Biomedical subjects

V Rivarola

Publications and source records attributed to V Rivarola.

At least 19 recordsLinked to original sources

PubMed2005

Asymmetry in the osmotic response of a rat cortical collecting duct cell line: role of aquaporin-2.

Transition from antidiuresis to diuresis exposes cortical collecting duct cells (CCD) to asymmetrical changes in environment osmolality, inducing an osmotic stress, which activates numerous membrane-associated events. The aim of the present work was to investigate, either in the presence or not of AQP2, the transepithelial osmotic water permeability (P(osm)) following cell exposure to asymmetrical hyper- or hypotonic gradients. For this purpose, transepithelial net volume fluxes were recorded every minute in two CCD cell lines: one not expressing AQPs (WT-RCCD(1)) and another stably transfected with AQP2 (AQP2-RCCD(1)). Our results demonstrated that the rate of osmosis produced by a given hypotonic shock depends on the gradient direction (osmotic rectification) only in the presence of apical AQP2. In contrast, hypertonic shocks elicit P(osm) rectification independently of AQP2 expression, and this phenomenon may be linked to modulation of basolateral membrane permeability. No asymmetry in transepithelial resistance was observed under hypo- or hypertonicity, indicating that rectification cannot be attributed to a shunt through the tight junction path. We conclude that osmotic rectification may be explained in terms of dynamical changes in membrane permeability probably due to activation/incorporation of AQPs or transporters to the plasma membrane via some mechanism triggered by osmolality.

Animals↗

PubMed2002

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line.

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.

Animals↗

PubMed2002

Physiological parameters and biodistribution of 5,10,15,20-tetra (4-methoxyphenyl) porphyrin in rats.

Physiological parameters on hepatic and renal functionality and biodistribution, accumulation and elimination, in different organs of the 5,10,15,20-tetra (4-methoxyphenyl) porphyrin (TMP) were determined in Wistar rats. The transport of TMP by low-density (LDL) and high-density lipoproteins (HDL) was also investigated. The photosensitizer is accumulated in the spleen, where its concentration is significantly increased 21 d post-injection; it also accumulates in the liver and in a lower proportion, in the duodenum, and poorly in brain and muscle. The urine and serum biochemical parameters reached normal values both in control and treated groups. The glomerular filtrate rate was not affected by the TMP treatment in any of the studied times. These results would indicate that the sensitizer does not modify the renal glomerular function. TMP is mainly eliminated from the organism via the bile-gut pathway. Considering the total amount of porphyrin bound to both lipoproteins (LDL and HDL) in comparison with the total value of the TMP in serum, it can be inferred that a large amount of the agent is transported by lipoproteins in the plasma. This study proves information about the behavior of TMP in vivo under dark conditions. The results can be used to design photodynamic treatments using this porphyrin model as the sensitizer.

Animals↗

PubMed2001

Vasopressin regulates water flow in a rat cortical collecting duct cell line not containing known aquaporins.

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD(1)). Transepithelial net water fluxes (J(w)) were recorded every minute in RCCD(1) monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10(-5) m), apical glibenclamide (10(-4) m) and apical BaCl(2) (5 x 10(-3) m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD(1) cells. AVP stimulates cAMP production and sodium reabsorption in RCCD(1) cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I(sc)) was paralleled by a simultaneous modification of the observed J(w): both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J(w) induced by serosal hypertonicity. We can conclude that: (i) transepithelial J(w) occurs in RCCD(1) cells in the absence of known renal aquaporins; (ii) the "water secretory component" observed could be linked to Cl- and K = secretion; (iii) the natriferic response to AVP, preserved in RCCD(1) cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost.

Animals↗

PubMed2001

Photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin on the Hep-2 human carcinoma cell line: effect of light dose and wavelength range.

The photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) has been investigated in two systems: reverse micelles of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water-bearing photooxidizable substrates and on a Hep-2 human carcinoma cell line. The effect of variation in the light dose and wavelength range (360-800, 455-800, and 590-800 nm) was compared in both media. The aerobic singlet oxygen-mediated photooxidation of L-tryptophan (Trp) was used as a model of biological substrate in a micellar system. A considerable increase of the observed rate constants of Trp (k(Trp)(obs)) was noted, increasing the irradiated area of the TMP spectrum. In vitro, the survival curves of Hep-2 cells, treated with TMP, were markedly dependent on the light wavelength ranges used for irradiation. A linear behavior between k(Trp)(obs) and the photoinactivation rate of Hep-2 cells was found, indicating that the singlet oxygen (1O2 ) is the main species responsible for cell inactivation. These results contributed to an understanding of the photodynamic process yielded by this porphyrin in vitro and the sensitivity of Hep-2 cells to photodamage.

Animals↗

PubMed2001

Synthesis and biological evaluation of methoxyphenyl porphyrin derivatives as potential photodynamic agents.

A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 2',6'-dinitro-4'-trifluoromethylphenyl group by an amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamic activities of 5 and 6 were evaluated in vitro on Hep-2 cells. A considerable increase in the photocytotoxic effect was found for 6, which has higher singlet molecular oxygen, O(2)((1)Delta(g)), production.

Humans↗

PubMed2001

Photodynamic studies of metallo 5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin: photochemical characterization and biological consequences in a human carcinoma cell line.

The photodynamic activities of the free-base 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) and their metal complexes with zinc(II) (ZnTMP), copper(II) (CuTMP) and cadmium(II) (CdTMP) have been compared in two systems: reverse micelle of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate/water bearing photooxidizable substrates and Hep-2 human larynx carcinoma cell line. The quantum yields of singlet molecular oxygen, O2(1 delta g), production (phi delta) of TMP, ZnTMP and CdTMP in tetrahydrofuran, were determined yielding values of 0.65, 0.73 and 0.73, respectively, while O2(1 delta g) formation was not detected for CuTMP. In the reverse micellar system, the amino acid L-tryptophan (Trp) was used as biological substrate to analyze the O2(1 delta g)-mediated photooxidation. The observed rate constants for Trp photooxidation (kobsTrp) were proportional to the sensitizer quantum yield of O2(1 delta g). A value of approximately 2 x 10(7) s-1 M-1 was found for the second-order rate constant of Trp (krTry) in this system. The response of Hep-2 cells to cytotoxicity photoinduced by these agents in a biological medium was studied. The Hep-2 cultures were treated with 1 microM of porphyrin for 24 h at 37 degrees C and the cells exposed to visible light. The cell survival at different light exposure levels was dependent on phi delta. Under these conditions, the cytotoxic effect increases in the order: Cu-TMP << TMP < ZnTMP approximately CdTMP, correlating with the production of O2(1 delta g). A similar behavior was observed in both the chemical and biological media indicating that the O2(1 delta g) mediation appears to be mainly responsible for the cell inactivation.

Cell Survival↗

PubMed2001

Photokilling of cultured tumour cells by the porphyrin derivative CF3.

We have analysed the photosensitizing properties of the new porphyrin 5-(4-N-(N-2',6'-dinitro-4'-trifluoromethylphenyl)aminophenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl) porphyrin (CF3) on HeLa cells. The fluorescence and singlet oxygen quantum yield for CF3 were, respectively, phiF = 0.032 and phidelta = 0.25. Cell treatments were done with 5 x 10(-6) M CF3 incorporated into liposome vesicles. Under violet-blue exciting light, the red fluorescence of CF3 was mainly detected in lysosome-like granules. No dark cytotoxicity was observed using high concentration (5 x 10(-6) M) and long incubation time (18 h). Cell cultures treated for 18 h with CF3 and exposed to light (360 < lambda < 460 nm; 8 mW/cm2) for 7 min revealed a great amount of apoptotic (75.8%) and detached cells (62%) 8 h later, leading to a cell lethality of 85% (LD85). Apoptosis was identified by chromatin fragmentation and DNA ladder in gel electrophoresis. Necrotic cells were found using 15 min irradiation (LD96) and showed first small and then giant bubbles at the cell surface, with homogeneous nuclear condensation. Incubation with CF3 for 3 h followed by 7 min irradiation (LD38) produced a mitotic arrest 18 h later (mitotic index: 25.1%). Forty-eight hours after this metaphase blockage, cultures showed a great number of apoptotic cells. Taking into account these results, CF3 could be a valuable photosensitizer for the photodynamic therapy of cancer.

Apoptosis↗

PubMed2000

Expression of Fas antigen and apoptosis caused by 5,10,15, 20-tetra(4-methoxyphenyl)porphyrin (TMP) on carcinoma cells: implication for photodynamic therapy.

The photodynamic effects of 5,10,15, 20-tetra(4-methoxyphenyl)porphyrin (TMP) on a Hep-2 cell line were investigated. TMP toxicity in the dark and in relation to illumination with visible light was examined. Hep-2 cells were treated with different TMP concentrations (1, 5 and 10 microM). The uptake of TMP by Hep-2 cells increased with TMP concentration and an increase of the initial uptake rate was observed with increasing TMP concentrations. However, after 24 h of incubation, a similar value of intracellular TMP concentration was reached at all three concentrations of TMP added. Cell toxicity induced by TMP was analyzed in the dark at different concentrations of the photosensitizer and at several incubation periods. The cell mortality obtained after exposure of the cell cultures to visible light was exclusively due to the photosensitization effect of TMP produced by light irradiation. Staining with the hematoxylin-eosin method demonstrated that treatment with TMP, followed by exposure to visible light, notably increased the apoptotic figures. Fas antigen was only expressed in these conditions. The results contribute to the understanding of the photodynamic therapy (PDT) mechanism produced by TMP on Hep-2 carcinoma cell line.

Apoptosis↗

PubMed2000

Effects of alpha-difluoromethylornithine on the cyclin A expression in Hep-2 cells.

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis. The goal of this study was to determine the effects of DFMO on the expression of cyclin A at different stages of the cell cycle of Hep-2 cells. The cell cycle analysis, done by measuring the incorporation of thymidine in the cell DNA, revealed that DFMO produced a lower and constant level of that incorporation; this effect is probably due to the incapacity of the cells to culminate the phase S of the cell cycle. The expression of cyclin A increased in the phases S and G2 in control cells, almost disappearing in phase M. However, in DFMO treated cultures, the expression of cyclin A was increased in M and this effect remained still after 48 h treatment. We conclude that polyamines could exert an effect on the cyclin destruction mechanism, and the depletion caused by DFMO would alter this mechanism.

Cyclin A↗

PubMed2000

Effects of alpha-difluoromethylornithine on the Fas expression and apoptosis in Hep-2 cells.

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.

Apoptosis↗

PubMed1999

Apoptosis is regulated by polyamines in the cell cycle of Chinese hamster ovary cells.

This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis.

Animals↗

PubMed1998

Reconstitution of a regulated transepithelial water pathway in cells transfected with AQP2 and an AQP1/AQP2 hybrid containing the AQP2-C terminus.

Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 +/- 8.2 vs. 12.5 +/- 3.3 cm.sec-1.10(-3)), but not in cells transfected with AQP1 (15.3 +/- 3.6 vs. 13.4 +/- 3.6 cm.sec-1.10(-3)). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 +/- 4.8 vs. 6.7 +/- 1.0 cm.sec-1.10(-3)). The increases in Posm values were not paralleled by increases in 14C-Mannitol permeability. HgCl2 inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2.

Animals↗

PubMed1998

Response of Azospirillum brasilense Cd to sodium chloride stress.

Growth of Azospirillum brasilense Cd in the presence of different NaCl concentrations showed that it tolerates up to 200 mM NaCl in the medium, without appreciable decline in growth rate. At 300 mM NaCl, a decrease of 66% in growth was observed at 24 h of culture. At 48 h of culture, bacteria in the presence of 300 mM NaCl reached the maximum optical density value that was attained at 12 h by control cultures. This investigation was designed to elucidate the effect of saline stress on Azospirillum brasilense Cd and the physiologic mechanism involved in its possible salinity tolerance. For this reason, studies of other osmolytes, as well as of putrescine metabolism and protein patterns were done with bacteria grown with this NaCl concentration in the medium, at 24 and at 48 hours. A. brasilense responded to saline stress elevating the intracellular concentration of glutamate at 24 h, and of K+ at 48 h. Glucan pattern, putrescine metabolism, and total and periplasmic protein patterns of the treated group showed several changes with respect to the control. In spite of the several cellular functions affected by saline stress, the results imply that A. brasilense Cd shows salinity tolerance in these experimental conditions.

Azospirillum brasilense↗

PubMed1996

2,4-Dichlorophenoxyacetic acid affects the attachment of Azospirillum brasilense Cd to maize roots.

2.4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide widely applied to forage, grain and cereals. We previously determined that 1 mM 2,4-D diminished cell growth and cellular activity of Azospirillum brasilense Cd. The present work was designed to determine the possible effect of this herbicide--at concentrations used on crops--on the attachment of the bacteria to maize roots, since this step is of prime importance for the growth stimulation of the plant obtained with Azospirillum brasilense. In this paper we demonstrate that 2,4-D alters the bacterial adhesion to maize roots.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1996

Characterization of 2,4-dichlorophenoxyacetic acid transport and its relationship with polyamines in Azospirillum brasilense.

We have previously shown that 2,4-dichlorophenoxyacetic acid (2,4-D) inhibits Azospirillum brasilense growth, the synthesis of DNA, RNA and proteins. These toxic effects are prevented when polyamines are added to the culture medium. The purposes of our research were to determine the effects of the herbicide on the number of viable Azospirillum brasilense cells, characterize the 2,4-D transport system and to study the effects of polyamines upon the latter in this microorganism. We found that 2,4-D reduced the number of viable cells and that 2,4-D transport is energy-independent, since it was not affected by metabolic inhibitors. Polyamines did not alter 2,4-D uptake, further supporting the hypothesis that the herbicide most likely produces its toxic effects by interfering with the polyamine metabolism.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1995

Effects of 2,4-dichlorophenoxyacetic acid on polyamine transport and metabolism in Azospirillum brasilense Cd.

We had previously demonstrated that 1 mM 2,4-D inhibited cell growth, nucleic acid synthesis and protein synthesis (at the ribosomal level) of Azospirillum brasilense. These alterations were prevented by the presence of polyamines in the culture medium. On the other hand, polyamines did not affect the 2,4-D uptake. In this paper we demonstrate that 2,4-D alters the metabolism of polyamines and increases their uptake.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1993

The interaction of 2,4-dichlorophenoxyacetic acid, ribosomes and polyamines in Azospirillum brasilense.

2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide used extensively in agriculture. We had previously determined that 1 mM 2,4-D could inhibit cell growth, DNA and protein synthesis of Azospirillum brasilense. The present work was designed to determine if these alterations are a consequence of 2,4-D action on polyamine biosynthesis and if the protein synthesis inhibition is a result of ribosomal impairment. In this paper we demonstrate that 2,4-D alters the metabolism of polyamines and, thus, affects protein synthesis at the ribosomal level.

2,4-Dichlorophenoxyacetic Acid↗