Jesus del Castillo - Academia.edu (original) (raw)
Papers by Jesus del Castillo
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1991
Neutral amino acid transport was examined by using isolated enterocytes. Cells transport L-alanin... more Neutral amino acid transport was examined by using isolated enterocytes. Cells transport L-alanine by at least three different mechanisms: two Na(+)-dependent systems (A and ASC) and one Na(+)-independent mechanism (system L), in addition to passive entry. System A was characterized acterized by measuring the Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) uptake. Na(+)-dependent MeAIB uptake was concentrative and saturable. Vmax was obtained at 80mM Na+ in the incubation medium and Kt app for Na+ was 21.5 mM. Kt app for MeAIB was 6.75 +/- 0.37 mM and the Vmax was 14.2 +/- 0.3 nmol.mg-1.min-1. System ASC was studied by evaluating the Na(+)-dependent L-alanine uptake, insensitive to MeAIB and inhibitable by L-serine and L-cysteine. Uptake by this mechanism was also concentrative and saturable. Maximal uptake was obtained with 80 mM Na+ in the incubation medium and Kt app for Na+ was 29.7 mM. Kt app for L-alanine was 7.02 +/- 0.61 mM and Vmax was 5.44 +/- 0.19 nmol.mg-1.min...
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1991
This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) ac... more This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) activity in the colonic mucosa of the rat distal colon. K(+)-activated ATPase activity was present in apical membranes but not in basolateral membranes. K(+)-activated ATPase activity in apical membranes represented an approximate 10-fold enrichment compared with that in the homogenate. Na(+)-K(+)-activated ATPase activity was also present in homogenate but was enriched less than fourfold in apical membranes. K(+)-activated ATPase activity in apical membranes had both ouabain-sensitive and ouabain-insensitive components. In contrast, Na(+)-K(+)-activated ATPase activity was completely inhibited by ouabain. Similar half-maximal concentrations for K+ and pH activation curves were found for both ouabain-sensitive and ouabain-insensitive fractions. In addition to K+, the ouabain-sensitive fraction of K(+)-activated ATPase activity was stimulated by Rb+, NH+4, and Cs+, whereas the ouabain-insen...
American Journal of Physiology-Cell Physiology, 1991
We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yi... more We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yield and high viability, as demonstrated by a 99% exclusion of trypan blue, only a 10% liberation of lactate dehydrogenase after 30-min incubation at 37 degrees C, and the inability of succinate to stimulate oxygen consumption before plasma membrane permeabilization. Oxygen consumption (QO2) measured after the sequential addition of the following drugs showed that oligomycin inhibited QO2 by 67%, carbonyl cyanide p-trifluoromethoxyphenylhydrazone increased QO2 by approximately 70% of the basal consumption, and rotenone inhibited QO2 by 90%. Cells at 37 degrees C for 30 min maintained intracellular Na+ and K+ concentrations of 25 and 120 mM, respectively. The ATP consumed by the Na(+)-K+ pump was derived from oxidative phosphorylation (79%) and from glycolysis (21%). Initial rates for Na+ and K+ transported by the pump were 105 +/- 10 and 65 +/- 5 nmol.mg protein-1.min-1, respectively. The...
Progress in clinical and biological research, 1986
Pflügers Archiv, 1993
The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. ... more The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na+-dependent system A and the Na+-independent system L without producing any changes in either the Na+-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in Vmax of system A without any change in Kt. Starvation produces an increase in Vmax of system L of 1.7 times without any change in K,. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na+-dependent L-alanine uptake and Na+-independent cycloleucine uptake by small-intestinal brushborder membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.
Journal of Biochemical and Biophysical Methods, 1991
A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer... more A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.
Immunology Letters, 2009
Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-sp... more Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
; (5) does not require K+; (6) is only stimulated by Na ÷ and Li* (in a lower extent); (7) is sim... more ; (5) does not require K+; (6) is only stimulated by Na ÷ and Li* (in a lower extent); (7) is similarly stimulated by the Na + salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47°C; (10) optimal pH: 6.9, (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na + + K +)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP= 1.25:1 and K, for Mg:ATP=0.50:0.40 raM; (4) K. for Na~-: 14 mM (data not shown); (5) needs K + together with Nat; (6) K + may be substituted by: Rb ÷ >NH~ >Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, IT]P, UTP, ADP, CTP; (9) optimal temperature: 52°C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.
Journal of General Virology, 1991
Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na ÷ and a decr... more Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na ÷ and a decrease in K ÷ intracellular concentrations, starting at 4 h postinfection. These changes were not related to an inhibition of the Na+/K+- pump since ouabain-sensitive
S6Rb uptake was augmented in rotavirus-infected cells compared to control cells, whereas the [3H]ouabain binding and Na÷/K + ATPase activity in the cell homogenate were unaffected. Furosemide-sensitive S6Rb uptake (Na+/K÷/2CI - cotransport) was not modified by the infection. Passive 86Rb efflux and 22Na influx were augmented in infected cells, suggesting increased plasma membrane permeability. The increased intracellular Na ÷ concentration might be responsible for the observed stimulation of the Na+/K + pump. This effect was dependent upon the synthesis of
viral proteins because it was abolished by the addition of cycloheximide up to 4 h post-infection. Prevention of the increase in intracellular Na ÷ by the use of low Na ÷-
containing media did not modify the pattern of protein synthesis. This suggests that changes in intracellular Na + and K ÷ concentrations were not related to the shutoff of cellular protein synthesis. Alterations of ion contents in the rotavirus-infected enterocytes might impair intestinal absorptive capacity before the appearance of histopathological lesions.
Del Castillo, Jesús R., Julio C. Arévalo, Luis Burguillos, and Marı́a C. Súlbaran-Carrasco. b-Adr... more Del Castillo, Jesús R., Julio C. Arévalo, Luis Burguillos, and Marı́a C. Súlbaran-Carrasco. b-Adrenergic agonists stimulate Na1-K1-Cl2 cotransport by inducing intracellular Ca21 liberation in crypt cells. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G563–G571, 1999.—Epinephrine and b-adrenergic agonists (b1 and b2 for isoproterenol, b1 for dobutamine, b2 for salbutamol) stimulated K1 (or 86Rb) influx mediated by the Na1-K1-2Cl2 cotransporter and the Na1-K1 pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na1-K1 pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 μM epinephrine with an EC50 of 91.6 6 9.98 nM. Epinephrineinduced K1 transport was blocked by propranolol with an IC50 of 134 6 28.2 nM. a-Adrenergic drugs did not modify K1 transport mechanisms. Neither Ba21 nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition...
American Journal of Physiology-Gastrointestinal and Liver Physiology
Epinephrine and β-adrenergic agonists (β1 and β2 for isoproterenol, β1 for dobutamine, β2 for sal... more Epinephrine and β-adrenergic agonists (β1 and β2 for isoproterenol, β1 for dobutamine, β2 for salbutamol) stimulated K+ (or86Rb) influx mediated by the Na+-K+-2Cl−cotransporter and the Na+-K+pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na+-K+pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 μM epinephrine with an EC50 of 91.6 ± 9.98 nM. Epinephrine-induced K+ transport was blocked by propranolol with an IC50 of 134 ± 28.2 nM. α-Adrenergic drugs did not modify K+ transport mechanisms. Neither Ba2+ nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition, epinephrine did not affect K+ efflux. Dibutyryl cAMP did not alter K+ transport. Reduction of extracellular Ca2+ to 30 nM did not influence the response to epinephrine. However, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM abolished epinephrine-induced K+ tra...
Biochemical and Biophysical Research Communications, 2010
P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the ... more P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H +), have been reported. They include reticulum and plasma-membrane Ca 2+-ATPases, Na + /K +-ATPase and H + /K +-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K +-ATPase a1-isoform, H + /K +-ATPase a2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K +-ATPase a2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.
Acta Cient Venez, 2005
Transepithelial Na + transport is mediated by passive Na + entry across the luminal membrane and ... more Transepithelial Na + transport is mediated by passive Na + entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na + /K + pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na + /K +-ATPase and the ouabain-insensitive, furosemideinhibitable Na +-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na +-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na +-and Na + / K +-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na +-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na +-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na +-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na + transport and cellular Na + homeostasis leads us to reconsider its role in health and disease.
Acta científica venezolana, 1993
The active ion transport is related to some vital functions for the normal eukaryotic cell metabo... more The active ion transport is related to some vital functions for the normal eukaryotic cell metabolism. The study of transport mechanism and its regulation is a fundamental problem in cellular biology. The production of monoclonal antibodies (AcM) let us to have a probe, with large specificity, for the study of the localization, distribution and function of carrier proteins in the epithelium, as well as the study of its molecular structure, synthesis and assembling in the cell. The quality of a monoclonal antibody depends of the proper selection of each step to follow in the course of its production. In this article, we examine the strategy more currently used in the production of monoclonal antibodies and its direct use in the ionics pump's morphological, biochemical and physiological study.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1991
Neutral amino acid transport was examined by using isolated enterocytes. Cells transport L-alanin... more Neutral amino acid transport was examined by using isolated enterocytes. Cells transport L-alanine by at least three different mechanisms: two Na(+)-dependent systems (A and ASC) and one Na(+)-independent mechanism (system L), in addition to passive entry. System A was characterized acterized by measuring the Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) uptake. Na(+)-dependent MeAIB uptake was concentrative and saturable. Vmax was obtained at 80mM Na+ in the incubation medium and Kt app for Na+ was 21.5 mM. Kt app for MeAIB was 6.75 +/- 0.37 mM and the Vmax was 14.2 +/- 0.3 nmol.mg-1.min-1. System ASC was studied by evaluating the Na(+)-dependent L-alanine uptake, insensitive to MeAIB and inhibitable by L-serine and L-cysteine. Uptake by this mechanism was also concentrative and saturable. Maximal uptake was obtained with 80 mM Na+ in the incubation medium and Kt app for Na+ was 29.7 mM. Kt app for L-alanine was 7.02 +/- 0.61 mM and Vmax was 5.44 +/- 0.19 nmol.mg-1.min...
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1991
This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) ac... more This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) activity in the colonic mucosa of the rat distal colon. K(+)-activated ATPase activity was present in apical membranes but not in basolateral membranes. K(+)-activated ATPase activity in apical membranes represented an approximate 10-fold enrichment compared with that in the homogenate. Na(+)-K(+)-activated ATPase activity was also present in homogenate but was enriched less than fourfold in apical membranes. K(+)-activated ATPase activity in apical membranes had both ouabain-sensitive and ouabain-insensitive components. In contrast, Na(+)-K(+)-activated ATPase activity was completely inhibited by ouabain. Similar half-maximal concentrations for K+ and pH activation curves were found for both ouabain-sensitive and ouabain-insensitive fractions. In addition to K+, the ouabain-sensitive fraction of K(+)-activated ATPase activity was stimulated by Rb+, NH+4, and Cs+, whereas the ouabain-insen...
American Journal of Physiology-Cell Physiology, 1991
We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yi... more We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yield and high viability, as demonstrated by a 99% exclusion of trypan blue, only a 10% liberation of lactate dehydrogenase after 30-min incubation at 37 degrees C, and the inability of succinate to stimulate oxygen consumption before plasma membrane permeabilization. Oxygen consumption (QO2) measured after the sequential addition of the following drugs showed that oligomycin inhibited QO2 by 67%, carbonyl cyanide p-trifluoromethoxyphenylhydrazone increased QO2 by approximately 70% of the basal consumption, and rotenone inhibited QO2 by 90%. Cells at 37 degrees C for 30 min maintained intracellular Na+ and K+ concentrations of 25 and 120 mM, respectively. The ATP consumed by the Na(+)-K+ pump was derived from oxidative phosphorylation (79%) and from glycolysis (21%). Initial rates for Na+ and K+ transported by the pump were 105 +/- 10 and 65 +/- 5 nmol.mg protein-1.min-1, respectively. The...
Progress in clinical and biological research, 1986
Pflügers Archiv, 1993
The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. ... more The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na+-dependent system A and the Na+-independent system L without producing any changes in either the Na+-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in Vmax of system A without any change in Kt. Starvation produces an increase in Vmax of system L of 1.7 times without any change in K,. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na+-dependent L-alanine uptake and Na+-independent cycloleucine uptake by small-intestinal brushborder membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.
Journal of Biochemical and Biophysical Methods, 1991
A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer... more A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.
Immunology Letters, 2009
Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-sp... more Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
; (5) does not require K+; (6) is only stimulated by Na ÷ and Li* (in a lower extent); (7) is sim... more ; (5) does not require K+; (6) is only stimulated by Na ÷ and Li* (in a lower extent); (7) is similarly stimulated by the Na + salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47°C; (10) optimal pH: 6.9, (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na + + K +)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP= 1.25:1 and K, for Mg:ATP=0.50:0.40 raM; (4) K. for Na~-: 14 mM (data not shown); (5) needs K + together with Nat; (6) K + may be substituted by: Rb ÷ >NH~ >Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, IT]P, UTP, ADP, CTP; (9) optimal temperature: 52°C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.
Journal of General Virology, 1991
Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na ÷ and a decr... more Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na ÷ and a decrease in K ÷ intracellular concentrations, starting at 4 h postinfection. These changes were not related to an inhibition of the Na+/K+- pump since ouabain-sensitive
S6Rb uptake was augmented in rotavirus-infected cells compared to control cells, whereas the [3H]ouabain binding and Na÷/K + ATPase activity in the cell homogenate were unaffected. Furosemide-sensitive S6Rb uptake (Na+/K÷/2CI - cotransport) was not modified by the infection. Passive 86Rb efflux and 22Na influx were augmented in infected cells, suggesting increased plasma membrane permeability. The increased intracellular Na ÷ concentration might be responsible for the observed stimulation of the Na+/K + pump. This effect was dependent upon the synthesis of
viral proteins because it was abolished by the addition of cycloheximide up to 4 h post-infection. Prevention of the increase in intracellular Na ÷ by the use of low Na ÷-
containing media did not modify the pattern of protein synthesis. This suggests that changes in intracellular Na + and K ÷ concentrations were not related to the shutoff of cellular protein synthesis. Alterations of ion contents in the rotavirus-infected enterocytes might impair intestinal absorptive capacity before the appearance of histopathological lesions.
Del Castillo, Jesús R., Julio C. Arévalo, Luis Burguillos, and Marı́a C. Súlbaran-Carrasco. b-Adr... more Del Castillo, Jesús R., Julio C. Arévalo, Luis Burguillos, and Marı́a C. Súlbaran-Carrasco. b-Adrenergic agonists stimulate Na1-K1-Cl2 cotransport by inducing intracellular Ca21 liberation in crypt cells. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G563–G571, 1999.—Epinephrine and b-adrenergic agonists (b1 and b2 for isoproterenol, b1 for dobutamine, b2 for salbutamol) stimulated K1 (or 86Rb) influx mediated by the Na1-K1-2Cl2 cotransporter and the Na1-K1 pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na1-K1 pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 μM epinephrine with an EC50 of 91.6 6 9.98 nM. Epinephrineinduced K1 transport was blocked by propranolol with an IC50 of 134 6 28.2 nM. a-Adrenergic drugs did not modify K1 transport mechanisms. Neither Ba21 nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition...
American Journal of Physiology-Gastrointestinal and Liver Physiology
Epinephrine and β-adrenergic agonists (β1 and β2 for isoproterenol, β1 for dobutamine, β2 for sal... more Epinephrine and β-adrenergic agonists (β1 and β2 for isoproterenol, β1 for dobutamine, β2 for salbutamol) stimulated K+ (or86Rb) influx mediated by the Na+-K+-2Cl−cotransporter and the Na+-K+pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na+-K+pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 μM epinephrine with an EC50 of 91.6 ± 9.98 nM. Epinephrine-induced K+ transport was blocked by propranolol with an IC50 of 134 ± 28.2 nM. α-Adrenergic drugs did not modify K+ transport mechanisms. Neither Ba2+ nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition, epinephrine did not affect K+ efflux. Dibutyryl cAMP did not alter K+ transport. Reduction of extracellular Ca2+ to 30 nM did not influence the response to epinephrine. However, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM abolished epinephrine-induced K+ tra...
Biochemical and Biophysical Research Communications, 2010
P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the ... more P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H +), have been reported. They include reticulum and plasma-membrane Ca 2+-ATPases, Na + /K +-ATPase and H + /K +-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K +-ATPase a1-isoform, H + /K +-ATPase a2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K +-ATPase a2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.
Acta Cient Venez, 2005
Transepithelial Na + transport is mediated by passive Na + entry across the luminal membrane and ... more Transepithelial Na + transport is mediated by passive Na + entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na + /K + pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na + /K +-ATPase and the ouabain-insensitive, furosemideinhibitable Na +-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na +-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na +-and Na + / K +-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na +-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na +-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na +-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na + transport and cellular Na + homeostasis leads us to reconsider its role in health and disease.
Acta científica venezolana, 1993
The active ion transport is related to some vital functions for the normal eukaryotic cell metabo... more The active ion transport is related to some vital functions for the normal eukaryotic cell metabolism. The study of transport mechanism and its regulation is a fundamental problem in cellular biology. The production of monoclonal antibodies (AcM) let us to have a probe, with large specificity, for the study of the localization, distribution and function of carrier proteins in the epithelium, as well as the study of its molecular structure, synthesis and assembling in the cell. The quality of a monoclonal antibody depends of the proper selection of each step to follow in the course of its production. In this article, we examine the strategy more currently used in the production of monoclonal antibodies and its direct use in the ionics pump's morphological, biochemical and physiological study.