Absence Of Bumetanide Research Articles

Contribution of Na+-H+ exchange to sodium reabsorption in the loop of henle: a microperfusion study in rats.

1. The contribution of apical Na+-H+ exchange to sodium reabsorption in the thick ascending limb of the loop of Henle (TALH) in vivo was examined in anaesthetized rats by perfusing loops of Henle of superficial nephrons with solutions containing the Na+-H+ exchange inhibitor, ethyl isopropyl amiloride (EIPA). 2. Using a standard perfusate, no statistically significant effect of EIPA on net sodium reabsorption (JNa) was detected. However, when sodium reabsorption in the pars recta of the proximal tubule was minimized by using a low-sodium perfusate, EIPA reduced JNa from 828 +/- 41 to 726 +/- 37 pmol min-1 (P < 0.05), indicating that apical Na+-H+ exchange can make a small contribution to net sodium reabsorption in the TALH in vivo. This contribution appears to be dependent on the bicarbonate load, since an increase in the latter led to an enhancement of EIPA-sensitive sodium transport. 3. Addition of the Na+-K+-2Cl- cotransport inhibitor, bumetanide, to the low-sodium perfusate reduced baseline JNa to 86 +/- 27 pmol min-1. In this setting, EIPA reduced JNa further, to -24 +/- 18 pmol min-1 (P < 0.05), an effect similar to that seen in the absence of bumetanide. This finding argues against previous suggestions (based on in vitro evidence) that inhibition of the Na+-K+-2Cl- cotransporter leads to an increase in apical Na+-H+ exchange in the TALH.

Kinetics of ATP-sensitive K+ channels in isolated rat hearts assessed by87Rb NMR spectroscopy

An experimental model was developed to evaluate the effects of activators and inhibitors of K(ATP) channels on unidirectional K+ fluxes in the whole heart. Isolated rat hearts perfused in the Langendorff mode were equilibrated with Pi-free Krebs-Henseleit buffer (KH buffer) containing 0.94-2.14 mM RbCl and 3.76 mM KCl (20-36% of K+ substituted by Rb+). Rb+ efflux was initiated by removing Rb+ from the perfusate and 87Rb spectra were acquired continuously with a 1-2 min time resolution. In hearts with normal energetics, the efflux of Rb+ fit a monoexponential function, and the rate constant did not depend on intracellular [Rb+]. Agents depressing excitability and heart rate (HR), such as 0.6 mM lidocaine (Lido), 10 microM carbachol (carb) and 20 mM MgSO4, inhibited Rb+ efflux such that the rate constant, k (10(3)/min), decreased from 50+/-1.2 in the beating heart to 26+/-1, 40+/-1.1 and 19+/-1.2, respectively. In contrast, high [K+] (21 mM) did not affect the k value (50+/-4.5), independently of the presence or absence of bumetanide (Bum, 30 microM) and glibenclamide (Glib, 5 microM). Dinitrophenol (DNP, 0.2 mM) added in the presence of high [K+] + Bum increased k three-fold, to 160+/-5. This effect was associated with a significant decrease in phosphocreatine (PCr, <10% of initial) and ATP ( 15%) levels, and a 7-fold increase in the Pi level, assessed by 31P-NMR spectroscopy. Glib completely reversed the effect of DNP. Pinacidil (Pin, 20-80 microM) did not affect the k value either in beating control hearts or in the presence of Carb or KCl + Bum. Moreover, under conditions of moderate metabolic stress induced by 0.05 mM DNP (PCr, 35%; ATP, 65%), where half-maximal activation of K(ATP) channels occurred, Pin did not further activate Rb+ efflux. We conclude that:(1) heart rate-independent Rb+ efflux accounts for 40-80% of the total Rb+ efflux in beating (300 bpm) rat hearts;(2) DNP-activated Rb+ efflux is a good model for testing inhibitors of KATP channels in whole hearts; and (3) Pin is not an effective K(ATP) channel opener in the rat heart model.

Nitric Oxide Reduces Na-K-2Cl Cotransport in Distal Lung Epithelial Cells from Fetal Sheep • 1621

Before birth secretion of Cl- into the lung lumen by the respiratory epithelium provides the osmotic force for transepithelial water movement and resultant filling of the fetal lung with liquid. Production of lung liquid is dependent on Na-K-2Cl cotransport, indicating that Cl- transport by this mechanism is important in fetal lung liquid secretion. After birth, Cl- secretion diminishes and Na+ transport-linked liquid reabsorption is established. The mechanism by which Cl-linked fluid secretion decreases after birth is uncertain, but we hypothesized that nitric oxide may be involved because it reduces lung liquid production in fetal lambs by a mechanism independent of Na+ reabsorption (J Appl Physiol 83: 1538, 1997). To test this notion, we studied the effect of nitric oxide on Na-K-2Cl cotransport in a line of cultured epithelial cells derived from the distal lung of a fetal lamb. After harvesting the cells into suspension, we incubated the cells for 5 min in a buffered salt solution saturated with nitrogen (control) or nitric oxide (≅6 × 10-7 M) and then calculated K+ uptake (using the K+ substitute86 Rb+) in the presence and absence of bumetanide (10-4 M) to determine the bumetanide-sensitive fraction of Rb uptake, an index of Na-K-2Cl activity. Bumetanide-sensitive Rb+ uptake averaged 58 ± 28 (nmol/million cells)/h under control conditions and was reduced (on average by 54%) to 30 ± 30 (nmol/million cells)/h when exposed to nitric oxide(n=6, mean ± SD, p < 0.002). To confirm this result, we used s-nitroso-n-acetylpenicillamine (SNAP, 250 μM) as an alternative method of generating nitric oxide. After harvesting the cells into suspension, we incubated the cells for 10 min with SNAP and then calculated Rb+ uptake in the presence and absence of bumetanide. SNAP reduced bumetanide-sensitive Rb+ uptake (on average by 47%) from 26 ± 8 (nmol/million cells)/h to 15 ± 10 (nmol/million cells)/h (control vs SNAP, n=6, p< 0.01). Thus, in fetal distal lung epithelial cells in culture, nitric oxide inhibits Na-K-2Cl cotransport, an ion transport mechanism that is important in the production of lung liquid in the fetus. We speculate that nitric oxide may play a role in reducing Cl- transport in the developing lung after birth.

Choroid Plexus Potassium Cotransport: Modulation by Osmotic Stress and External Potassium

The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide-sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 +/- 0.02, 0.44 +/- 0.04, and 0.84 +/- 0.06 min-1 in 240, 300, and 424 mOsm/kg solutions, respectively (p < 0.001). This increase in efflux with osmolality, an opposite effect to that found in many cells, was solely due to enhanced K cotransport. The increased cotransport may be involved in limiting brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide-sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 +/- 1, 19 +/- 3, 13 +/- 2, and 6 +/- 1 nmol/mg/min after 0, 10, 30, and 60-min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide-sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.

Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes

The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net salt transport in a variety of cells. As a first step in obtaining structural information on this transport system, we attempted to express it in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney. Four days after injection, batches of oocytes were tested for 86Rb uptake in presence and absence of 10 microM bumetanide to distinguish that fraction of influx mediated by NKCCT. In the absence of bumetanide, the oocytes formed a bimodal distribution with respect to 86Rb uptake, with some oocytes accumulating significantly more 86Rb than others. In the presence of bumetanide, or when Na was replaced with choline, that group of oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not accumulate sufficient 86Rb to be distinguishable above background. Taken together, these data suggest that some of the mRNA-injected oocytes expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by acid-urea agarose gel electrophoresis, and fractions were injected into oocytes. One fraction corresponding to messages of approximately 7 kilobases in length induced a bumetanide-sensitive 86Rb influx resembling that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density gradients gave similar results. It is concluded that the rat kidney NKCCT has been expressed in Xenopus oocytes from a high molecular weight fraction of poly(A)+ mRNA.

Volume and ion transport by fetal rat alveolar and tracheal epithelia in submersion culture.

The complex architecture of the mammalian lung has hindered measurements of permeability of and transport by the intact alveolar epithelium. We compared properties of fetal rat alveolar buds and tracheas in submersion culture by microelectrode and micropuncture techniques. Both alveolar buds and tracheas form cysts that accumulate liquid for up to 3 wk in culture. The transepithelial electric potential difference (PD) of alveolar buds and tracheas (lumen negative, 1.2-3.5 and 8-21 mV, respectively) was abolished by metabolic inhibitors or ouabain in the bath. Despite the difference in PDs, both epithelial regions, after 1 or 2 wk in culture, exhibited the following. 1) Cl- activities and concentrations in luminal liquid were 13 and 34% higher than the bathing medium; Na+ and K+ concentrations were not different. 2) Bumetanide inhibited PD by 70%, whereas terbutaline increased PD by 45%. 3) Amiloride injected into the cyst lumen induced a 20% decrease in the PD of 1-wk explants but did not affect 2-wk preparations. Replacement of bath Na+ by choline decreased the PD of tracheas by 85% but did not change alveolar PD in the presence or absence of bumetanide. The pattern of cyst liquid composition and PD responses to drugs suggests that fetal rat alveolar and tracheal epithelia secrete Cl-, which drives volume flow.

8-BrcAMP does not affect Na-K-2Cl cotransport in winter flounder intestine.

The flounder intestinal epithelium possesses luminal Na-K-2Cl cotransport and K secretory mechanisms that account for the short-circuit current (Isc) across the tissue. This epithelium is also highly cation selective. Bumetanide or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) completely inhibit Isc, Na-K-2Cl cotransport, and K secretion, whereas 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibits K secretion, partially inhibits Isc, and greatly increases Cl permeability. Although the direct effects of adenosine 3',5'-cyclic monophosphate (cAMP) on other Na-K-2Cl cotransport systems have been examined, the effects of 8-BrcAMP on flounder intestinal Na-K-2Cl cotransport have not been directly measured. In this study, the effects of 8-BrcAMP and bumetanide, either alone or in combination, on the influx (initial rates of uptake) of Cl, Rb, and Na across the luminal surface of the flounder intestine were examined. Bumetanide significantly inhibited Na (50%), Cl, and Rb influx (70% each). In contrast, 8-BrcAMP significantly increased Cl influx in the presence or absence of bumetanide and thereby did not effect the bumetanide-sensitive Cl influx. The nucleotide neither altered bumetanide-sensitive Rb influx nor net transpithelial Na absorption measured under short-circuit conditions but caused a 51-60% decrease in Isc. Measurements of bumetanide-sensitive Na influx exhibited large experimental variability but showed no statistically significant effects of 8-BrcAMP. Prostaglandin E1 (PGE1, 10 microM) and forskolin (10 microM) but not atrial natriuretic factor (1 microM) increased flounder intestinal [cAMP] 200 and 237%, respectively, and, like 8-BrcAMP, increased tissue conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

36Cl − and 86Rb + uptake in rat parotid acinar cells

36Cl − uptake was markedly (85 per cent) inhibited by the loop diuretics, furosemide and bumetanide. Partial replacement of Na + in the incubation medium (from 137 to 50 mM) reduced 36Cl − uptake 30 per cent; total replacement reduced uptake to 40–45 per cent of control values. Partial replacement of K + (from 5.8 to 1 mM) decreased 36Cl − uptake approx. 45 per cent; complete replacement resulted in minimal levels of 36Cl − uptake (< 10 per cent of controls). Ouabain reduced 36Cl − uptake approx. 55 per cent in the absence of bumetanide, but was without effect in its presence. 86Rb + uptake was reduced approx. 85 per cent with bumetanide; complete replacement of Cl − with I − or gluconate −, decreased 86Rb + uptake by 55 or 40 per cent respectively. The results support the notion that the bulk of Cl − influx in rat parotid acinar cells is via a loop diuretic-sensitive Na +/K +/Cl − co-transport mechanism as may occur in other secretory epithelia.

Chloride transport in the thick ascending limb of Henle's loop: potassium dependence and stoichiometry of the NaCl cotransport system in plasma membrane vesicles

Cells were isolated from the thick ascending limb of Henle's loop of rabbit kidney outer medulla and a plasma membrane fraction was prepared by differential centrifugation. Sodium and rubidium uptake into the plasma membrane vesicles were determined by a rapid filtration technique. In the presence of a 100 mM KCl gradient and 0.5 mM sodium the vesicles took up 252 +/- 82 pmoles sodium/mg protein X 15s; 52% of the uptake was dependent on the presence of chloride or inhibited by 10(-3) M bumetanide. If KCl was stepwise replaced by choline chloride, sodium uptake decreased in the absence of bumetanide but was only insignificantly altered in the presence of bumetanide. Potassium exerted a halfmaximum stimulation at 22.3 +/- 9.7 mM. In tracer exchange experiments under zero salt gradient conditions, sodium uptake was also strongly reduced in the absence of potassium. Rubidium uptake into the same membrane fraction was highest in the presence of a NaCl gradient, and decreased 41% when sodium was replaced by choline or when 10(-3) M bumetanide was present. Replacement of sodium chloride by sodium nitrate also inhibited rubidium uptake. When the sodium, chloride and potassium dependence of the bumetanide sensitive sodium uptake was investigated in more detail, Hill coefficients for sodium of 1.0 +/- 0.03, for chloride of 1.8 +/- 0.2 and for potassium of 0.98 +/- 0.03 were obtained. These results are consistent with the presence of a Na-Cl-K cotransport system in the medullary thick ascending limb of Henle's loop which may operate with a stoichiometry of 1/2/1.