Biomedical subjects

A G Lopes

Publications and source records attributed to A G Lopes.

At least 19 recordsLinked to original sources

PubMed1999

Bradykinin modulates the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule.

This paper studies the modulation by bradykinin of the ouabain-insensitive Na+-ATPase activity in both renal cortex homogenate and basolateral membrane from proximal tubule. The increase in bradykinin concentration from 10-14 to 10-10 M stimulated the ouabain-insensitive Na+-ATPase activity in cortex homogenates about 2.2-fold, but inhibited the enzyme activity of basolateral membrane preparations by 60%. In both preparations, the maximal effect was obtained with 10-10 M bradykinin. Further increase in the concentration of bradykinin completely abolished these effects. The antagonist of the B2 receptor, Hyp3, completely abolished the effect of 10-10 M bradykinin on the Na+-ATPase activity in the basolateral membrane preparation in a dose-dependent manner, but had no effect on the bradykinin stimulated enzyme activity of the cortex homogenate. Furthermore, in the presence of 10-7 M Hyp3, 10-10 M bradykinin stimulated the Na+-ATPase activity by 45% in the basolateral membrane preparations. The increase in des-Arg9-bradykinin concentration from 10-12 to 10-7 M, an agonist of the B1 receptor, stimulated the Na+-ATPase activity of the cortex homogenates and of the basolateral membrane preparations by 105 and 148%, respectively. In the presence of 25 microM mergetpa, an inhibitor of kininase I, the increase in bradykinin concentration from 10-12 to 10-10 M promoted similar inhibition of the Na+-ATPase activity of both cortex homogenates and basolateral membrane preparations. These results suggest that bradykinin stimulated the Na+-ATPase activity of proximal tubule through the interaction with B1 receptors and inhibited the enzyme through the interaction with B2 receptors. Furthermore, the cortex homogenate expresses a kininase I activity that cleaves bradykinin to des-Arg9-bradykinin.

Adenosine Triphosphatases↗

PubMed1999

Angiotensin II activates the ouabain-insensitive Na+-ATPase from renal proximal tubules through a G-protein.

Angiotensin II (AG II) stimulates the ouabain-insensitive, furosemide- sensitive Na+-ATPase present in the basolateral membrane of pig renal proximal tubules in a dose dependent manner. Maximum effect was obtained with 10-8 M AG II, which corresponded to an activity 134% higher than control. Half of the maximum effect was observed between 10-11 M and 10-10 M, corresponding to physiological hormone levels. Saralasin, an AG II peptide analogue receptor antagonist, abolished the phenomenon, demonstrating that AG II interacts with specific sites in pig proximal tubules. The AG II stimulatory effect was also prevented by dithiothreitol (DTT), a reducing compound, and by 10 nM losartan, a non-peptide antagonist highly specific for AT1 receptors, characterizing AG II binding to AT1 receptors. GTPgammaS, a non-hydrolysable GTP analogue, increased by 159% the enzyme activity as compared to the control values. The simultaneous addition of 10-5 M GTPgammaS and 10-8 M AG II did not have additive effects. Furthermore, the stimulatory action of AG II was completely abolished by 0.1 microM GDPbetaS, a non-hydrolysable GDP analogue. Two microgram ml-1 pertussis toxin, an inhibitor of Gi-protein, did not modulate the AG II stimulatory effect. On the other hand, the Na+-ATPase activity was enhanced 100% in the presence of cholera toxin and 85% in the presence of both AG II and cholera toxin. Taken together, these data suggest that AG II activates the Na+-ATPase activity through AT1 receptors coupled to a pertussis-insensitive and cholera-sensitive G-protein.

Adenosine Triphosphatases↗

PubMed1998

Ouabain-insensitive Na(+)-ATPase activity of Malpighian tubules from Rhodnius prolixus.

In the present paper, we show the existence of a furosemide-sensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na(+)-pump. The main characteristics of this activity are: (1) K0.5 for Na+ = 1.49 +/- 0.18 mM, (2) Vmax = 2.8 +/- 0.1 nmol inorganic orthophosphate (Pi).mg prot-1.min-1, (3) it is fully abolished by 2 mM furosemide, (4)it is insensitive to ouabain concentrations up to 10(-2) M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca2+ in the incubation medium.

Adenosine Triphosphatases↗

PubMed1997

Effect of adenosine on the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule.

The regulation of the furosemide-sensitive Na+-ATPase activity and ouabain-sensitive (Na+ + K+)ATPase activities from proximal tubules by adenosine was investigated. When the concentration of adenosine was increased the furosemide-sensitive ATPase activity decreased with maximal inhibition at 10(-8) M (56% of inhibition). However, the (Na+ + K+)ATPase activity was not affected by adenosine. Theophylline, an antagonist of P1 adenosine receptor, completely reversed the effect of adenosine on the furosemide-sensitive ATPase activity in a dose-response manner. The adenosine effect was mimicked by N6-cyclohexyladenosine (CHA), an agonist for A1 adenosine receptor. 5'-N-ethylcarboxamideadenosine (NECA), an agonist for A2 adenosine receptor, did not affect the furosemide-sensitive ATPase activity. When adenosine was used in the presence of 1 microg ml(-1) pertussis toxin, a Gi protein inhibitor, no change in the furosemide-sensitive ATPase activity was observed. The addition of 1 nM cholera toxin increased the Na+-ATPase activity by 60%. Adenosine decreased the cholera toxin stimulated Na+-ATPase in 42%, similar to the effect observed in the absence of cholera toxin. Dibutyryl-cAMP reversed the effect of adenosine in a dose dependent manner while the protein kinase A peptide inhibitor mimicked it. These data are compatible with a modulatory effect of adenosine on the Na+-ATPase activity via A1 subtype receptor.

Adenosine↗

PubMed1996

Both the wild type and a functional isoform of CFTR are expressed in kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

Animals↗

PubMed1995

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus.

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using video-optic techniques. When the medium osmolality was increased with addition of 100 mM mannitol the cells shrank to a minimum of 16.84 +/- 2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K+ or Cl- or HCO(3-) by Na+, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na+ by tetramethylammonium (TMA+) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10(-4) M, anthracene-9-COOH 5 x 10(-4) M, furosemide 5 x 10(-4) M or ethacrynic acid 5 x 10(-5) M, also abolished RVI. On the other hand, addition of either Ba2+ 10(-3) M, SITS 5 x 10(-4) M, ouabain 10(-3) M or vanadate 10(-3) M, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mM or verapamil 10(-6) M, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mM induced a cell swelling to a maximum of 33.11 +/- 1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na+, K+, Cl-, Ca2+ and HCO(3-) transport pathways and a ouabain-insensitive ATPase stimulated by Na+.

Animals↗

PubMed1992

Cell volume regulation in rat thin ascending limb of Henle's loop.

Thin ascending limb cells of Henle's loop from Wistar rats were studied with in vitro microperfusion and video-optical techniques to investigate their ability in regulating cell volume during osmotic shock and to identify mechanisms of ion transport involved in the process. These cells showed a clear volume regulatory decrease (VRD) response in hyposmotic medium, but no volume regulatory increase in hyperosmotic medium. The presence of barium in the bath abolished VRD. Removal of K+ from bath and perfusate also inhibited the VRD response. Reintroduction of K+ in hyposmotic conditions reestablished cell volume regulation. Introduction of anthracene-9-COOH to the basolateral medium blocked cell volume regulatory response. Cl- removal from perfusate and bath solutions also inhibited VRD, probably because of a significant intracellular Cl- depletion. Exposure of cells to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid in perfusate and bath solutions reduced significantly Ca2+ concentration and impaired VRD. Reintroduction of Ca2+ in hyposmotic conditions restored volume regulation. The presence of ouabain in basolateral medium also inhibited VRD. These data suggest that the following mechanisms in the basolateral membrane are involved in VRD response: K+ and Cl- conductive pathways, which might be Ca2+ dependent for activation, and an Na(+)-K(+)-adenosinetriphosphatase.

Animals↗

PubMed1990

pH-stat experiments in proximal renal tubules.

The pH-stat technique has been used to measure H+ fluxes in gastric mucosa and urinary bladder "in vitro" while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1 N HC1-filled microelectrodes to inject OH- or H+ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H(+)-ion conductance). These ratios were reduced after luminal 10(-4) M acetazolamide, during injection of OH-, but they increased during injection of H+. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H+ fluxes per cm2 epithelium. The relation between JH+ and pH gradient showed saturation kinetics of H fluxes, both during OH- and H+ injection. This kinetic behavior is compatible with inhibition of JH by luminal H+. It is also compatible with dependence on Na+ and H+ gradients of a saturable Na/H exchanger. H(+)-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H+ flow is mediated by a membrane component, most likely the Na(+)-H+ exchanger.

Acetazolamide↗

PubMed1990

Ionic dependence of cell volume regulation by the thin ascending limb of Henle's loop.

Thin ascending limb cells from Henle's loop were studied with optical and video techniques to evaluate cell volume regulation in response to anisoosmotic media and its ionic dependence. Cell volume regulation was observed when these cells were exposed to hypoosmotic solutions. Under hyperosmotic conditions only an osmometric response was found, with no volume regulatory increase (VRI). The removal of Cl- or HCO3- abolished the volume regulatory decrease (VRD) normally observed during exposure to hypoosmotic solutions. Re-addition of these ions did not elicit the VRD response. The removal of K+ from hypoosmotic solutions abolished VRD but its re-introduction restored the volume regulatory response. In the absence of Na+, a partial inhibition of VRD was found; re-addition of Na+ completely restored the regulatory response. These indicate that cells from the thin ascending limb of Henle's loop regulate their volume under hypoosmotic conditions, and that this process is dependent upon Cl-, HCO3-, Na+ and K+, with different patterns of response being observed upon addition or deletion of these ions.

Animals↗

PubMed1988

Cell volume regulation by the thin descending limb of Henle's loop.

Thin descending limb cells from Henle's loop (from the inner strip of the outer medulla of long loops) were studied with optical and video techniques to identify the mechanisms of ion transport and cell volume regulation. Increasing the K+ concentration in the basolateral solution from 5 to 90 mM caused the cells to swell. This K+-induced swelling was inhibited by exposure of the basolateral membrane to 9 mM Ba2+ and was abolished by removing Cl- from the perfusion solutions. Decreasing the perfusion osmolality caused an increase in cell volume followed by a return to the preexposure volume. The latter regulatory decrease in hypoosmolality was slowed by basolateral Ba2+ and the removal of HCO-3 from the solutions. Further slowing occurred when both HCO-3 and Cl- were removed. Exposure of cells to ouabain abolished volume regulation. These data suggest that the basolateral cell membrane of the thin descending limb has a Cl- -dependent K+ permeability, which is important in cell volume regulation. The cells also possess Cl- and HCO-3 transport pathways that participate in volume regulation. Finally, volume regulation is dependent upon the operation of the Na/K pump.

Animals↗

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↗