Biomedical subjects

A G Cabado

Publications and source records attributed to A G Cabado.

17 recordsLinked to original sources

PubMed2003

Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells.

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.

Actins↗

PubMed2003

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Actins↗

PubMed2000

Hypertonicity-induced intracellular pH changes in rat mast cells.

In a non-isotonic environment, cells can shrink or swell and return to their normal shape by activating ion transport pathways. Changes in intracellular pH (pHi) after osmotic stress have been identified in several cells. In order to study the mechanisms that regulate cytosolic pH of rat mast cells in a hypertonic medium, we used the pH sensitive dye, BCECF. Under these hypertonic conditions, pHi undergoes an alkalinization following an initial acidification. The alkalinization is mediated by a Na+/H+ exchanger, since it is inhibited by amiloride and lack of extracellular sodium. Under these conditions, the alkalinization is increased with the PKC activators, TPA and OAG, and partially blocked with trifluoperazine, an unspecific protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinases (Ca2+/CaM K) inhibitor. There is also an anion exchanger, blocked with DIDS but not activated by PKC, that participates in the observed alkalinization. However, Na+/H+ exchanger is the main mechanism involved in the alkalinization of pHi of mast cells in a hyperosmotic environment.

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

PubMed2000

Functional compartments in rat mast cells for cAMP and calcium on histamine release.

The crosstalk between 3', 5'-cyclic adenosine monophosphate (cAMP), intracellular calcium, and histamine release in rat mast cells using the stimulatory effect of three different drugs, thapsigargin, sodium fluoride (NaF), and compound 48/80 were studied. Each of these drugs induces histamine release by different mechanisms. The transducting pathways modulating cAMP and intracellular calcium levels were modified by using, cholera toxin (CTX) which ADP-rybosylates Gs-protein, pertussis toxin (PTX) which ADP-rybosylates Gi-protein, and okadaic acid (OA) which inhibits phosphatases 1 and 2a. Our results show that CTX increased cAMP levels and inhibited histamine release elicited by thapsigargin and compound 48/80. The inhibitory effect of CTX on histamine release was potentiated by OA in the presence of compound 48/80 but was decreased in the presence of thapsigargin. Calcium uptake was stimulated by NaF and compound 48/80. The previous treatment with OA increased calcium uptake when combined with compound 48/80 but not with NaF. Treatment with NaF highly stimulated calcium uptake and cAMP levels only when combined with OA and CTX. These results suggest that the modulatory effect of intracellular calcium and cAMP on histamine release depend more on the crosstalk of the activated signal transducting pathway than on the final level of calcium or cAMP, further supporting the theory that rat mast cells are divided into functionally distinct compartments.

Animals↗

PubMed2000

Crosstalk between cytosolic pH and intracellular calcium in human lymphocytes: effect of 4-aminopyridin, ammoniun chloride and ionomycin.

Stimulation of lymphocytes by specific antigens is followed by the activation of different signal transduction mechanisms, such as alterations in the cytoplasmic levels of Ca(2+), H(+) and variations in membrane potential. To study interrelationships among these parameters, changes in pHi and Ca(2+) were measured with the fluorescent probes BCECF and Fura-2 in freshly isolated blood human lymphocytes. Moreover, membrane potential qualitative alterations were recorded with the fluorescent dye bis-oxonol. In a bicarbonate-free medium, cell alkalinization with NH(4)Cl slightly decreased intracellular Ca(2+) concentration ([Ca(2+)](i)) due to efflux of Ca(2+) from the cell. In contrast, an elevation of pHi induced with 4-AP increased [Ca(2+)](i), either in the presence or absence of external Ca(2+). The increase in Ca(2+)-free medium is likely to be due to Ca(2+) release from thapsigargin and caffeine-independent intracellular stores. Both 4-AP or NH(4)Cl induced a plasma membrane depolarisation, although with different kinetics. The ionosphere ionomycin increased pHi, Ca(2+) levels and also induced membrane depolarisation. Together, these observations demonstrate a lack of correlation between the magnitude of changes in pHi and Ca(2+).

4-Aminopyridine↗

PubMed2000

Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation.

The aim of this work was to study the relationship between intracellular alkalinization, calcium fluxes and histamine release in rat mast cells. Intracellular alkalinization was induced by nigericin, a monovalent cation ionophore, and by NH(4)Cl (ammonium chloride). Calcium cytosolic and intracellular pH were measured by fluorescence digital imaging using Fura-2-AM and BCECF-AM. In rat mast cells, nigericin and NH(4)Cl induce a dose-dependent intracellular alkalinization, a dose-dependent increase in intracellular calcium levels by releasing calcium from intracellular pools, and an activation of capacitative calcium influx. The increase in both intracellular calcium and pH activates exocytosis (histamine release) in the absence of external calcium. Under the same conditions, thapsigargin does not activate exocytosis, the main difference being that thapsigargin does not alkalinize the cytosol. After alkalinization, histamine release is intracellular-calcium dependent. With 2.5 mM EGTA and thapsigargin the cell response decreases by 62%. The cytosolic alkalinization, in addition to the calcium increase it is enough signal to elicit the exocytotic process in rat mast cells.

Ammonium Chloride↗

PubMed1999

Evidence for an electrogenic, negatively protein-kinase-A-modulated, Na+-dependent HCO3- transporter in human lymphocytes.

We studied the effects of external HCO3- on pHi regulation in human lymphocytes after an acid load. Cells were acidified by preincubation with NH4Cl and pHi recovery was measured with the fluorescent dye BCECF. Cells recovering in HCO3--containing medium reached a higher final pHi, the H+ efflux rate was increased and shifted to alkaline pHi compared to that of cells recovering in HCO3--free solution. The resting pHi was higher in a HCO3--containing solution. Experiments carried out in the presence of amiloride, DIDS and in the absence of external Na+ suggest the existence of two major mechanisms acting in the pHi recovery of lymphocytes after an acid load: an amiloride-sensitive Na+/H+ exchanger and a DIDS-sensitive Na+-dependent HCO3- transporter. The last mechanism could be a Na+/HCO3- cotransporter based on membrane potential changes determined with the potential-sensitive fluorescent probe bis-oxonol. Preincubation of cells with forskolin and H-89 showed protein-kinase-A-dependent downregulation of the amiloride-insensitive recovery of pHi in human lymphocytes. In summary, this paper provides functional evidence for the existence of a Na+/HCO3--dependent mechanism involved in pHi recovery in human lymphocytes following an acid load, that is electrogenic and downregulated by PKA.

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

PubMed1999

Membrane potential changes associated with calcium signals in human lymphocytes and rat mast cells.

Human lymphocytes and rat mast cells, two non-excitable cellular models, were used to investigate membrane potential changes accompanying Ca2+ signals. Cells were stimulated with agents known to induce both Ca2+ release from internal stores and influx of extracellular Ca2+, namely thapsigargin, ionomycin and compound 48/80. Thapsigargin and ionomycin were used to activate lymphocytes, while compound 48/80 was used to stimulate mast cells. Membrane potential changes and Ca2+ concentration were monitored with the fluorescent dyes bis-oxonol and fura-2, respectively. In lymphocytes, thapsigargin induced a hyperpolarization temporally correlated with the increase in intracellular Ca2+ concentration. This hyperpolarization is due to activation of a K+ conductance which consists of two phases, a first phase independent on external Ca2+ and a second one blocked in a Ca2+-free medium. Ionomycin induced a Ca2+-dependent depolarization attributed to a massive influx of external Ca2+. On the other hand, stimulation of mast cells with compound 48/80 produced a fast hyperpolarization and an increase in intracellular Ca2+ levels. Besides different time-courses, this hyperpolarization differs from that induced by thapsigargin in lymphocytes in two aspects, it is mainly due to a Cl(-)-entry current and exit of K+ and it is completely inhibited in the absence of extracellular Ca2+. Compound 48/80-induced histamine release is not related to membrane potential changes.

Animals↗

PubMed1998

Inhibition of Na+/K+ ATPase under hypertonic conditions in rat mast cells.

Ionic fluxes that contribute to changes in membrane potential and variations of pHi (intracellular pH) are not well known in mast cells, although they can be important in the stimulus-secretion coupling. Cellular volume regulation implies changes in the concentration of intracellular ions, such as sodium and potassium and volume changes can be imposed varying the tonicity of the medium. We studied the physiology of sodium and examined the effect of ouabain on [22Na] entry in mast cells in isotonic and hypertonic media. We also recorded changes in membrane potential and pHi using the fluorescent dyes bis-oxonol (Bis-(1,3-diethylthiobarbituric acid) trimethineoxonol) a n d BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) in hypertonic conditions. The results show that [22Na] influx increases four fold in hypertonic solutions and it is mediated mainly by an amiloride-sensitive Na+/H+ exchanger. This transporter is involved in the shrinkage-activated cellular alkalinization and the pHi recovery is accelerated by inhibition of the Na+/K+ ATPase with ouabain in the absence of extracellular calcium. Under hypertonic conditions 22Na influx is apparently not increased by ouabain, while the Na+/K+ ATPase inhibitor clearly increases [22Na] uptake and also induces membrane depolarization in isotonic conditions. All together, these findings suggest that Na+/K+ ATPase is partially inhibited in hypertonic conditions.

Animals↗

PubMed1997

Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. phosphorylation-dependent and -independent mechanisms.

We recently identified a region within the cytoplasmic C-terminal tail of the Na+/H+ exchanger NHE3 isoform (residues 579 to 684) which is essential for inhibition of transport activity by cAMP-dependent protein kinase (PKA) (Cabado, A. G., Yu, F. H., Kapus, A., Gergely, L., Grinstein, S., and Orlowski, J. (1996) J. Biol. Chem. 271, 3590-3599). To further define determinants of PKA regulation, six serine residues located in potential recognition sequences for PKA within, or adjacent to, this region (positions 552, 605, 634, 661, 690, and 691) were altered either independently or in various combinations using site-directed mutagenesis. Wild type and mutant NHE3s tagged with the influenza virus hemagglutinin epitope were stably expressed in exchanger-deficient Chinese hamster ovary cells (AP-1) for functional studies. Of the individual mutations examined, only substitutions at Ser605 or Ser634 affected sensitivity to forskolin, an activator of adenylate cyclase, although partial inhibition of NHE3 activity by forskolin remained. By contrast, simultaneous mutation of both these serines completely abolished cAMP-mediated inhibition of NHE3 without greatly affecting basal transport activity. Two-dimensional analysis of tryptic digests of immunoprecipitated NHE3 labeled in vivo with [32P]orthophosphate revealed several phosphopeptides under basal conditions. Phosphorylation was increased approximately 3-fold in one of these peptides following forskolin treatment, and this change was eliminated by mutation of residue Ser605. Thus, phosphorylation of Ser605 is essential for cAMP-mediated inhibition of NHE3. In addition, Ser634 is also required for the effect of cAMP, even though this residue does not become phosphorylated upon activation of PKA.

Amiloride↗

PubMed1996

Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform.

Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N-terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.

Adenosine Triphosphate↗

PubMed1994

Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells.

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na+/K+ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium.

Animals↗

PubMed1994

Effect of lyophilization on the stability of gonyautoxins obtained from contaminated mussels.

This study describes the stability of gonyautoxins (GTX) and C toxins obtained from contaminated mussels and stored at different temperatures in lyophilized samples. Analyses of extracts from mussels contaminated with paralytic shellfish poison (PSP) indicated the presence of gonyautoxins as the major component in red tides of the North-West coast of Spain. These GTX and C toxins were extracted from contaminated mussels (Mytilus galloprovincialis Lmk) and partially purified by chromatography on Bio-Gel P-2 and Bio-Rex 70. The stability of these toxins was analysed by high performance liquid chromatography. GTX 4 and GTX 6 are the most stable toxins among GTX. We conclude that the lyophilization procedure is not the safest way to process most of the gonyautoxins. However, the lyophilization procedure made the C toxins unstable, so clearly this procedure must be rejected.

Animals↗

PubMed1993

Solid-phase radioreceptor assay for paralytic shellfish toxins.

Sodium channels obtained from rat brain membrane preparations were coated onto microtiter plates and used to develop a direct solid-phase binding assay. The tritiated sodium channel blocker saxitoxin ([3H]-saxitoxin; STX) was used to detect toxins in paralytic shellfish poisoning (PSP) by measuring the competitive displacement of other toxins. With this assay the amount of STX and tetrodotoxin needed to displace 50% of bound [3H]STX was 1.7 and 1.76 ng/ml for buffer samples, respectively. In the direct solid-phase binding assays, the PSP toxins were effectively bound to the rat brain membranes. The IC50 of this assay for different PSP toxin solutions obtained from mussels contaminated in red tides ranged from 0.03 to 0.30 ng/ml. Therefore, this assay represents a potentially useful method for the detection of toxin-contaminated mussels.

Animals↗

PubMed1993

Amiloride-dependent transport is the main mechanism implicated in sodium influx regulation in rat mast cells.

Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by which mast cells regulate sodium levels, we have examined the effect of amiloride and ouabain on 22Na entry in rat mast cells in isotonic and hypertonic conditions. Ouabain (0.5 mM) enhances sodium uptake by 32% in isotonic conditions. Hypertonicity increases by 400% the uptake of sodium through an amiloride (1 mM) dependent mechanism. Ouabain has no appreciable effect on the entry of 22Na in hypertonic conditions.

Amiloride↗

PubMed1993

Rat pleural and peritoneal mast cells stimulated at different cellular levels: difference in and influence of purification media.

We checked the effect that purification through different media can produce on rat mast cell response, considering different populations of pleural and peritoneal mast cells. We compared the media Ficoll, Percoll, bovine serum albumin and sucrose with the seldom used Nycodenz. Histamine release was elicited with compound 48/80, polymyxin B, ionophore A23187 and calcium on cells preincubated with FNa. High osmolarity media induced the most important changes on the cell response as compared to low osmolarity media. Our results show that Ficoll and sucrose induced the most notable changes in the response. Also, the more sensitive stimulus to the purification procedure is sodium fluoride-calcium. The response to ionophore A23187 did not show any important alteration.

Animals↗

PubMed1992

Influence of protein kinase C, cAMP and phosphatase activity on histamine release produced by compound 48/80 and sodium fluoride on rat mast cells.

We have studied the effect of protein kinase C and protein kinase A activation, and phosphatase inhibition on two different stimuli with distinct mechanisms of action. The first stimulus is compound 48/80, and its action is mediated probably by a Gi-protein, while the other is sodium fluoride, which unspecifically activates G-proteins. We established a comparative study because the action of compound 48/80 is calcium-independent, while fluoride is strictly calcium-dependent. The activation of protein kinase C was attained with the phorbol esther 12-O-tetradecanoylphorbol-13-acetate, protein kinase A was activated by increasing cAMP levels with forskolin or rolipram, and the phosphatase activity was inhibited with okadaic acid (OA), which inhibits phosphatases type 1 and 2A. Our results show that OA enhances the response to fluoride and compound 48/80 in the absence of calcium, and we conclude that calcium has a negative feedback role on the cell response. Protein kinase A activation strongly inhibits the response to fluoride, and the results show a positive regulation of protein kinase C and a negative regulation of protein kinase A over fluoride response. As previously reported by other authors for the ionophore A23187, TPA notably potentiates the response to fluoride, which supports its possible modulatory role on extracellular calcium-dependent stimuli.

Animals↗