharides and related compounds also permeated liver microsomes in a fashion similar to D-glucose. These data indicate the existence of a facilitative transport system(s) for glucose in the membrane of liver endoplasmic reticulum vesicles.

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

Inhibition of p-nitrophenol glucuronidation by calcium mobilizing hormones.

1. Vasopressin and phenylephrine markedly inhibited the glucuronidation of p-nitrophenol in isolated murine hepatocytes. 2. After longer preincubation of hepatocytes in the presence of vasopressin or phenylephrine the rate of conjugation began to return to the control values indicating the reversibility of the inhibition caused by these agents. 3. The inhibitory effect of both agents was dependent on the Ca2+ filled state of the intracellular stores. 4. The inhibition caused by the alpha 1 receptor agonist phenylephrine was receptor mediated because it could be prevented by the addition of alpha 1 antagonist prazosin. 5. The data support the theory that the maintenance of the intralumenal Ca2+ concentration is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyl-transferase.

Adrenergic alpha-1 Receptor Agonists↗
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

Latency is the major determinant of UDP-glucuronosyltransferase activity in isolated hepatocytes.

The glucuronidation of p-nitrophenol was measured in intact, saponin- and alamethicin-treated isolated mouse hepatocytes. In saponin-permeabilized cells the elevation of extrareticular UDP-glucuronic acid concentration enhanced the rate of glucuronidation threefold. When intracellular membranes were also permeabilized by alamethicin, a further tenfold increase in the glucuronidation of p-nitrophenol was present. Parallel measurements of the ER mannose 6-phosphatase activity revealed that saponin selectively permeabilized the plasma membrane, whereas alamethicin permeabilized both plasma membrane and ER membranes. The inhibition of p-nitrophenol glucuronidation by dbcAMP in intact hepatocytes was still present in saponin-treated cells and disappeared in alamethicin-permeabilized hepatocytes. It is suggested that the permeability of the endoplasmic reticulum membrane is a major determinant of glucuronidation not only in microsomes but in isolated hepatocytes as well.

Alamethicin↗
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↗
PubMed1993

Intraluminal calcium of the liver endoplasmic reticulum stimulates the glucuronidation of p-nitrophenol.

The relationship between the intraluminal Ca2+ content of endoplasmic reticulum and the rate of the glucuronidation of p-nitrophenol was investigated in isolated rat hepatocytes. Different agents which decrease the Ca2+ level in the endoplasmic reticulum [calcium ionophores (A23187, ionomycin) or Ca(2+)-ATPase inhibitors(thapsigargin,2,5-di-(t-butyl)-1,4-benzohydroquinone+ ++)] inhibited the conjugation of p-nitrophenol. Depletion of intracellular Ca2+ stores by preincubation of hepatocytes in the absence of free Ca2+ (in the presence of excess EGTA) also decreased the rate of glucuronidation; Ca2+ re-admission to EGTA-treated hepatocytes restored glucuronidation. In intact liver microsomes the p-nitrophenol UDP-glucuronosyl-transferase activity was not modified by varying the external free Ca2+ concentrations within a cytosol-like range. Emptying of the Ca2+ from the lumen of microsomal vesicles by A23187, after MgATP-stimulated Ca2+ sequestration, decreased the glucuronidation of p-nitrophenol. A similar effect was observed in filipin-permeabilized hepatocytes. In native and in detergent-treated microsomes, Ca2+ (1-10 mM) increased the p-nitrophenol UDP-glucuronosyltransferase activity. It is suggested that the physiological concentration of Ca2+ in the lumen of the endoplasmic reticulum is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyltransferase; the depletion of Ca2+ decreases the activity of the enzyme.

Animals↗
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↗