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J A Arruda

Publications and source records attributed to J A Arruda.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of the renal Na-HCO3 cotransporter by cAMP and Ca-dependent protein kinases.

Changes in the activity of the brush-border Na-H antiporter are accompanied by parallel changes in the activity of the Na-HCO3 cotransporter. Adenosine 3',5'-cyclic monophosphate (cAMP) and calmodulin inhibit the Na-H antiporter, whereas protein kinase C (PKC) stimulates it. We hypothesized that cAMP, calmodulin, and PKC should have similar effects on the Na-HCO3 cotransporter activity. Phosphorylated renal basolateral membranes were treated with either cAMP, calmodulin, or phorbol ester. cAMP, 1 microM, inhibited HCO3-dependent 22Na uptake without affecting 22Na uptake in presence of gluconate, suggesting that cAMP inhibits Na-HCO3 cotransporter activity without altering diffusive 22Na uptake. The effect of cAMP to inhibit the Na-HCO3 cotransporter could also be elicited by the catalytic subunit of cAMP, and this inhibitory effect was prevented by the protein kinase A (PKA) inhibitor. Calmodulin (1 microM), in presence of Ca, also inhibited HCO3-dependent 22Na uptake in presence of HCO3, whereas 22Na uptake in the presence of gluconate was unchanged. The inhibitory effect of calmodulin on HCO3-dependent 22Na uptake was prevented by N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13), an inhibitor of calmodulin. Phorbol 12-myristate 13-acetate and PKC stimulated Na-HCO3 cotransporter activity, whereas the inactive analogue, 4 alpha-phorbol, failed to elicit such a stimulation. Basolateral membranes displayed cAMP-dependent and Ca-dependent protein kinase activities. Thus PKA and Ca-dependent protein kinases regulate the activity of the Na-HCO3 cotransporter and suggest that hormones that act through these systems modulate the activity of the Na-HCO3 cotransporter.

Animals↗
PubMed1992

ATP-dependent renal H+ translocation: regional localization, kinetic characteristics, and chloride dependence.

We characterized Mg(2+)-dependent ATPase activity in membranes from the renal cortex, the outer and inner stripes of the outer medulla, and papillary vesicles. In all regions, there was Mg(2+)-dependent ATPase activity that was resistant to oligomycin and vanadate and sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide, and filipin. DCCD-Sensitive Mg(2+)-ATPase activity was highest in the inner stripe of the outer medulla and lowest in the cortex, with intermediate values in the outer stripe of the outer medulla and papilla. The Km for ATP, however, was similar among the different regions of the kidney. DCCD-Sensitive Mg(2+)-ATPase activity was critically dependent upon chloride with Km for Cl- in the range of 2-5 mM. In the presence of ATP, this ATPase was capable of H+ translocation, as assessed by acridine orange quenching. Inhibitors of ATPase activity prevented H+ translocation, which suggests that the Mg(2+)-ATPase represents, at least in part, an H(+)-ATPase. H+ transport was likewise critically dependent upon chloride, with similar Km. The effect of chloride on H+ translocation was blocked by the chloride channel inhibitor, diphenylamine-2 carboxylic acid. In the absence of chloride, H+ transport was abolished, but it could be partially restored by the creation of a favorable electric gradient by K+ and valinomycin. These studies demonstrate that the renal H(+)-ATPase exhibits different activities in various regions of the kidney. The ATPase activity and H+ translocation are critically dependent upon the presence of chloride, which suggests that chloride influences H+ translocation by dissipating the H+ gradient and acting at the catalytic site of the ATPase.

Acridine Orange↗
PubMed1992

Anion channel in basolateral cortical membranes of the rabbit kidney.

The presence of chloride conductance in basolateral membranes of proximal tubule is controversial. We measured 36Cl uptake in basolateral membranes loaded with KCl and suspended in a K(+)-free solution to create a positive intravesicular potential difference. Under these conditions, 36Cl uptake was maximal at 1 min, remained stable for at least 10 min and decreased to equilibrium levels by 60-120 min. Collapse of the voltage by valinomycin decreased 36Cl uptake by 46%, indicating the presence of K(+)-gradient-dependent chloride uptake. The chloride channel inhibitor diphenylamine-2-carboxylic acid inhibited 36Cl uptake in a dose-dependent fashion. 36Cl uptake was inhibited equally by unlabeled chloride, iodide and nitrate but not by sulfate or gluconate, indicating that the basolateral anion conductance is relatively selective. 36Cl uptake was pH independent but was calcium dependent. Phosphorylation of basolateral membranes with ATP significantly decreased 36Cl uptake, but the inhibitory effect of ATP was not further altered by exogenous cyclic AMP or the active phorbol ester PMA. These data demonstrate the presence of a relatively selective basolateral anion conductance which is regulated by pH, calcium and ATP.

Adenosine Triphosphate↗
PubMed1991

Epidermal growth factor binding to cortical basolateral membranes in compensatory renal hypertrophy.

We studied epidermal growth factor (EGF) binding to renal basolateral membranes before and following unilateral nephrectomy. After 48 h unilateral nephrectomy there was a small increase in kidney cortex weight but EGF binding was unchanged, suggesting that alterations in EGF binding do not play a role in early renal hypertrophy. In contrast, 3 week unilateral nephrectomy was associated with a significant decrease in the Bmax of the high affinity binding sites for EGF without a change in the affinity constant. The changes in EGF binding seemed specific since binding for insulin was not changed by 3 week unilateral nephrectomy. The changes in EGF binding were not correlated with changes in Na-H antiporter activity elicited by unilateral nephrectomy but seemed inversely correlated with changes in renal cortical weight. Our results demonstrate that unilateral nephrectomy is not associated with changes in EGF binding in early stages, but is associated with a decrease in the number of high affinity binding sites after 3 weeks. This suggests that in the steady state, compensatory renal hypertrophy is associated with 'down regulation' of the EGF receptor.

Animals↗
PubMed1991

High-affinity binding sites for VIP in renal cortical membranes: possible role of VIP in renal transport.

We studied binding and degradation of vasoactive intestinal peptide (VIP) by highly purified brush border and basolateral membranes from rabbit kidney cortex. Brush border and basolateral membranes were capable of 73 and 49% degradation of VIP after 20 minutes, and the degradation was totally prevented by bacitracin. There was 66 and 87% specific binding of 125I-VIP to brush border and basolateral membranes, respectively. 125I-VIP binding to renal membrane was displaced in a dose dependent fashion by unlabeled VIP with half maximal displacement at 2 x 10(-7) M. Other related peptides failed to displace VIP. Scatchard analysis showed one single class of receptors for VIP in both membranes with similar Kd (0.5 x 10(-7) M), but higher number of binding sites (Bmax) in the basolateral membranes than in the brush border membranes (22.0 vs. 4.4 pmol/mg protein), respectively. Forty-eight percent of VIP binding to brush border membranes could be explained by cross contamination of these membranes with basolateral membranes. We examined the effect of VIP on Na-H antiporter, Na-dependent glucose uptake and Na-dependent phosphate uptake by isolated proximal tubule suspension. In acid loaded proximal tubules VIP (10(-6) M) inhibited total and amiloride-sensitive 22Na uptake by 35 and 75%, respectively, as compared to control. On the other hand VIP failed to inhibit Na-dependent methyl alpha-14C-glucopyranoside and Na-dependent 32phosphate uptake. VIP failed to stimulate cyclic AMP generation by proximal tubule suspension while PTH showed the expected stimulation. Our results demonstrate the presence of specific binding for VIP in highly purified cortical membranes and suggest an effect of VIP to inhibit the Na-H antiporter by a mechanism independent of cyclic AMP.

Animals↗
PubMed1991

Adaptation to metabolic alkalosis by the turtle urinary bladder.

We studied the mechanism of adaptation to metabolic alkalosis by the turtle urinary bladder in vitro. Turtles were made alkalotic by administration of oral NaHCO3. Bladders removed from alkalotic turtles had an increased rate of HCO3- secretion in vitro as compared with that of control. H+ secretion, however, was not different, indicating that metabolic alkalosis selectively increases HCO3- secretion. Fluorescence microscopy was used to quantify the carbonic anhydrase cells. The total number of carbonic anhydrase cells was determined by mucosal staining of the bladder with 6-carboxyfluorescein diacetate. The number of HCO3(-)-secreting cells (beta cells) was quantified by mucosal staining with NBD-taurine and the number of H(+)-secreting cells (alpha cells) was calculated from the difference between the two. Metabolic alkalosis significantly increased the total number of 6-carboxyfluorescein positive cells and NBD-taurine-positive cells. The increase in the number of 6-carboxyfluorescein positive cells was totally accounted for by the increase in the NBD-taurine-positive cells without change in the number of alpha cells. If NBD-taurine accurately reflects the number of beta cells, these studies show that the adaptation to metabolic alkalosis is mediated, at least in part, by an increase in the number of HCO3(-)-secreting (beta) cells.

Adaptation, Physiological↗
PubMed1991

Dual effect of cyclic GMP on renal brush border Na-H antiporter.

The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 microM) increased the amiloride-sensitive 22Na uptake in control from 1.26 +/- 0.13 to 1.54 +/- 0.12 nmol/mg/protein/10 sec, P less than 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 microM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.

Adenosine Triphosphate↗
PubMed1990

Stimulation of H+ secretion by CO2 in turtle bladder: role of intracellular pH, exocytosis, and calcium.

We studied the interaction of intracellular pH, exocytosis, and cell calcium on the stimulation of H+ secretion by CO2 in turtle bladder. Intracellular pH was continuously monitored by the fluorescent dye 6-carboxyfluorescein and exocytosis was monitored by the release of mucosal fluorescein dextran. The initial stimulation of H+ secretion by 1 or 5% CO2 added to the serosal solution was accompanied by a similar and temporally related increase in exocytosis. Furthermore, a decrease in intracellular pH seems necessary for the early increase in H+ secretion and exocytosis. Because calcium plays an important role in exocytosis, we measured intracellular calcium in isolated cells with the fluorescent dye quin2. An increase in intracellular calcium (from 50 to 100 nM) was observed in isolated turtle bladder epithelial cells gassed with 5% CO2. To further evaluate the role of intracellular calcium on H+ secretion and exocytosis we utilized agents that alter cell calcium such as trifluoperazine and lanthanum. In the presence of CO2 these agents blocked partially the increase in H+ secretion and exocytosis but did not affect the decrease in intracellular H+. In conclusion, exocytosis, intracellular pH, and intracellular calcium play a key role in mediating CO2-stimulated H+ secretion in the turtle bladder.

Animals↗
PubMed1990

Regional localization of renal Na(+)-H+ antiporter: response to respiratory acidosis.

The Na(+)-H+ antiporter of renal brush-border membranes has been well characterized and plays a role in adaptation to acidosis. Na(+)-H+ antiporter activity has been described in other renal regions, but its kinetics as well as its role in adaptation to acidosis are unclear. Thus we measured Na(+)-H+ antiporter activity in membrane vesicles of outer and inner stripes of outer medulla (OSOM and ISOM, respectively) and in plasma membranes from papilla and compared it to Na(+)-H+ antiporter activity of the cortex in control and hypercapnic rabbits. Chronic hypercapnia (induced by exposure to CO2 for 48 h) was associated with significantly higher PCO2 and plasma HCO3- and lower urine pH than controls. In control animals, magnitude of Vmax of amiloride-sensitive component of Na(+)-H+ antiporter (expressed as fluorescence units.300 micrograms protein-1.min-1) was 392.2 +/- 32 in cortex, 115 +/- 9.7 in OSOM, 66.1 +/- 9.4 in 15-25% (F1) fraction and 118.7 +/- 16.8 in 25-40% (F2) fraction of ISOM, respectively, and 79.3 +/- 5.2 in papilla. These values were significantly different from each other except between F1 and papilla and F2 and OSOM. The Km for Na, however, was not different, suggesting that the renal Na(+)-H+ antiporter is basically the same in different renal regions but displays different activity. Hypercapnia for 48 h increased significantly the amiloride-sensitive component of Na(+)-H+ antiporter by 60% in cortex, 43% in F1, and 29% in papilla but failed to alter Vmax in OSOM.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis, Respiratory↗
PubMed1990

Adaptation to respiratory acidosis in the turtle bladder.

The effect of in vivo respiratory acidosis for 4 and 48 hr was examined in the turtle bladder by placing turtles in hypercapnic chambers. Blood pH was significantly lowered and pCO2 was significantly elevated over control values both 4 and 48 hr, while blood bicarbonate was only increased after 48 hr. In vitro rates for H+ secretion determined by the reverse short-circuit current were significantly greater in bladders from 48 hr of respiratory acidosis than those of controls (27.3 +/- 2.7 vs 20.6 +/- 1.7 microA, P less than 0.05). In vitro rates for HCO3- secretion determined by pH stat were not altered. Fluorescence microscopy was used to study cell morphology. The number of carbonic anhydrase cells (corrected for the total number of cells) as determined by four different fluorescence stains (6-carboxyfluorescein, rhodamine 123, acridine orange, and 3,3'-diethyloxacarbocyaninine iodide) was increased both after 4 and 48 hr of respiratory acidosis. However, the number of HCO3(-)-secreting (beta subtype) carbonic anhydrase cells, determined by a probe for the anion exchanger, NBD-taurine, was not increased. In vitro 1% CO2 for 4 hr also resulted in an increase in H+ secretion and in the number of 6-carboxyfluorescein-positive cells, both of which could be blocked with SITS pretreatment. We conclude that CO2 changes the mucosal cells more toward the carbonic anhydrase phenotype, and that if NBD-taurine accurately identifies the beta cells, that the adaptation produces or recruits more alpha-carbonic anhydrase cells.

Acidosis, Respiratory↗
PubMed1989

Methyl isobutyl amiloride: a new probe to assess the number of Na-H antiporters.

We measured the binding of [3H]-5-(N-methyl-N-isobutyl) amiloride (MIA) to purified rabbit renal brush border membranes. MIA binding was protein, temperature and time dependent with optimal binding at pH 8.0 or above. At low pH MIA binding was inhibited, suggesting competition between H+ ions and MIA for the MIA binding site. There was 70-80% specific binding which reached a plateau at 30 min and remained stable thereafter for 150 min. Scatchard analysis revealed one family of binding sites with Bmax of 3.4 +/- 0.4 pmoles/mg protein and Kd of 30.5 +/- 2.3 nM. MIA inhibited the Vmax of the Na-H antiporter (assessed by acridine orange quenching) in a dose dependent fashion with 100% inhibition at MIA concentration of 10(-3) M and this inhibition was greater than that of amiloride. We conclude that MIA, a potent inhibitor of the Na-H antiporter, displays a high percentage of specific binding to renal brush border membranes and can be used to assess the number of the Na-H antiporters.

Amiloride↗
PubMed1989

Permeability of the urinary bladder of the turtle, Pseudemys scripta elegans, to ammonia.

1. The permeability of the isolated turtle urinary bladder to ammonia was investigated by varying the ratio of NH3 to NH4+ concentration in the serosal bath and measuring the flux of ammonia into the mucosal bath. 2. The permeability of the turtle bladder to NH3 was in the range of 5.4-6.1 x 10(-3) cm/sec and the permeability of the unstirred layer to NH4+ was in the range of 1.1-2.1 x 10(-5) cm/sec. 3. The flux of ammonia calculated from the permeability of NH3 can account for the decrease in mucosal H+ secretion observed with addition of NH4Cl to the serosal bath.

Ammonia↗
PubMed1989

Turtle urinary bladder epithelial cell line.

1. We compared enzymatic and functional properties of a turtle bladder cell line to those of turtle bladder epithelial cells. Like the original epithelium, the cell line displays carbonic anhydrase activity and acetazolamide inhibits O2 consumption in isolated cells and acidification by cells grown in monolayers. 2. Staining with acridine orange revealed the presence of cytoplasmic orange red vesicles which could be dissipated by NH4Cl or protonophores indicating that these vesicles represent areas of low pH. 3. Addition of ATP to cells permeabilized by digitonin led to reappearance of the red granules suggesting that acidification of the vesicles is mediated by H+-ATPase.

Animals↗
PubMed1989

pH regulation of calcium efflux by turtle liver mitochondria.

1. Turtle liver mitochondria are capable of taking up calcium in a pattern similar to that described from rat liver mitochondria. 2. Turtle liver mitochondria also possess a system for calcium efflux which is extremely sensitive to changes in extramitochondrial pH. A decrease in extramitochondrial pH by addition of HCl or by gassing with CO2 caused a rapid release of calcium. 3. The profound changes in pH and pCO2 during deep diving likely affects Ca efflux from mitochondria in the turtle liver.

Animals↗
PubMed1989

Na-HCO3 cotransport and Na-H antiporter in chronic respiratory acidosis and alkalosis.

Renal acidification in renal proximal tubule is thought to be mediated by luminal Na-H antiporter and the HCO3- generated by this antiporter is removed from the cell by a basolateral Na-HCO3 cotransporter. To study the effect of respiratory acid-base disorders on these transport systems, we have measured the Na-HCO3 cotransport in basolateral membranes and Na-H antiporter in luminal membranes in control rabbits, rabbits exposed to 10% CO2 (chronic hypercapnia), and rabbits exposed to 10% O2-90% N2 (chronic hypocapnia). The Vmax of HCO3(-)-dependent 22Na uptake was significantly higher in chronic hypercapnia than controls (2.54 +/- 0.03 vs. 1.18 +/- 0.21 nmol.mg protein-1.3 s-1, P less than 0.001). Likewise, the Vmax of the Na-H antiporter was also increased compared with controls (924.9 +/- 42.1 vs. 549.1 +/- 62.8 fluorescence units (FU).300 micrograms protein-1.min-1). In chronic hypocapnia, the Vmax of Na-HCO3 cotransport was lower than controls (0.72 +/- 0.11 vs. 1.18 +/- 0.21 nmol.mg protein-1.3 s-1, P less than 0.05). There was no difference, however, in the Vmax of the Na-H antiporter between hypocapnia and control (524.2 +/- 24.3 vs. 549.1 +/- 62.8, FU.300 micrograms protein-1.min-1). The Vmaxs of the Na-HCO3 cotransport and of the Na-H antiporter in hypocapnic, control, and hypercapnic rabbits were linearly related (r = 0.81), suggesting a simultaneous adaptation of the two systems in respiratory acid-base disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis, Respiratory↗
PubMed1988

Effect of metabolic or respiratory acidosis on rabbit renal medullary proton-ATPase.

Distal urinary acidification is thought to be mediated by an H+-ATPase sensitive to N-ethylmaleimide and dicyclohexyl-carbodiimide. We have studied the effect of chronic metabolic acidosis (NH4Cl for 3 days) or respiratory acidosis (inhalation of 10% CO2 for 2 days) on the H+-ATPase of plasma membranes prepared from the medulla. The enzymatic assay for the H+-ATPase was performed in the presence of ouabain and oligomycin and in the absence of Ca. H+-transport activity was assessed by the quenching of acridine orange in the presence of ATP. The 15-25% sucrose gradient fraction was enriched 40-fold in enzymatic activity over the homogenate, and 8-fold in enzymatic activity and 4-fold in H+-transport activity over the fluffy fraction (38,000 X g). Metabolic acidosis (pH less than 7.31) or chronic hypercapnia (PCO2 greater than 66 mmHg; 1 mmHg = 133.3 Pa) was induced for 2-3 days. Both groups showed the same enrichment factor in enzymatic and H+-transport assays as the control rabbits. Enzymatic and H+-transport activities, however, were not different between animals with respiratory acidosis and controls. Kinetic studies failed to disclose an increase in Vmax (673 vs. 702 mumol/(mg protein.min] or a decrease in Km (0.43 vs. 0.48 mM) in chronic hypercapnia as compared with controls. Metabolic acidosis also failed to increase H+-ATPase activity. These data demonstrate that the H+-ATPase of renal medulla does not display the expected increase in activity during acidosis. The role of this H+-ATPase in the adaptation to acidosis remains to be determined.

Acidosis↗