Exchange In Brush-border Membrane Vesicles Research Articles

K+‐H+ Exchange Activity in Brush‐Border Membrane Vesicles Isolated from Chick Small Intestine

The purpose of this study was to investigate the presence of a K(+)-H+ exchanger in brush-border membrane vesicles (BBMV) isolated from chick small intestine. 86Rb+, as a tracer for K+ transport, was used to probe for the exchange mechanism. An outwardly directed proton gradient (pH 5.5 inside, pH 7.5 outside) stimulated 86Rb+ uptake into voltage-clamped BBMV. H(+)-driven 86Rb+ uptake was only weakly inhibited by 5-(N-ethyl-N-isopropyl)amiloride, whereas this agent strongly inhibited H(+)-driven Na+ uptake. At initial rates, proton-driven 86Rb+ uptake was significantly reduced by external K+ but it was not significantly affected by external Na+. Conversely, extravesicular Na+ inhibited proton-driven Na+ uptake, whilst K+ had little effect. H(+)-driven K+ uptake tended to saturate with increasing external K+ concentrations and Lineweaver-Burk analysis of the data revealed a Km for external K+ of 2 mM. These findings are consistent with the presence of K(+)-H+ exchange activity in the chicken jejunal brush-border membrane.

Effect of acute changes in glomerular filtration rate on Na+/H+ exchange in rat renal cortex.

Studies were undertaken in Munich-Wistar rats to assess the influence of changes in filtered bicarbonate (FLHCO3), induced by changes in GFR, on Na+/H+ exchange activity in renal brush border membrane vesicles (BBMV). Whole-kidney and micropuncture measurements of GFR, FLHCO3, and whole-kidney and proximal tubule HCO3 reabsorption (APRHCO3) were coupled with BBMV measurements of H+ gradient-driven 22Na+ uptake in each animal studied. 22Na+ uptake was measured at three Na+ concentration gradients to allow calculation of Vmax and Km for Na+/H+ exchange. GFR was varied by studying animals under conditions of hydropenia, plasma repletion, and acute plasma expansion. The increase in GFR, FLHCO3, and APRHCO3 induced by plasma administration correlated directly with an increase in the Vmax for Na+/H+ exchange in BBMV. The Km for sodium was unaffected. In the plasma-expanded rats, the Vmax for Na+/H+ exchange was 22% greater than in the hydropenic rats (P less than 0.025) whereas APRHCO3 was 86% greater (P less than 0.001). These results indicate that increases in FLHCO3, induced by acute increases in GFR, stimulate Na+/H+ exchange activity in proximal tubular epithelium. This stimulation is a mechanism which can, in part, account for the delivery dependence of proximal bicarbonate reabsorption.

Uricosurics inhibit urate transporter in rat renal brush border membrane vesicles

It has been proposed that a urate-anion exchanger system in brush border membrane vesicles (BBMV), which mediates hydroxyl ion (OH −) gradient-dependent urate uptake, is the most likely route for the mediation of urate transport in the first step of urate reabsorption in the proximal tubules. Luminal drugs which inhibit urate reabsorption would inhibit the transport of urate into the cell by blocking the urate-anion exchanger. To confirm this hypothesis, we investigated the inhibitory effects of well-known uricosuric drugs on the OH −/urate exchange in BBMV. The rank order of potency was benzbromarone > tienilic acid > sulfinpyrazone > probenecid, which is consistent with clinical doses in man. AA-193 (5-chloro-7,8-dihydro-3-phenylfurol[2,3-g]-1,2-benzisoxazole-7-carboxylic acid), an excellent candidate for a uricosuric, exerted the most potent inhibition on urate uptake (K i = 0.12 μM). In contrast with that by stilbene disulfonates, the inhibition by AA-193 or benzbromarone was reversible.

Activation of Cl-/OH- exchange by parachloromercuribenzoic acid in rabbit renal brush-border membranes.

The effect of the sulfhydryl reagent parachloromercuribenzoic acid (PCMB) on chloride transport was examined in rabbit renal brush-border membrane vesicles (BBMV). PCMB had no effect on the chloride conductive pathway. In the presence of an inside-alkaline pH gradient and a K-/valinomycin voltage clamp, the addition of PCMB stimulated 36Cl uptake and induced a threefold overshoot above the equilibrium value, indicating Cl/OH exchange. The effect of PCMB was reversed by dithiothreitol. Cl/OH exchange was not observed in the absence of PCMB. PCMB-activated Cl/OH exchange persisted even when the membrane potential was made inside-negative relative to the controls, thus, demonstrating that PCMB's effect on 36Cl uptake under pH-gradient conditions is not mediated by parallel Cl- and H+ conductive pathways. PCMB-activated Cl/OH exchange was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) with IC50 values of 290 and 80 microM, respectively. These results demonstrate that modification of sulfhydryl groups by PCMB activates Cl/OH exchange in BBMV.

Adaptability of marine teleost renal inorganic sulfate excretion: evidence for glucocorticoid involvement.

The marine winter flounder, Pseudopleuronectes americanus, ingests seawater (SW) and excretes most of the resultant divalent ion load by renal tubule secretion. The site of secretion is generally thought to be the proximal tubule. Although sulfate clearance ratios (C ratio-sulfate clearance/polyethylene glycol clearance) have been reported as high as 12 in SW-acclimated animals, the present study shows that net secretion ceases after acclimation to 10% SW (SO4 free). Intravenous infusion of sulfate into the latter induced net secretion within 5 h (C ratio increased from 0.83 +/- 0.23 to 2.12 +/- 0.33). Increased sulfate secretion coincided with an increase in the magnitude of HCO3-SO4 exchange in brush-border membrane vesicles (BBMV) isolated from flounder renal tubules. Treatment of 10% SW-acclimated flounder with 60 micrograms dexamethasone/100 g body wt also caused an increase in the HCO3-SO4 exchange in BBMV to a level comparable to that of BBMV from SW fish. The glucocorticoid effect was further tested in flounder renal tubule primary monolayer cultures. Measurement of unidirectional sulfate fluxes (J) showed that in the presence of cortisol net secretion (serosal-to-mucosal flux) dominated (Js----m = 111 +/- 6.5, Jm----s = 9 +/- 4.3, Jnet = 102 +/- 2.2 nmol.h-1.cm-2). Removal of cortisol from the culture medium significantly reduced net sulfate secretion to one-third of control (Js----m = 39 +/- 13.9, Jm----s = 6 +/- 1.0, Jnet = 33 +/- 14.1 nmol.h-1.cm-2).

Short-term and long-term stimulation of Na+-H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney.

The effect of unilateral nephrectomy on Na+-H+ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70-75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted to Vmax, whereas the Km for Na+ was similar in control and compensatory growing kidney. The increase of Na+-H+ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na+-H+ exchange. The late, but not the initial stimulations of Na+-H+ exchange were associated with elevated activities of cortical (Na++K+)-ATPase indicating that changes in antiporter activity precede those in the (Na++K+)-pump. The early stimulation of Na+-H+ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na+-H+ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na+-H+ exchanger may be regulated by phosphorylation-dephosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of increased electroneutral Na +-H + exchange in renal cortical brush-border membranes from hyperthyroid rats

The fluorescence quenching of acridine orange was used to compare Na +-H + exchange and ion conductances in renal cortical brush-border membrane vesicles (BBMV) isolated from euthyroid and hyperthyroid rats. In BBMV from euthyroid animals, Na +-H + exchange was entirely electroneutral. In BBMV from hyperthyroid rats, the total rates of Na +-H + exchange were about 30% higher than in BBMV from euthyroid animals. However, the electroneutral exchange in these membranes was similar to that in BBMV from euthyroid rats; the observed increase in exchange was due to electrically coupled Na + and H + movements through conductive pathways in the membranes. Ion conductances in isolated BBMV were tested with outwardly directed K + gradients in the presence of carbonyl cyanide m-chlorophenylhydrazone (K + conductance) or valinomycin (H + conductance). The K + conductance was negligible and similar in BBMV from both groups of rats. A significant H + conductance was present in both kinds of membrane preparations and was by 37% higher in BBMV from hyperthyroid animals. Therefore, our experiments failed to demonstrate an increased electroneutral Na +-H + exchange in BBMV from hyperthyroid rats. Instead, a finding of a significant electrically coupled Na +-H + antiport in the presence of increased H + conductance in BBMV from hyperthyroid rats indicates that these membranes may also have increased Na + conductance.

DEFECTIVE JEJUNAL BRUSH-BORDER Na +/H + EXCHANGE: A CAUSE OF CONGENITAL SECRETORY DIARRHOEA

In a child with congenital secretory diarrhoea, jejunal perfusion studies showed net water and electrolyte secretion and suggested a defect in sodium/proton exchange. A technique was developed for investigation of such exchange in brush-border membrane vesicles from jejunal biopsy specimens. Vesicles from control specimens displayed proton-gradient-dependent sodium uptake, whereas those from the patient did not. These findings, as well as pointing to a cause of congenital secretory diarrhoea, confirm the existence and importance of jejunal sodium/proton exchange in man.