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Biomedical subjects

A G Lopes

Publications and source records attributed to A G Lopes.

52 records · Page 3Linked to original sources
PubMed1988

Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.

Ammonia↗
PubMed1987

Volume regulation in the early proximal tubule of the Necturus kidney.

The ability of early proximal tubule cells of the Necturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes. Necturus proximal tubule cells regulate volume in both hyper- and hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO3- dependent and is associated with a decrease in the relative K+ conductance of the basolateral cell membrane and a decrease in the resistance ratio, Ra/Rbl. Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO3- but is also inhibited by 2 mM Ba2+ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K+ conductance of the basolateral cell membrane and an increase in Ra/Rbl. Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO3- removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO3(-)-dependent transport systems and the baso-lateral K+ conductance.

Animals↗
PubMed1987

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule.

This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane potential (Vbl) and intracellular pH (pHi) of 1) lowering basolateral [HCO3-] at constant PCO2, and 2) replacing Na+ with N-methyl-D-glucamine. Vbl and pHi were measured with Ling-Gerard and liquid-membrane pH microelectrodes, respectively, in isolated tubules perfused in vitro. We found that decreasing basolateral [HCO3-] from 10 mM (pH 7.5) to 2 mM (pH 6.8) resulted in an immediate depolarization of 14.9 mV, and a pHi decrease of 0.35. SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulfonic acid, 0.5 mM) inhibited the HCO3-induced depolarization by 87% and inhibited the initial rate of the pHi decrease by 79%. Replacement of basolateral Na+ with N-methyl-D-glucamine resulted in an immediate depolarization of 11.3 mV, and a pHi decrease of 0.36. SITS inhibited the zero Na-induced depolarization by 86% and the initial rate of the pHi decrease by 81%. Nominal removal of basolateral HCO3- (replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) inhibited the zero Na-induced depolarization by 64%, whereas nominal removal of Na+ inhibited the 2 mM HCO3-induced depolarization by 67%. Replacement of all basolateral Cl- with glucuronate did not inhibit the changes in Vbl induced by changing [HCO3-] or [Na+]. Observations similar to those described above have been made previously on Ambystoma proximal tubules, and attributed to an electrogenic Na/HCO3 cotransport mechanism that carries HCO3-, Na+, and net negative charge in the same direction. We conclude that Necturus proximal tubules possess a similar, if not identical, electrogenic Na/HCO3 cotransport mechanism.

Animals↗
PubMed1987

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence.

When the filtered load of buffers like bicarbonate or phosphate is increased by elevating GFR or buffer concentration in plasma, the overall renal reabsorption of bicarbonate or the formation of titratable acidity are markedly increased. The same happens when buffer concentration or flow rate are varied during proximal microperfusion. We have recently studied the mechanisms of this functional dependence. We have observed that the rate of bicarbonate reabsorption is always proportional to luminal buffer concentration when a stationary fluid column is injected into the proximal lumen. H-ion secretion is also proportional to luminal levels of non-bicarbonate buffers. Using a pH-stat technique adapted to renal tubules, we have shown that H-ion secretion is dependent on proximal pH independently of the used buffer species. A kinetic analysis of these data shows a non-linear relationship between luminal H+ and H+ secretion, compatible with carrier mediated transport.

Animals↗
PubMed1986

Regulation of single potassium ion channels from apical membrane of rabbit collecting tubule.

The regulation of K+-channel activity from the apical membrane of the rabbit cortical collecting tubule was studied using the patch-clamp technique. Using inside-out patches, channel open probability was determined as a function of calcium and barium concentration and transmembrane potential. Channel open probability was increased by raising bath (cytoplasmic) calcium concentration, with an apparent Ka of 2.4 microM. Mean channel open time also increased during this maneuver. The channel was reversibly inhibited by barium, applied to the cytoplasmic face, with an apparent Ki of 12 microM. Depolarization of the transmembrane potential increased channel open probability. With 1 mM calcium in the bath solution, the open probability was one-half maximal at -55 mV. It is concluded that this channel is the probable route for transcellular K+ secretion by the cortical collecting tubule and that procedures likely to increase intracellular calcium and/or depolarize the apical membrane will cause an increased potassium secretion.

Animals↗
PubMed1985

Basolateral Na-H exchange in the rabbit cortical collecting tubule.

We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and -6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na-dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT.

Amiloride↗
PubMed1985

Dinitrophenol effect on proximal tubular acidification in the rat.

The effect of 10(-3) M-dinitrophenol (DNP) on renal tubular acidification was studied in proximal tubules of rat kidneys perfused with mammalian Ringer solution. Alkaline (pH 7.8) or acid (pH 5.8) phosphate-buffered solutions were injected into the lumen, and pH changes recorded with antimony micro-electrodes. Luminal perfusion with DNP caused complex acidification or alkalinization curves, an initial rapid shift toward a higher than control pH being followed by a slower acidification. Acidification half-times of the initial phase (t1/2 = 1.6 s) were markedly shorter than controls (6.2 s). This response was probably due to transient action of DNP, since keeping constant peritubular DNP levels by capillary perfusion caused simple exponential pH curves. In such experiments luminal pH increased from pH 6.6-6.8 to 7.1-7.2, while acidification and alkalinization t1/2 decreased from about 7 s to 3-5 s. Secretory H-ion fluxes increased transiently and then fell below controls after a few minutes of perfusion, while H-ion efflux from the lumen increased progressively. These data suggest that, besides its known effect on cell metabolism, DNP acts directly on proximal tubular cell membranes, increasing the rate of passive H-ion equilibration, both mechanisms impairing the tubular capacity to maintain normal proximal pH gradients and fluxes.

2,4-Dinitrophenol↗
PubMed1984

Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules.

Recordings of single potassium channels from the apical membrane of rabbit cortical collecting tubule have been achieved using the patch-clamp technique. The conductive properties of the channel have been studied in inside-out patches. The slope conductance of the open channel is approximately equal to 90 pS. The channel is selective to potassium over sodium, with a selectivity ratio of 9:1. Decreasing the calcium concentration of the solution bathing the cytoplasmic face of the patch results in a decrease of the open-channel probability. Decreasing the calcium concentration to 10 nM or less completely inhibited channel activity. The channel is also inhibited by barium in a dose-dependent fashion.

Animals↗
PubMed1982

Voltage transients during ionic substitution in renal cortical tubules.

A voltage transient is described which is found during proximal tubular perfusion with impermeant cation or anion salt solutions in the rat. It was shown that the magnitude of transepithelial diffusion potentials depended on luminal hydrostatic pressure, suggesting that the observed transients might be the consequence of the enlargement of ionic pathways by tubular dilatation. Thus, when reporting PD values, care should be taken to define the pressure levels at which measurements were performed.

Animals↗
PubMed1976

Conductances, diffusion and streaming potentials in the rat proximal tubule.

1. Transtubular potential differences and specific resistances were measured in rat proximal tubules by means of single and double barrelled glass micro-electrodes. 2. Tip localization was made by observation of effective resistance changes measured with double barrelled micro-electrodes upon passage of oil droplets, and by perfusion with choline C1. 3. Mean early proximal p.d.s. of the order of -1 to -2 mV, and late values of +0-5 to +1mV were found. Mean specific resistances ranged from 12 to 15 omega cm2. 4. Diffusion potentials and single ion relative conductances were evaluated, perfusing the lumen with solutions differing only with respect to one salt concentration. Na and K conductances were similar and greater than those of C1. 5. Luminal and peritubular perfusions with hypotonic solutions showed the occurrence of streaming potentials in this structure suggesting the existence of pores lined with negative charges. The effective diameter of these pores appeared to be reduced by hypotonic perfusion, as evidenced by a significant increase in resistance, indicating that the main ion path across this structure is represented by intercellular spaces.

Animals↗
PubMedDate not supplied

Vanadate is toxic to adherent- growing multidrug-resistant cells.

The development of multidrug resistance (MDR) is the primary cause of failure of cancer chemotherapy and circumventing this problem is a major challenge in oncology. Vanadate is known to inhibit the ATPase activity of the P-glycoprotein and multidrug-resistant associated protein. In the present study we show that adherent MDR cells are more sensitive to vanadate than adherent non-MDR ones, but the same is not true for suspension-growing cells. Vanadate induced stress fiber in the non-MDR adherent MDCK cell line, but destroyed the actin fibers of MDCK/60 and MA104 cells, two adherent MDR cell lines, suggesting that the sensitivity of these cells to vanadate is related to their actin cytoskeleton. The results suggest that vanadate may be used as an adjuvant in the chemotherapy of solid tumors, not only as an ATPase inhibitor but also because of its effect in the MDR cell cytoskeleton.

ATP Binding Cassette Transporter, Subfamily B↗
PubMedDate not supplied

Cisplatinum-induced lesion of proximal tubule acidification in the rat.

Rats were given a 4- to 6-mg/kg body weight intraperitoneal injection of the antitumor drug, Cisplatin, 5-7 days prior to experiments to study tubule acidification by clearance and stationary microperfusion techniques. Cisplatin reduced the glomerular filtration rate markedly and caused a moderate degree of metabolic acidosis, but urine acidification (pH) was well maintained. Proximal tubule stationary pH and bicarbonate concentrations, as measured by pH microelectrodes, were significantly increased. The defect of proximal H+ secretion is reflected by increased acidification half-times (from 4.44 to 10.2 s) and reduced bicarbonate reabsorption to 37% of control values. H-ion back flux, measured during tubule and capillary perfusions with Ringer's bicarbonate- and CO2-free phosphate solutions, was reduced to 68% of control values. The apparent H-ion permeability was lowered from 0.79 to 0.54 cm/s. These results indicate that proximal acidification is reduced by impairment of H+ transport and not by increased transepithelial H+ shunting. Blunted acidification is compatible with a reduction in the number of Na/H exchangers in the proximal brush border and/or a decrease in the apical sodium gradient, the driving force for proximal H-ion secretion. Cortical distal tubule acidification, measured by double-barreled ion-exchange resin/PD microelectrodes, was not significantly affected by Cisplatin. This accounts for the observation that, in spite of the impaired proximal acidification, urine pH is kept within the normal range.

Acid-Base Equilibrium↗
PubMedDate not supplied

Trypanosoma cruzi epimastigotes express the ouabain- and vanadate-sensitive (Na(+)+K+)ATPase activity.

The presence of (Na(+)+K+)ATPase activity in CL14 clone and NIH NTY strain of Trypanosoma cruzi epimastigotes is demonstrated. A Na+ plus K+ stimulated ATPase activity is found in both strains. The optimal Na+/K+ ratio is 5:1 and 9:1 in CL14 clone and NIH NTY strain, respectively. In both strains, vanadate completely inhibits the ouabain-sensitive ATPase activity indicating that it belongs to the P-type (E1/E2) family of ion-transporting ATPases. The I50 for vanadate is 0.66 +/- 0.04 and 0.04 +/- 0.02 microM in CL14 clone and NIH NTY strain, respectively. These data indicate that both strains of T. cruzi epimastigotes express the ouabain- and vanadate-sensitive (Na(+)+K+)ATPase activity. On the other hand, the discrepancy between the parameters analyzed for the inhibitors suggests that they express different isoforms of this enzyme.

Animals↗
PubMedDate not supplied

Ouabain-insensitive Na(+)-ATPase activity in Trypanosoma cruzi epimastigotes.

In the present paper, the presence of a ouabain-insensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in T. cruzi epimastigotes CL14 clone and Y strain was investigated. The increase in Na+ concentration (from 5 to 170 mM), in the presence of 2 mM ouabain, increases the ATPase activity in a saturable manner along a rectangular hyperbola. The Vmax was 18.0 +/- 1.0 and 21.1 +/- 1.1 nmoles Pi x mg-1 x min-1 and the half-activation value (K50) for Na+ was 34.3 +/- 5.8 mM and 37.7 +/- 5.3 in CL14 clone and in Y strain, respectively. The Na(+)-stimulated ATPase activity was inhibited by 5-[aminosulfonyl]-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid (furosemide) in a dose-dependent manner. The half-inhibition value (I50) was 0.22 +/- 0.03 and 0.24 +/- 0.07 mM, and the Hill number (n) was 0.99 +/- 0.2 and 2.16 +/- 0.29 for CL14 clone and Y strain, respectively. These data indicate that both cell types express the ouabain-insensitive Na(+)-ATPase activity, which might be considered the biochemical expression of the second Na+ pump.

Analysis of Variance↗
PubMedDate not supplied

Expression of functionally P-glycoprotein in MA104 kidney cells.

Rhesus monkey kidney MA104 cells are a polarized epithelium with some unusual characteristics, including a resistance to ouabain, although their Na(+)-K(+)-ATPase has normal affinity with this drug. This work suggests that MA104 cells have high expression of functionally P-glycoprotein in their membranes. This was established using four complementary methods to investigate the expression and function of P-glycoprotein in these cells. MA104 cells were strongly resistant to vincristine, which could be reversed by three known P-glycoprotein modulators: verapamil, cyclosporin A and trifluoperazine. In addition, MA104 cells accumulate little rhodamine 123, and the incubation with verapamil increased this accumulation. The mdr1-mRNA was detected by reverse transcription-polymerase chain reaction and a subcloned 283-bp product was identified. Its nucleotide sequence was compared with the related region of human mdr1, showing a high identity (96%) between the two sequences. The expression of P-glycoprotein in the cell membrane was observed by Western blot and immunofluorescence. The results taken together suggest that MA104 cells intrinsically have a high expression of functionally P-glycoprotein in their membranes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗