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Biomedical subjects

Luis M Botana

Publications and source records attributed to Luis M Botana.

At least 19 recordsLinked to original sources
PubMed2007

Effects of azaspiracid-1, a potent cytotoxic agent, on primary neuronal cultures. A structure-activity relationship study.

Azaspiracids (AZAs) are marine phycotoxins with an unknown mechanism of action, implicated in human intoxications. We investigated the effect of azaspiracid-1 (AZA-1) on the cytosolic calcium concentration ([Ca2+]c), intracellular pH (pHi), and neuron viability in neuronal cultures. AZA-1 increased [Ca2+]c and decreased neuronal viability. The effects of several fragments of the AZA-1 molecule (13 different chemical structures) were examined. The ent-ABCD-azaspiracid-1 (2) showed similar potency to AZA-1 (1) in increasing [Ca2+]c but higher cytotoxity than AZA-1. The chemical structures containing only the ABCD or the ABCDE ring domains (3-8) caused a [Ca2+]c increase but did not alter cell viability. The compounds containing only the FGHI ring domain of AZA-1 (9-14) did not modify the [Ca2+]c or the cell viability. Therefore, the effect of AZA-1 on [Ca2+]c depends on the presence of the ABCD or the ABCDE-ring structure, but the complete chemical structure is needed to produce neurotoxic effects.

Actins↗
PubMed2007

Effects of the marine phycotoxin palytoxin on neuronal pH in primary cultures of cerebellar granule cells.

Palytoxin (PTX) is a potent marine phycotoxin that binds to the Na,K-ATPase, converting this pump into an open channel. We have recently shown (Vale et al., 2006) that PTX causes an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)) in primary cultures of cerebellar granule cells (CGC). In this work, we investigated the effect of PTX on the intracellular pH (pH(i)) in the same cellular model. PTX-induced changes in pH(i) were studied in CGC by using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). PTX caused an irreversible intracellular acidification of CGC. This acidification was due to an influx of extracellular calcium, inasmuch as it was completely abolished by the use of Ca(2+)-free medium. Different mechanisms that could be involved in the PTX-induced pH(i) decrease such as displacement of H(+) by Ca(2+) from a common intracellular binding site, PTX-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by an increase of mitochondrial Ca(2+) uptake by PTX were excluded. PTX-induced intracellular acidification was completely prevented by several inhibitors of the plasma membrane calcium ATPase (PMCA), including orthovanadate, lanthanum, high extracellular pH, and caloxin 2A1. Our results indicate that the PMCA is involved in the PTX-induced intracellular acidification in primary cultures of CGC. The PTX-evoked increase in [Ca(2+)](c) will activate the calcium extrusion mechanisms through the PMCA, which, in turn, will decrease pH(i) by countertransport of H(+) ions. The effect of PTX on neuronal pH could be a potential factor to contribute to the high cytotoxicity of this toxin in cultured cerebellar neurons.

Acrylamides↗
PubMed2006

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1.

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.

Alkalies↗
PubMed2006

Calcium-pH crosstalks in the human mast cell line HMC-1: intracellular alkalinization activates calcium extrusion through the plasma membrane Ca2+-ATPase.

The human mast cell line (HMC-1) has been used to study the relationship between intracellular pH and cytosolic calcium (Ca2+) in mast cells. Thapsigargin (TG) caused store-operated Ca2+ entry, that is enhanced by the PKC activator PMA. NH4Cl-induced alkalinization showed an inhibitory effect on TG-sensitive stores depletion (not on TG-insensitive stores), and also on final cytosolic Ca2+ levels reached in response to both TG and the ionophore ionomycin. Loperamide, a positive modulator of store-operated channels, induced a slight Ca2+ entry by itself, and also increased TG-induced Ca2+ entry. This enhancement was not enough to reverse the inhibitory effect of NH4Cl-induced alkalinization. When comparing the effect of NH4Cl-induced alkalinization on Ca2+ levels, with those observed using Ca2+ channel blockers (namely Ni2+ and SKF-96365), cytosolic profiles for this ion are different, either in modified saline solution or in HCO3(-)-free medium. Thus, it seems unlikely that the inhibitory effect of NH4Cl-induced alkalinization on Ca2+ is taking place by blockage of Ca2+ entry. Furthermore, inhibition of the plasma membrane Ca2+-ATPase (an important mechanism for Ca2+ efflux) with sodium orthovanadate (SO) matches with the inhibition of the negative effect on Ca2+ levels elicited by NH4Cl. Data indicate that NH4Cl-induced alkalinization might be activating Ca2+ efflux from the cell, by stimulation of the plasma membrane Ca2+-ATPase, and also confirm our previous finding that Ca2+ is a secondary signal to activate HMC-1 cells.

Ammonium Chloride↗
PubMed2006

Role of the plasma membrane calcium adenosine triphosphatase on domoate-induced intracellular acidification in primary cultures of cerebelar granule cells.

Changes in intracellular pH (pH(i)) and cytosolic calcium concentration ([Ca(2+)](c)) caused by the glutamate agonist domoate (DOM) were studied in single cultured mouse cerebellar granule cells (CGC) by using the fluorescent probes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and simultaneous evaluation of cytosolic calcium concentration with the fluorescent dye Fura-2 acetoxymethyl ester (Fura-2 AM). DOM caused a concentration-dependent increase in [Ca(2+)](c) and a concentration-dependent intracellular acidification of CGC. DOM-induced intracellular acidification was completely abolished by the use of Ca(2+)-free medium, suggesting that it was due mostly to an influx of extracellular calcium. The pH(i) decrease caused by DOM was also completely blocked in the presence of the AMPA/kainate receptor antagonist CNQX, indicating that the DOM-induced intracellular acidification was caused by DOM activation of the AMPA/kainate subtype of glutamate receptors. Different mechanisms that could be involved in DOM-induced pH(i) decrease, such as displacement of H(+) by Ca(2+) from a common intracellular binding site, DOM-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by DOM-induced increase of mitochondrial Ca(2+) uptake, were excluded. DOM-induced intracellular acidification was completely prevented by inhibitors of the plasma membrane calcium adenosine triphosphatase (ATPase) (PMCA), including orthovanadate, lanthanum extracellular pH of 8.5, and the specific PMCA inhibitor caloxin 2A1. Our results therefore indicate that PMCA is involved in DOM-induced intracellular acidification in primary cultures of CGC. Simultaneous recording of [Ca(2+)](c) and pH(i) indicates that the increase in intracellular calcium evoked by DOM will activate the calcium extrusion mechanisms through the calcium pump, which, in turn, will decrease intracellular pH by countertransport of H(+) ions.

Acid-Base Equilibrium↗
PubMed2006

The sodium channel of human excitable cells is a target for gambierol.

BACKGROUND: Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized. METHODS: We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium. RESULTS: We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxin-induced depolarization and iv) increased cytosolic calcium in neuroblastoma cells. CONCLUSION: These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.

Calcium↗
PubMed2006

KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients.

Despite the relevance of the c-kit/stem cell factor (SCF) signaling pathway in mast cell (MC) diseases, the exact frequency of KIT mutations in different compartments of bone marrow (BM) hematopoietic cells of individuals with systemic mastocytosis (SM), and its different diagnostic categories, remains unknown. In this study, we prospectively analyzed the presence of KIT mutations in fluorescence-activated cell-sorting (FACS)- purified populations of BM MCs (n = 113) and other BM cell compartments (n = 67) from adults with SM. Our results show the presence of D816V KIT mutation in virtually all adults (93%) with indolent and aggressive forms of SM, except well-differentiated SM (29%), while other KIT mutations were rarely (< 3%) detected. In around one-third of patients with mutated MCs, the KIT mutation was also detected in CD34+ hematopoietic cells and eosinophils, and, to a lesser extent, in monocytic, neutrophil-lineage BM precursor cells and lymphocytes. Most patient with poor-prognosis SM (81%) carried the KIT mutation in 2 or more BM myeloid cell populations, while this was detected in a smaller proportion (27%) of indolent cases. These results would support the notion that KIT mutation is a hallmark of adult SM where it targets a pluripotent hematopoietic stem cell, and may contribute to explaining previously observed discrepancies in the literature.

Adolescent↗
PubMed2006

Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin.

A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na(+). Li(+) decreased the PTX-evoked rise in [Ca(2+)](c); replacement of Na(+) by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca(2+)](c) increase by PTX was strongly reduced after inhibition of the reverse operation of the Na(+)/Ca(2+) exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca(2+)-ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na(+)-dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na(+)/Ca(2+) exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space.

Acrylamides↗
PubMed2006

Study of the interaction between different phosphodiesterases and yessotoxin using a resonant mirror biosensor.

Yessotoxins (YTXs) are disulfated polyether toxins that were first isolated from scallops in Japan. It has been proposed that these toxins activate cellular phosphodiesterases (PDEs). The interaction between YTX and PDEs was confirmed by resonant biosensor and fluorescence polarization studies. The aim of this work is to study the specificity of different PDEs for YTX binding. Association measurements are done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers from the sensor surface where the association PDEs-YTX takes place. We use aminosilane cuvettes, where exonuclease Phosphodiesterase I from Crotalus atrox (PDE I), exonuclease Phosphodiesterase II from bovine spleen (PDE II), or phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) are immobilized. Over immobilized exonuclease PDE I and exonuclease PDE II are added different amounts of YTX, and typical association curve profiles are observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration, the association rate constant (k(ass)) and the dissociation rate constant (k(diss)) are obtained. From these values, the kinetic equilibrium dissociation constant (K(D)) of the YTX-PDE association can be calculated, indicating the affinity between them. The specificity of cyclic nucleotide PDE families is studied using different inhibitors that are added over immobilized cyclic nucleotide PDEs. In these conditions, changes in the association PDEs-YTX curves are detected. The results show YTX affinity by cyclic nucleotide PDE 1, PDE 3, PDE 4, and exonuclease PDE I.

Animals↗
PubMed2006

Cell growth inhibition and actin cytoskeleton disorganization induced by azaspiracid-1 structure-activity studies.

Azaspiracid-1 (AZA-1) is a marine toxin discovered 10 years ago. Since then, toxicologic studies have demonstrated that AZA-1 targets several organs in vivo, including the intestine, lymphoid tissues, lungs, and nervous system; however, the mechanism of action of AZA-1 remains unknown. Studies in vitro suggest that AZA-1 affects the actin cytoskeleton in nonadherent cells. We characterized the effects of AZA-1 on the cytoskeleton of adherent cells and on cell growth, an adhesion-dependent process in many cell types, and analyzed the structure dependency of this toxicity. Confocal and TIRF imaging of fluorescently labeled cytosketon showed that AZA-1 induced the rearrangement of stress fibers (actin filament bundles) and the loss of focal adhesion points in neuroblastoma and Caco-2 cells, without affecting the amount of polymerized actin. AZA-1 did not seem to alter the microtubule cytoskeleton, but it changed the cell shape and internal morphology observed by phase contrast imaging. Cell growth of lung carcinoma and neuroblastoma cells was inhibited by the toxin, as measured by a sulforhodamine B assay and BrdU incorporation to newly synthesized DNA. Fifteen different fragments and/or stereoisomers of AZA-1 were tested for cytoskeletal rearrangement and cell growth inhibition. Results showed that no fragment or stereoisomer had any activity, except for ABCD-epi-AZA-1, which conserved toxicity. AZA-1-induced reorganization of the actin cytoskeleton concurred with detachment and growth inhibition, three events that are probably related.

Actins↗
PubMed2005

Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure.

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Azaspiracid-4 inhibits Ca2+ entry by stored operated channels in human T lymphocytes.

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6-11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca2+]i in freshly human lymphocytes. Cytosolic Ca2+ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca2+ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca2+ levels induced by thapsigargin (Tg). AZ-4 decreased Ca2+-influx induced by Tg but did not affect the Ca2+-release from internal stores induced by this drug. The effects of AZ-4 on Ca2+-influx induced by Tg were reversible and not regulated by adenosine 3',5'-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca2+ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca2+-influx by 5-10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca2+ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.

Calcium Channel Blockers↗
PubMed2005

Kinetic analysis of the interaction between yessotoxin and analogues and immobilized phosphodiesterases using a resonant mirror optical biosensor.

Yessotoxin (YTX) is a disulfated polyether toxin produced by phytoplanktonic microalgae from the dinoflagellates group. YTX has structural similarities to maitotoxin or brevetoxins, and the mechanism of action for YTX is specific and related to calcium modulation. Some studies show that YTX decreases adenosine 3',5'-cyclic monophosphate (cAMP) levels in human lymphocytes. This effect is calcium-dependent, and phosphodiesterases (PDEs) seem to be involved. This point was recently confirmed by studying kinetic constants of YTX-PDEs binding. The aim of this work is to compare the ability of YTX and analogues to bind immobilized PDEs by using a resonant mirror biosensor. These instruments measure biomolecular interactions in real time, without labeling, and allow detailed investigation of the reaction kinetics by analysis of the resultant signals. YTX and the derivatives, 45-hydroxyyessotoxin, carboxyyessotoxin, and yessotoxin-45-(S)-a-methoxy-a-trifluormethylphenylacetate, were added over immobilized PDEs. The kinetic constants obtained from these bindings indicate a relationship between toxin structure and affinity. Since the presence of the functional groups in the side chain decreased the toxic YTX effect and the PDEs-YTX association, our results show that the side chain plays an important role in the YTX affinity for PDEs.

Biosensing Techniques↗
PubMed2004

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions.

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2004

A rapid microplate fluorescence method to detect yessotoxins based on their capacity to activate phosphodiesterases.

This paper describes an easy and fast assay with enough sensitivity to detect yessotoxin (YTX) in shellfish samples. YTX decreases intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels by increasing the activity of phosphodiesterases (PDEs). Looking for new methods to detect YTXs, we developed a technique based on this effect. We use the fluorescent derivative of cAMP, anthranyloyl-cAMP, whose fluorescence decreases in time by hydrolysis effect of PDEs. The fluorescence fall is quantified in a plate reader. PDEs induce an anthranyloyl-cAMP hydrolysis rate that is increased in the presence of YTX. This effect is dose dependent, and the representation of YTX concentration versus rate of hydrolysis follows a lineal regression. The measurable range of YTX in this assay is 0.1 to 10microM, while by mouse bioassay, the official method to detect YTXs, the detection limit is 2microM. We determined by this method the concentration of YTX from alcoholic extracts whose concentrations were first determined by high performance liquid chromatography and the variation of concentration was from 5.26microM by fluorescence to 6microM by high performance liquid chromatography and from 3.16 by fluorescence to 3microM by HPLC.

Animals↗
PubMed2004

Effects of Azaspiracids 2 and 3 on intracellular cAMP, [Ca2+], and pH.

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4, and AZ-5 and the recently described AZ-6-11. Toxic episodes of AZs show gastrointestinal illness as in diarrhetic shellfish poisoning, but neurotoxic symptoms are also observed in a mouse bioassay. Despite their great importance in human health, so far, its mechanism of action is largely unknown. In this report, we present the first data of AZ-2 and AZ-3 effects on intracellular cyclic adenosine monophosphate (cAMP), intracellular calcium ([Ca(2+)](i)), and cytosolic pH levels (pH(i)) in freshly human lymphocytes. The variations of cAMP, calcium, and pH were determined by fluorescence digital imaging microscopy using recombinant fluorescein- and rhodamine-labeled protein kinase A, Fura2-AM, and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, respectively. Our experiments show that both analogues, AZ-2 and AZ-3, clearly increase cytosolic cAMP levels of human lymphocytes. In calcium studies, we found that only if cells are initially in a calcium-free medium, AZ-2 increases the intracellular calcium concentration with two components: Ca(2+) release from internal stores and Ca(2+) influx from extracellular medium. AZ-2 sensitive Ca(2+) stores seem to be different from the thapsigargin sensitive one. AZ-2-induced Ca(2+) influx is mediated through Ni(2+) and SKF96365 blockable channels, and it is additive with Tg-induced Ca(2+) influx. Surprisingly, AZ-3 does not empty intracellular stores but also increases cytosolic calcium levels. This AZ-3-induced Ca(2+) influx is mediated through Ni(2+) blockable channels, and it is not additive with Tg-induced Ca(2+) influx. In addition, AZ-3 slightly alkalinizes cytosol. In accordance with cAMP studies, we found that adenylyl cyclase (AC) modulation inhibits AZ-2- and AZ-3-evoked Ca(2+) increase and AZ-3-induced pH(i) rise. Thus, both analogues seem to involve an AC pathway, although its effects on [Ca(2+)](i) and pH(i) are quite different.

Calcium↗
PubMed2003

The Cl-HCO3- exchanger slows the recovery of acute pHi changes in rat mast cells.

The CO(2)/HCO(3)(-) buffering system is one of the main mechanisms implicated in cytosolic pH (pH(i)) regulation. We studied this pH(i)-regulatory system in rat mast cells using a fluorescent dye. Mast cells had a more alkaline pH(i) in the presence of HCO(3)(-) than in its absence. The recovery from an acid load was faster in HCO(3)(-)-free conditions than in HCO(3)(-)-containing media. In HCO(3)(-)-buffered conditions the increase of the recovery rate of an acidification in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-incubated cells suggested the implication of a Na(+)-independent Cl(-)/HCO(3)(-) exchanger. This HCO(3)(-) transport acidified the cytosol and was also partially responsible for the recovery of intracellular alkalinizations. Moreover, regulation of the recovery rate of an acidification by protein kinase C and calcium signaling pathways depended on the presence or absence of HCO(3)(-). The presence of HCO(3)(-) limits the recovery of acute intracellular acidifications probably through the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and modulates the regulation of pH(i) by protein kinase C and calcium.

Animals↗