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R Caviedes

Publications and source records attributed to R Caviedes.

At least 19 recordsLinked to original sources
PubMed2002

Neurotoxicity of some MAO inhibitors in adult rat hypothalamic cell culture.

Monoamine oxidase-A (MAO-A) [amiflamine (AMF) and 4-methylthioamphetamine (MTA)] and MAO-B (L-deprenyl) inhibitors were found to be cytotoxic in a concentration-dependent manner for RCHT cells derived from adult rat hypothalamus. The cytotoxic effects were increased when the inhibitors were co-incubated with dicoumarol and especially with 25 micro M AMF+100 micro M dicoumarol (2.5-fold; P <0.001). The treatment of RCHT cells solely with AMF induced a marked decrease in the expression of DT-diaphorase mRNA.

Journal Article↗
PubMed2001

Possible role of salsolinol quinone methide in the decrease of RCSN-3 cell survival.

The endogenous dopamine-derived neurotoxin salsolinol was found to decrease survival in the dopaminergic neuronal cell line RCSN-3, derived from adult rat substantia nigra in a concentration-dependent manner (208 microM salsolinol induced a 50% survival decrease). Incubation of RCSN-3 cells with 100 micro;M dicoumarol and salsolinol significantly decreased cell survival by 2.5-fold (P < 0.001), contrasting with a negligible effect on RCHT cells, which exhibited nearly a 5-fold lower nomifensine-insensitive dopamine uptake. The levels of catalase and glutathione peroxidase mRNA were decreased when RCSN-3 cells were treated with 100 microM salsolinol alone or in the presence of 100 microM dicoumarol. In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinoline diol as final products of salsolinol oxidation as determined by NMR analysis. Evidence of the formation of salsolinol o-semiquinone radical has been provided by ESR studies during one-electron oxidation of salsolinol catalyzed by lactoperoxidase.

Animals↗
PubMed2001

Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line.

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu-dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu-dopamine complex mediates the uptake of (64)CuSO(4) into the Raúl Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m M) (63%, p < 0.001) and nomifensine (2 microM) (77%, p < 0.001). Copper sulfate (1 m M) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO(4) and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO(4), dopamine, or CuSO(4) and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO(4), dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO(4) increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu-dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome.

Animals↗
PubMed2000

Studies of aminochrome toxicity in a mouse derived neuronal cell line: is this toxicity mediated via glutamate transmission?

Aminochrome was found to be toxic in a mouse-derived neuronal cell line (CNh). The effect was concentration dependent (10-150microM). The issue whether aminochrome toxicity involves glutamate transmission was studied with several glutamate receptors antagonists. Incubation of the cells with aminochrome (150microM) in the presence of 100microM of the AMPA antagonist, NBQX resulted in an increase of cell survival, from 52 to 73%. However, this protective effect did not seem to be related to activation of ionotropic glutamate receptors since incubation of CNh cells with 200microM of glutamate resulted in only 10% decrease of cell survival. However, NBQX was found to inhibit in vitro the autoxidation process. One hundred microM AP-5 did not have any effect on aminochrome toxicity. The toxic effect of aminochrome on CNh cells seems to be dependent of extracellular activation since addition of dicoumarol, a specific inhibitor of DT-diaphorase, did not affect that toxicity, which can be explained perhaps by a lack of a transport system for aminochrome into the CNh cells.

Animals↗
PubMed2000

Impaired cholinergic function in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

Murine trisomy 16 is an animal model of human Down's syndrome. We have successfully established permanently growing cell lines from the cerebral cortex of normal and trisomy 16 foetal mice using an original procedure. These lines, named CNh (derived from a normal animal) and CTb (derived from a trisomic foetus), express neuronal markers. Considering that Down's syndrome exhibits cholinergic deficits, we examined cholinergic function in these lines, using incorporation of [3H]-choline and fractional release studies. After 1, 3 and 5 min of [3H]-choline incubation, CTb cell uptake was lower by approximately 50% compared to controls. Hemicholinium-3 significantly reduced the incorporation of [3H]-choline in both CNh and CTb cells at high concentration (10 microM), suggesting high-affinity choline transport. However, CTb cells exhibited greater sensitivity to the blocker. For fractional release experiments, the cells were stimulated by K+ depolarization, glutamate or nicotine. When depolarized, CTb cells showed a 68% reduction in fractional release of [3H]-acetylcholine compared to CNh cell line, and a 45% reduction when stimulated by nicotine. Interestingly, glutamate induced similar levels of release in both cell types. The results indicate the existence of cholinergic dysfunction in CTb cells when compared to CNh, similar to that reported for primary cultures of trisomy 16 brain tissue (Fiedler et al. 1994, Brain Res., 658, 27-32). Thus, the CTb cell line may serve as a model for the study of Down's syndrome pathophysiology.

Acetylcholine↗
PubMed1999

Calcium signals in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.

Animals↗
PubMed1998

Differences in both inositol 1,4,5-trisphosphate mass and inositol 1,4,5-trisphosphate receptors between normal and dystrophic skeletal muscle cell lines.

Human normal (RCMH) and Duchenne muscular dystrophy (RCDMD) cell lines, as well as newly developed normal and dystrophic murine cell lines, were used for the study of both changes in inositol 1,4,5-trisphosphate (IP3) mass and IP3 binding to receptors. Basal levels of IP3 were increased two- to threefold in dystrophic human and murine cell lines compared to normal cell lines. Potassium depolarization induced a time-dependent IP3 rise in normal human cells and cells of the myogenic mouse cell line (129CB3), which returned to their basal levels after 60 s. However, in the human dystrophic cell line (RCDMD), IP3 levels remained high up to 200 s after potassium depolarization. Expression of IP3 receptors was studied measuring specific binding of 3H-IP3 in the murine cell lines (normal 129CB3 and dystrophic mdx XLT 4-2). All the cell lines bind 3H-IP3 with relatively high affinity (Kd: between 40 and 100 nmol/L). IP3 receptors are concentrated in the nuclear fraction, and their density is significantly higher in dystrophic cells compared to normal. These findings together with high basal levels of IP3 mass suggest a possible role for this system in the deficiency of intracellular calcium regulation in Duchenne muscular dystrophy.

Actinin↗
PubMed1997

Expression of ion channels during differentiation of a human skeletal muscle cell line.

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.

Biomarkers↗
PubMed1994

Regional alteration of cholinergic function in central neurons of trisomy 16 mouse fetuses, an animal model of human trisomy 21 (Down syndrome).

The trisomy-16 (TS16) mouse is considered to be a model of human trisomy 21 (Down syndrome) because of genetic homology between mouse chromosome 16 and human chromosome 21. We examined cholinergic function of brain and spinal cord tissue and in cultured neurons from TS16 mouse compared with that of age matched controls. Mean acetylcholinesterase activity in both tissue types did not differ between trisomic and control conditions. Acetylcholine (ACh) synthesis, measured as choline O-acetyltratransferase (acetyl-CoA) activity, was reduced to 67% of control in TS16 brain but not in TS16 spinal cord. Steady-state accumulation of ACh precursor, [3H]choline, was measured in primary cell cultures. Steady-state choline uptake was reduced to 35% and to 61% in neurons of TS16 brain and spinal cord, respectively, when compared with controls. Kinetics experiments in TS16 brain cells showed a 50% reduction of the maximal velocity of choline uptake when compared to controls. Further, the ACh release induced by KCl depolarization in TS16 spinal cord neurons did not differ from control neurons but was reduced in TS16 brain neurons. This effect cannot be explained solely by a reduction in ACh synthesis. The results indicate that the TS16 condition in mice significantly modified the cholinergic function in brain, and to a lesser degree in spinal cord, suggesting that the higher gene dosage inherent to the trisomic condition affects cholinergic neurons in different regions of the central nervous system in a differential fashion.

Acetylcholine↗
PubMed1994

Ion channels in a skeletal muscle cell line from a Duchenne muscular dystrophy patient.

A cell line (RCDMD), derived from a muscle biopsy taken from a 7-year-old patient with Duchenne muscular dystrophy (DMD), was established in vitro using conditioned media from the UCHT1 thyroid cell line as described elsewhere (Biochim Biophys Acta 1992; 1134:247-255). Unlike other cell lines established by the same procedure, RCDMD cells were highly refractory to transformation and the resulting cell line grew slowly with a doubling time of approximately 72 h. Further, cells continue to grow after more than 20 doublings and 15 passages. Some of the characteristics of the cell line include lack of reaction with antidystrophin antibodies and the presence of receptors for the dihydropyridine PN200-110 (Kd) = 0.3 +/- 0.05 nmol/L and Bmax = 1.06 +/- 0.03 pmol/mg protein) and for alpha-bungarotoxin (Kd = 1.02 +/- 0.17 nmol/L and Bmax = 4.2 +/- 0.37 pmol/mg protein). Patch clamped cells in the voltage clamp configuration lack ion currents when growing in complete medium with high serum, but they can be induced to differentiate by serum deprivation and addition of hormones and trace elements. After 5 days in differentiating medium, noninactivating, delayed rectifier potassium currents are seen. At day 12, A-type, inactivating potassium currents as well as transient inward currents are seen. In conditions in which sodium and potassium currents are absent, a very fast activating and fast inactivating calcium current was evident. The cell line offers the possibility of studying cellular mechanisms in the pathophysiology of DMD.

Bungarotoxins↗
PubMed1993

Calcium fluxes, ion currents and dihydropyridine receptors in a new immortal cell line from rat heart muscle.

A cell line (RCVC) in permanent culture was developed from adult rat ventricular cells; transformation was attained by incubation with conditioned media from UCHT1, a rat thyroid cell line. Immortalized ventricular cells have a doubling time of 20 h, contact inhibition of growth, and display some muscle markers such as a high glycogen content and positive immunoreaction for myoglobin, alpha-sarcomeric actin, alpha-actinin and desmin. A microsomal fraction from these cells was shown to bind 3H-nitrendipine with a maximal capacity of 295 fmol/mg protein and an equilibrium dissociation constant of 0.7 nM. Nifedipine-sensitive 45Ca2+ influx was evident in partially depolarized cells (40 mM K+ in the incubation medium). An equivalent influx, induced by the calcium channel agonist BAYK-8644 and CGP-28392, was obtained in normally polarized cells. Patch clamp studies show slow inward currents that can be completely blocked by 5 microM nifedipine; cells were induced to further differentiation by culturing in a hormone supplemented medium for 30 days. Under this condition, fast, inactivating inward currents and a large outward current became apparent. After 40-60 days, the cells exhibit La(3+)-sensitive fast and slow inactivating inward currents that resemble T and L-type Ca2+ currents. This cell line appears to be a good model system for the investigation of cardiomyocyte differentiation in situ.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

A human skeletal muscle cell line obtained from an adult donor.

A cell line (RCMH) in permanent culture was established from surgically removed adult normal human skeletal muscle by exposure to conditioned media obtained from thyroid cells. Cells proliferated indefinitely but displayed density inhibition of growth while maintaining some differentiated markers. Under certain incubation conditions, cells fused into myotube-like structures, with a concomitant increase in muscle specific proteins, such as human myoglobin, skeletal muscle myosin, desmin and dystrophin, as identified using immunocytochemical procedures. In addition, RCMH cells displayed high affinity receptors for alpha-bungarotoxin (Bmax = 0.7 pmol/mg protein, Kd = 1.5 nM) and dihydropyridines (Bmax = 0.3 pmol/mg protein, Kd = 0.5 nM for [3H]PN200-110); these values are comparable to those reported for muscle cells in primary culture. Patch-clamp studies showed the presence of 42 pS carbachol gated channels and of 5 pS calcium channels (current carried by barium); chloride and potassium channels were also seen. This new cell line appears to be a convenient model system to study skeletal muscle function.

Adult↗
PubMed1987

Identification of kallikrein in cultures of adult renal cells.

Cortical tubular cultures enriched with distal segments were prepared from the kidney of adult Fisher 344 rats bearing thyroid tumors. After two years of cultivation (20 passages) cell monolayers contained immunocytochemically detectable cytokeratin and kallikrein material in their cytoplasm. Furthermore cell pellets showed a true renal type of kininogenase activity corresponding to active kallikrein which ranged from 22.3 to 1.5, and total Kallikrein from 29.9 to 2.8 pg kinins/min per mg of protein.

Animals↗
PubMed1986

Tetrodotoxin-sensitive sodium channels in a continuously cultured cell line derived from the adult rat cerebellum.

A method to establish continuously cultured cell lines from adult cerebellar cortex is reported. Clones (prepared by this procedure) were isolated from cerebellar established cultures at the 25th passage and after 15 months in vitro. One clone (UCHCC1) was maintained in culture and studied while the others were frozen. The cerebellar cell line UCHCC1 retained a neuronal-like morphology; the addition of dimethylsulfoxide (DMSO) to the culture medium elicited a reproducible morphological 'differentiation' event, characterized mainly by process extension. In 'differentiated' cells, veratridine significantly increased the uptake of 22Na. Such enhanced uptake was blocked by tetrodotoxin (TTX) with a half-maximal inhibitory concentration of 0.9 nM. Binding of an [3H]ethylenediamine derivative of TTX ([3H]en-TTX) to the microsomal fraction prepared from same DMSO-treated cells, showed a single class of receptors with a maximal binding (Bmax) of 173 fmol/mg protein and a Kd of 1.1 nM. Thyroid UCHT1 cells and 'undifferentiated' (cultured without DMSO) cerebellar cells, did not show significant effects of veratridine on 22Na-uptake, or [3H]en-TTX binding. The 'differentiated' nerve-like properties, induced by appropriate environmental manipulation, demonstrate the usefulness of cerebellar UCHCC1 cells as a model system for the developing central neuron. On the other hand, the novel transforming procedure opens new possibilities for obtaining permanent cell lines from other regions of the adult CNS.

Animals↗
PubMed1985

Effects of factors derived from a tumor clonal cell line on DNA synthesis of transformed and non transformed cells.

Conditioned medium from neoplastic thyroid cell cultures, extracts of tumors developed by identical cells in isogeneic Fisher 344 rats and serum from those tumor-bearing animals, were tested in pulse thymidine labelled experiments on a transformed and two non transformed cell lines. Tumor extract and conditioned medium inhibited DNA synthesis. Tumor-bearing rat serum increased DNA synthesis in a cerebellar transformed cell line, but no in chick embryo fibroblasts or in aorta non transformed cells.

Animals↗
PubMed1976

Studies on a cell line from a functional rat thyroid tumor in continuous culture.

A clonal cell line derived from a functional and transplantable rat thyroid tumor was established in continuous monolayer culture by the use of enzymatic dissociation followed by an alternate culture-animal passage procedure. After being implanted backin the animal and again plated in culture, epithelial-like cells aggregated and rearranged themselves over the bottoms of dishes in structures resembling cross sections of a normal thyroid gland. The same morphology and growth pattern were maintained after innumerable subcultures and freeze/thawing periods. Cells grew with a population-doubling time of about 24 h in serum-supplemented synthetic medium. Cell monolayers stained with periodic acid-Schiff (PAS) showed a uniformly epithelial-like morphology; their cytoplasm contained abundant PAS-positive material that was resistant to enzymatic digestion with amylase. Thin-layer chromatography of acid-butanol cell extracts in primary and clonal cultures, followed by a specific and sensitive staining method for iodine compounds, demonstrated the presence of monoiodotyrosine (MIT), diiodotyrosine, and triiodothyronine-thyroxine. In contrast, in similar extracts obtained from a cultured liver cell line, the only iodinated amino acid was MIT. Thus, with regard to the above criteria, this cell line preserved specialized thyroid cell morphology and function.

Animals↗