Calcium-activated chloride currents and non-selective cation channels in a novel cystic fibrosis-derived human pancreatic duct cell line.

The modulation of ion fluxes across the plasma membrane of epithelial cells is central for fluid secretion and absorption. Their disruption can lead to pathological states. An example is cystic fibrosis (CF), a disease characterized by abnormal functioning of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-modulated chloride channel. Here we report the characterization of calcium-activated, DIDS sensitive chloride current and non-selective calcium-activated cation channels in a novel human pancreatic duct cell line (YHV-1) derived from a non-delta F508 mutation CF patient bearing a severe phenotype. Southern blot analysis of the CFTR gene indicates a distinct electrophoretic pattern for the region spanned by exons 15-24, a result presumably related to a mutation which has yet to be identified. In contrast to large calcium-activated chloride currents there were no cAMP-dependent CFTR-type chloride currents. Non-selective cation channels were blocked by intracellular ATP and activated by intracellular calcium and cAMP. We propose the cell line YHV-1 as a suitable model for studying pancreatic ion and fluid secretion alterations in CF.

CFTR mutations in Chilean cystic fibrosis patients.

An analysis of five of the most common cystic fibrosis (CF) mutations worldwide (delta F-508, R-553X, G-551D, N-1303K and G-542X) was performed in 36 Chilean patients. Polymerase chain reaction (PCR) amplification of the DNA followed by allele specific restriction enzyme analysis was used for detection. The overall frequencies of the mutations in the chromosomes analyzed were 29.2% for delta F-508 and 4.2% for R-553X (n = 72). The G-542X, G-551D and N-1303 K mutations were absent in the Chilean sample. Our data suggest however that delta F-508 is not the most common CF mutation in Chilean patients. delta F-508 and R-553X account for only 33.4% of the alleles; 66.6% of them do not respond to the probes used and still remain uncharacterized.

[Molecular genetic analysis of 2 Chilean cystic fibrosis patients and their families]. / Análisis genético molecular de la fibrosis quística en dos pacientes chilenos y sus familias.

As a contribution to establish the real incidence of cystic fibrosis (CF) and the prevalent mutations in the Chilean population a method for the detection of delta F-508 and R-553X, two of the most frequent mutations described worldwide, has been implemented. The method is based on the polymerase chain reaction (PCR) amplification of DNA followed by allele specific restriction enzymatic digestion. The application of this techniques allowed to confirm CF diagnosis in two patients and to detect asymptomatic carriers in both families. One of the patients showed normal sweat electrolyte concentration.

[Molecular genetic study of cystic fibrosis in the Chilean population. Relationship to its clinical expression]. / Estudio genético molecular de la fibrosis quística en la población Chilena. Relación con su expresión clínica.

Aiming to establish a genotype-phenotype relationship and to search a clinical expression in heterozygotes, 25 Chilean subjects with Cystic Fibrosis and 165 relatives were subjected to a clinical-molecular study. The most common mutations found worldwide were studied: delta F-508, G-542X, N-1303K, R-553X and G551D. Clinical and laboratory assessment comprised chest X-rays, spirometry, clinical evaluation, nutritional assessment, sweat test and carotenemia. Age at diagnosis was lower among homozygotes for the mutation delta F-508. In this group, Brasfield and Schawchman scores were better, probably due to an earlier initiation of treatment. No other differences were found among genotypic groups or relatives. Genetic markers indicated a higher european component of the sample, compared to the general Chilean population.

Neuropathological changes in the rat brain cortex in vitro: effects of kainic acid and of ion substitutions.

The ionic mechanisms that may contribute to the neurotoxicity of kainic acid, were studied in a system of rat thin neocortical slices superfused in vitro. Slices superfused for 3 h under control conditions showed an essentially normal aspect when studied by light microscopy. Presence of 30 microM kainate in the superfusion fluid induced neuronal swelling, nuclear condensation and signs of necrosis in some cells, while other neurons, especially in deeper layers, appeared dark and condensed, with microvacuolation. The neuropil presented numerous profiles of swollen dendrites. When the slices were superfused with chloride-free medium, a large number of pyknotic neurons was seen. This was further enhanced by 30 microM kainate, which produced no swelling in this medium. These effects of Cl-free medium were almost entirely prevented in Cl-free medium without calcium and with 0.1 mM of EGTA. Sodium-free medium induced a marked neuronal swelling that was not much changed by kainate. When calcium in an otherwise normal superfusion fluid was reduced to 0.1 mM, a large number of pyknotic neurons, some with incrustations, were seen. Kainate (30 microM) in this low calcium medium led to a very large swelling and destruction of neurons, and to a spongy neuropil. These effects of kainate were greatly intensified in calcium-free-EGTA (0.1 mM) medium. Ca-free-EGTA medium by itself induced considerable neuronal and neuropil swelling. It is concluded that kainate induces neuronal swelling by a sodium- and chloride-dependent mechanism, and the enhancement of swelling in low calcium is due to an increased sodium uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure.

Rat brain cortex synaptic vesicles have been isolated by 3 different procedures. The one of Hata et al. (J. Neurochem., 27 (1976) 139) gave synaptic vesicles with a high glutamate content, but also, as judged by [3H]ouabain binding and electron microscopy, with considerable contamination by plasma membrane vesicles. This did not allow a precise estimation of the glutamate content of each synaptic vesicle. The second procedure used (Life Sci., 21 (1977) 1075), in which the tissue is homogenized with an all glass homogenizer, yielded vesicles of higher purity, but with no glutamate. A slightly modified Kadota and Kadota procedure (J. Cell Biol., 58 (1973) 135) gave synaptic vesicles of a very high purity that were filtered on a Sepharose 4B column, and there, the synaptic vesicle fraction of highest purity was estimated to contain 3640 glutamate molecules in each glutamatergic vesicle. This is equivalent to an intravesicular concentration of 0.21 M, that is, at least 10 times higher than the glutamate concentration in the rat brain cortex.

N-methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances.

N-Methyl-DL-aspartate, L-glutamate, kainate and DL-homocysteate were found to increase the initial rate and the maximal uptake of 45Ca into the non-inulin space of rat brain cortex slices incubated in vitro. The N-methylaspartate-stimulated calcium uptake was blocked by cadmium and cobalt ions, but not by the organic calcium channel blocker nifedipine or by tetrodotoxin, both of which stimulated the N-methylaspartate-independent calcium influx. gamma-Aminobutyrate increased the spontaneous calcium influx, and also reduced that stimulated by N-methylaspartate to the same level, as found with gamma-aminobutyrate alone. Adenosine (1-100 microM), ethanol (0.1 M), pentobarbital (10-100 microM) and morphine (0.2 mM), were unable to inhibit the N-methylaspartate-activated calcium influx. Ethanol (0.1 M), had no effect on the glutamate- or kainate-activated calcium influx. These findings suggest that the excitatory amino acids, because of their neuronal depolarizing action in brain cortex, lead to the opening of voltage-sensitive calcium channels, which may be blocked by cadmium, but not by the organic calcium channel antagonist, nifedipine. The activation of calcium channels by the excitatory amino acid N-methylaspartate, was entirely unaffected by the depressants ethanol, pentobarbital or morphine, or by the endogenous inhibitory substance, adenosine, thus suggesting that their inhibitory or depressant effects occur through interference with a neuronal mechanism unrelated to the one studied here. gamma-Aminobutyrate, on the other hand, considerably inhibited N-methylaspartate-induced calcium uptake, an effect interpreted as due to a gamma-aminobutyrate-induced increase in chloride conductance, that "clamps" the membrane potential and does not allow further depolarization by N-methylaspartate.

A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations.

The possible excitatory effect of N-acetyl-alpha- aspartylglutamate ( NAAG ) was studied in 3 different systems. First on the increase in 45Ca2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM L-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of L-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 micrograms) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro.

Kainate (0.62-5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl-DL-aspartate (50-1000 microM), L-glutamate (0.62-5 mM), DL-homocysteate (0.62-2.5 mM), and ibotenate (6-170 microM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino acids N-acetyl-L-aspartate, and alpha-methyl-DL-aspartate (both 1.25-5 mM), did not increase such influx. We suggest that enhanced calcium uptake may represent the basis for the neurotoxic effects of these compounds.

A search in rat brain cortex synaptic vesicles for endogenous ligands for kainic acid receptors.

Conditions were found for stabilizing rat brain cortex kainic acid (KA) receptors. Such receptors had the same Hill number (about 0.6) for KA and for glutamate. The receptors were then used as detectors for endogenous ligands present in brain cortex synaptic vesicle (SV) soluble extracts. When these SV extracts were fractionated by gel filtration on Sephadex G-10, by thin-layer chromatography, or by high voltage electrophoresis, a single endogenous component, that in all cases comigrated with glutamic acid, was found.