Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis, Argentina.

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis: Argentina / Caracterización molecular por ERIC-PCR de cepas de Listeria spp. aisladas de diversos orígenes en San Luis: Argentina

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

En este estudio se analizaron 24 cepas de Listeria spp. De ellas, 22 fueron obtenidas en San Luis (Argentina), a partir de muestras humanas, de animales y alimentos. Se incluyeron 2 cepas de referencia Listeria monocytogenes CLIP 22762 y Listeria innocua CLIP 74915. Todos los aislamientos fueron identificados bioquímicamente y caracterizados por serotipificación, fagotipificación y detección del gen flaA por reacción en cadena de la polimerasa (PCR). Se generaron perfiles de bandas de ADN mediante la amplificación de secuencias repetitivas de consenso intergénico de enterobacterias (ERIC-PCR). De acuerdo a los resultados obtenidos por ERIC-PCR, las cepas de Listeria spp. fueron divididas en 3 grupos según su origen. Los perfiles de los aislamientos humanos y animales fueron distintos de los correspondientes a alimentos. Por otra parte, dentro de los grupos I y II se incluyeron 10 cepas de L. monocytogenes y solamente una de Listeria seeligeri. Dentro del grupo III no sólo estuvieron incluidas las 9 cepas de L. innocua sino también 4 de L. monocytogenes. La evaluación de los perfiles de bandas obtenidos por ERIC-PCR permitió la discriminación entre los serotipos ensayados 1/2b, 4b, 6a y 6b dentro de cada grupo. El índice de discriminación calculado para ERIC-PCR fue de 0,94. Los resultados sugieren que la técnica de ERIC-PCR provee un método alternativo válido para la identificación de especies de Listeria y, asimismo, permite la diferenciación de cepas dentro de una misma especie.

Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic beta-cells.

Glucose-induced insulin secretion by pancreatic beta-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic beta-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual beta-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.

Volume changes and whole cell membrane currents activated during gradual osmolarity decrease in C6 glioma cells: contribution of two types of K+ channels.

Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca(2+) dependent. In nominally Ca(2+)-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial -79 mV to a steady state of -54 mV reached at 18% osmolarity reduction [hyposmolarity of -18% (H-18%)]. Recordings of Cl(-) and K(+) currents showed activation at H-3% of an outwardly rectifying Cl(-) current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K(+) current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K(+) channel blockers. With 200 nM Ca(2+) in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from -77 to -63 mV. A K(+) current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K(+) channels in response to GOR in the same cell type, activated by Ca(2+) and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl(-) and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions.

Modulation of the maitotoxin response by intracellular and extracellular cations.

The aim of the present study was to characterize the role played by intracellular and extracellular calcium and sodium on the maitotoxin (MTX) response in Chinese hamster ovary (CHO) cells. The results presented here indicated that: (1) MTX activates calcium and sodium influx in a concentration-dependent manner; (2) extracellular calcium is required for the sodium influx; (3) removal of the extracellular sodium did not prevent the MTX-induced calcium influx; (4) elevation in the intracellular calcium concentration potentiates the MTX response; and (5) MTX, at the concentrations tested, did not compromise cell viability.

Isovolumetric regulation mechanisms in cultured cerebellar granule neurons.

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).

Functional expression of recombinant type 1 ryanodine receptor in insect cells.

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence.

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: a possible role for tyrosine kinases.

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.

SITS blockade induces multiple subconductance states in a large conductance chloride channel.

The effect of the chloride channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) on the gating and amplitude of an endothelial chloride channel was explored using the outside-out configuration of the patch-clamp technique. Under control conditions the channel displayed two main gating modes: shut and fully open. Transitions to equally spaced subconductance states were rarely observed (less than 10 events/minute). At low concentrations (<45 microm), SITS increased the number of transitions to the three subconductance states in a concentration-dependent manner, while reducing the number of transitions to the fully open state. This effect was maintained after removing SITS from the bath solution, suggesting that the modifications in the channel induced by SITS were irreversible. All four conducting states had similar current-voltage relationships. At higher concentrations (>45 microm), SITS reduced the amplitude of all conducting states (three subconductances and fully open). This effect was fully reversible upon SITS removal from the bath solution. A half-inhibitory concentration (IC50) of 55.6 +/- 2.7 microm (+60 mV) and 66.7 +/- 2.2 (-60 mV) was obtained from the fitting to a Langmuir function. All these results are compatible with the existence of two SITS binding sites in the chloride channel: one of high affinity responsible for the increment in the number of transitions to subconductance states, and one low affinity binding site involved in the reduction of the amplitude of all conducting states.

Functional uncoupling between intracellular calcium dynamics and secretion in the alphaT3-1 gonadotropic cell line.

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.

[Listeria spp. in food of animal origin]. / Listeria spp. en alimentos de origen animal.

Different food samples of animal origin were analyzed for Listeria spp. Five L. innocua strains, one L. monocytogenes strain and one L. welshimeri strain were obtained from 208 samples of raw milk. The strains were typified by biochemical and serologic tests. The shortened enrichment method was chosen for isolations; Palcam and Oxford agar also permitted the growth of the seven strains. L. monocytogenes was recovered from milk of an animal with subclinical mastitis. No Listeria strains were isolated from pasteurized milk, chocolate milk or cheese samples. One L. welshimeri strain was detected in ice cream. In the case of meat food samples, the employment of two-step enrichment methods facilitated the detection of Listeria spp. A prevalence of L. ivanovii was observed in 2.5% of these samples.

Modulation of a calcium-activated chloride current by Maitotoxin.

The effect of Maitotoxin (MTX) on the calcium-activated chloride current (ICl-Ca) from Xenopus oocytes was studied, applying the two-electrode voltage clamp technique. MTX increased the current amplitude at all the voltages explored and reduced the time to reach the maximum current level (time to peak). At low toxin concentrations (15 pM), both effects were fully reversible. Activation of ICl-Ca by MTX was secondary to the increment in the intracellular Ca2+ concentration induced by this toxin, since incubation of the oocytes with the cell-permeant Ca2+ chelator BAPTA-AM, greatly reduced the effect of MTX on ICl-Ca. Furthermore, external chloride ions removal also diminished the MTX effect on the current, strongly suggesting that the main current activated by MTX is ICl-Ca. Subsequent applications of a fixed toxin concentration after toxin washout resulted in enhanced ICl-Ca, suggesting that the toxin effect potentiates.

Listeria spp. en alimentos de origen animal / Listeria spp. in food of animal origin

Se analizaron diferentes alimentos de origen animal para detectar la presencia de Listeria spp. De 208 muestras de leche cruda de tambo, se obtuvieron 5 capas de Listeria innocua, 1 de L. monocytogenes y 1 de L. welshimeri, tipificadas por pruebas bioquímicas y serológicas. El método de enriquecimiento rápido resultó el de elección y tanto el agar Palcam como el agar Oxford permitieron el crecimeitno de las 7 cepas. L. monocytogenes se recuperó de la leche de un animal con mastitis subclínica. Ninguna de las muestras analizadas de leches pasteurizadas o chocolatada ni de quesos contenía listeria, en cambio en las de helados se recuperó una cepa de L. welshimeri. Para los alimentos cárnicos, el empleo de enriquecimiento en 2 etapas facilitó la detección de Listeria spp. Se observó un predominio de L. ivanovii en el 2,5 por ciento de las muestras

Listeria spp. en alimentos de origen animal / Listeria spp. in food of animal origin

Se analizaron diferentes alimentos de origen animal para detectar la presencia de Listeria spp. De 208 muestras de leche cruda de tambo, se obtuvieron 5 capas de Listeria innocua, 1 de L. monocytogenes y 1 de L. welshimeri, tipificadas por pruebas bioquímicas y serológicas. El método de enriquecimiento rápido resultó el de elección y tanto el agar Palcam como el agar Oxford permitieron el crecimeitno de las 7 cepas. L. monocytogenes se recuperó de la leche de un animal con mastitis subclínica. Ninguna de las muestras analizadas de leches pasteurizadas o chocolatada ni de quesos contenía listeria, en cambio en las de helados se recuperó una cepa de L. welshimeri. Para los alimentos cárnicos, el empleo de enriquecimiento en 2 etapas facilitó la detección de Listeria spp. Se observó un predominio de L. ivanovii en el 2,5 por ciento de las muestras(AU)

Listeria spp. en alimentos de origen animal. / [Listeria spp. in food of animal origin]

Different food samples of animal origin were analyzed for Listeria spp. Five L. innocua strains, one L. monocytogenes strain and one L. welshimeri strain were obtained from 208 samples of raw milk. The strains were typified by biochemical and serologic tests. The shortened enrichment method was chosen for isolations; Palcam and Oxford agar also permitted the growth of the seven strains. L. monocytogenes was recovered from milk of an animal with subclinical mastitis. No Listeria strains were isolated from pasteurized milk, chocolate milk or cheese samples. One L. welshimeri strain was detected in ice cream. In the case of meat food samples, the employment of two-step enrichment methods facilitated the detection of Listeria spp. A prevalence of L. ivanovii was observed in 2.5

of these samples.

Osmotic swelling-induced changes in cytosolic calcium do not affect regulatory volume decrease in rat cultured suspended cerebellar astrocytes.

Hyposmotic swelling-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+]i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+]i elevation. Omission of external Ca2+ or addition of Cd2+, Mn2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1-1 mM). Verapamil did not affect either the swelling-evoked [Ca2+]i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+-free medium) with 0.2 microM thapsigargin (Tg), 10 microM 2,5-di-tert-butylhydroquinone, 1 microM ionomycin, or 100 microM ATP abolished the increase in [Ca2+]i but did not affect RVD. However, prolonged exposure to 1 microM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 microM) and caffeine (10 mM) did not modify [Ca2+]i or RVD. BAPTA-acetoxymethyl ester (20 microM) abolished [Ca2+]i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+]i rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.

Maitotoxin, a cationic channel activator.

Maitotoxin (MTX), a water soluble polyether obtained from the marine dinoflagellate Gambierdiscus toxicus is one of the entities responsible for Ciguatera, a form of seafood poisoning. This toxin is a potent activator of changes in the intracellular Ca2+ concentrations of cells from a wide variety of organisms. Evidence published in the last few years strongly suggests that this toxin has no ionophoretic activity. Molecular mechanics studies, shown for the first time in this review, models MTX as a molecular 'wire'. The present work compiles the few studies developed with electrophysiological techniques. All these reports indicate that MTX is activating a voltage independent, nonselective cationic channel, which in some preparations requires the presence of extracellular Ca2+ for channel activation. The conductance estimated from a variety of tissues is in the order of 12-40 pS. Thus far, no specific blocker has been identified for this channel. The nature of the MTX receptor remains a mistery.