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C Burnham

Publications and source records attributed to C Burnham.

12 recordsLinked to original sources
PubMed2000

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells.

Thickening of airway mucus and lung dysfunction in cystic fibrosis (CF) results, at least in part, from abnormal secretion of Cl(-) and HCO(3)(-) across the tracheal epithelium. The mechanism of the defect in HCO(3)(-) secretion is ill defined; however, a lack of apical Cl(-)/HCO(3)(-) exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO(3)(-) exchangers in tracheal epithelial cells exhibiting physiological features prototypical of cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1 cells transfected with functional wild-type CFTR, termed CFT-WT). Cells were grown on coverslips and were loaded with the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was monitored. Cl(-)/HCO(3)(-) exchange activity increased by approximately 300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1 cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited by DIDS. The mRNA expression of the ubiquitous basolateral AE-2 Cl(-)/HCO(3)(-) exchanger remained unchanged. However, mRNA encoding DRA, recently shown to be a Cl(-)/HCO(3)(-) exchanger (Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274: 22855-22861, 1999.) was abundantly expressed in cells expressing functional CFTR but not in cells that lacked CFTR or that expressed mutant CFTR. In conclusion, CFTR induces the mRNA expression of "downregulated in adenoma" (DRA) and, as a result, upregulates the apical Cl(-)/HCO(3)(-) exchanger activity in tracheal cells. We propose that the tracheal HCO(3)(-) secretion defect in patients with CF is partly due to the downregulation of the apical Cl(-)/HCO(3)(-) exchange activity mediated by DRA.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Functional characterization of a cloned human kidney Na+:HCO3- cotransporter.

Functional properties of a cloned human kidney Na+:HCO3- cotransporter (NBC-1) were studied in cultured HEK-293 cells that were transiently transfected with NBC-1 cDNA. The Na+:HCO3- cotransporter activity was assayed as the Na+ and HCO3-dependent pHi recovery from intracellular acidosis with the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In acid-loaded cells and in the presence of amiloride (to block Na+/H+ exchange), switching to a Na+-containing solution (115 mM) resulted in rapid pHi recovery only in the presence of HCO3-. This recovery was completely abolished by 300 microM 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. Replacing the Na+ with Li+ (115 mM) caused significant HCO3--dependent, DIDS-sensitive pHi recovery from intracellular acidosis, with Li+ showing lower affinity than Na+. Potassium (K+) had no affinity for the Na+:HCO3- cotransporter. The Na+-dependent HCO3- cotransport was abolished in the presence of 0.2 mM harmaline. The Na+:HCO3- cotransporter could also function in Na+:OH- cotransport mode, although only at high external pH (7.8). Based on functional similarities with the mammalian kidney experiments, we propose that NBC-1 is the proximal tubule Na+:HCO3- cotransporter.

Bicarbonates↗
PubMed1997

Ischemic-reperfusion injury in the kidney: overexpression of colonic H+-K+-ATPase and suppression of NHE-3.

Ischemic renal injury is associated with changes in the expression of a number of genes. Although pH regulation is undoubtedly important during the recovery from ischemia, the expression of acid-base transporters during acute ischemic renal failure has not been studied. In the present study, levels of mRNA encoding the colonic H+-K+-ATPase and four isoforms of the Na+/H+ exchanger (NHE-1, NHE-2, NHE-3 and NHE-4) were measured by quantitative Northern analysis in rat renal cortex and medulla following ischemia-reperfusion injury. Rats were subjected to 30 minutes of renal artery occlusion and then sacrificed either 12 or 24 hours after the occlusion was released. The most striking changes followed 30 minutes of occlusion and 12 hours of reperfusion and involved the mRNA for NHE-3 (involved in HCO3- reabsorption in proximal tubule and thick limb) and colonic H+-K+-ATPase (involved in HCO3- reabsorption in collecting duct). These changes were: (1) a approximately 75% decrease in NHE-3 mRNA in both cortex and medulla; and (2) an approximately 8-fold increase in colonic H+-K+-ATPase mRNA in the cortex. At 12 hours of reperfusion, there was a 66% reduction in the Na+/H+ exchanger (NHE-3) activity as assayed by acid-stimulated 22Na+ influx into brush border membrane vesicles (P < 0.01). After 24 hours of reperfusion, NHE-3 mRNA remained suppressed while cortical colonic H+-K+-ATPase mRNA declined to only twice the control level. Medullary colonic H+-K+-ATPase mRNA did not change significantly. Gastric H+-K+-ATPase mRNA in cortex or medulla remained the same at 0, 12, and 24 hours after reperfusion. Cortical NHE-1 increased mildly at 12 and 24 hours of reperfusion whereas a moderate decrease in NHE-2 and NHE-4 mRNAs was observed in cortex and medulla after both 12 and 24 hours of reperfusion. We suggest that overexpression of colonic H+-K+-ATPase in the early phase of renal reperfusion injury may be responsible for compensatory reabsorption of increased HCO3- load resulting from suppression of NHE-3. This was supported by a fourfold increase in colonic H+-K+-ATPase mRNA in rats treated with acetazolamide, which causes renal HCO3-wasting. Rapid decline in colonic H+-K+-ATPase expression at 24 hours after reperfusion is likely due to reduced HCO3- delivery to distal tubules resulting from decreased GFR. Overexpression of H+-K+-ATPase may be vital to acid-base homeostasis in the early phase of acute ischemic renal failure.

Animals↗
PubMed1990

Local gas transport in eucapnic ventilation: effects of gravity and breathing frequency.

The effects of body position and respiratory frequency (f) on regional gas transport during eucapnic conventional ventilation (CV) and high-frequency ventilation (HFV) were assessed from the washout of nitrogen 13 (13NN) using positron-emission tomography. In one protocol, six dogs were ventilated with CV or HFV at f = 6 Hz and tidal volume (VT) selected supine for eucapnia. A coronal cross section of the lung base was studied in the supine, prone, and right and left lateral decubitus positions. In a second protocol, six dogs were studied prone: apical and basal cross sections were studied in CV and in HFV with f = 3 and 9 Hz at eucapnic VT. Regional alveolar ventilation per unit of lung volume (spVr) was calculated for selected regions and analyzed for gravity-dependent cephalocaudal and right-to-left gradients. In both CV and HFV, nonuniformity in spVr was highest supine and lowest prone. In CV there were vertical gradients of spVr in all body positions: nondependent less ventilated than dependent regions, particularly in the supine position. In HFV there was a moderate vertical gradient in spVr in addition to a preferentially ventilated central region in all body positions. Overall lung spV was unaffected by body position in CV but in HFV was highest supine and lowest prone. Nonuniformity in eucapnic prone HFV was unaffected by f and always higher than in CV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Regional mapping of gas transport during high-frequency and conventional ventilation.

The effects of changing tidal volume (VT) and frequency (f) on the distribution of ventilation during high-frequency ventilation (HFV) were assessed from the washout of nitrogen-13 by positron emission tomography. Six dogs, anesthetized and paralyzed, were studied in the supine position during conventional ventilation (CV) and during HFV at f of 3, 6, and 9 Hz. In CV and HFV at 6 Hz, VT was selected to achieve eucapnic arterial partial pressure of CO2 (37 +/- 3 Torr). At 3 and 9 Hz, VT was proportionally changed so that the product of VT and f remained constant and equal to that at 6 Hz. Mean residence time (MRT) of nitrogen-13 during washout was calculated for apical, midheart, and basal transverse sections of the lung and further analyzed for gravity-dependent, cephalocaudal and radial gradients. An index of local alveolar ventilation per unit of lung volume, or specific ventilation (spV), was calculated as the reciprocal of MRT. During CV vertical gradients of regional spV were seen in all sections with ventral (nondependent) regions less ventilated than dorsal (dependent) regions. Regional nonuniformity in gas transport was greatest for HFV at 3 and 6 Hz and lowest at 9 Hz and during CV. During HFV, a central region at the base of the lungs was preferentially ventilated, resulting in a regional time-averaged tracer concentration equivalent to that of the main bronchi. Because the main bronchi were certainly receiving fresh gas, the presence of this preferentially ventilated area, whose ventilation increased with VT, strongly supports the hypothesis that direct convection of fresh gas is an important mechanism of gas transport during eucapnic HFV. Aside from the local effect of increasing overall lung ventilation, this central area probably served as an intermediate shuttle station for the transport of gas between mouth and deeper alveoli when VT was less than the anatomic dead space.

Animals↗
PubMed1986

A Ca-dependent K channel in "luminal" membranes from the renal outer medulla.

This paper describes properties of 86Rb fluxes through K channels in "luminal" membrane vesicles prepared from rabbit renal outer medulla. By measuring 86Rb uptake against an opposing chemical gradient of K ions, using membranes loaded with KCl, a transient accumulation of isotope is observed, which is blocked by Ba ions. This is the behavior expected of a conductive Rb flux through a Ba-sensitive K channel. The 86Rb accumulation is driven by an electrical diffusion potential as shown in experiments using either vesicles loaded with different anions, or an outwardly directed Li gradient with a Li ionophore. The vesicles containing the channel show a cation selectivity with the order Rb greater than K greater than Cs greater than Li greater than Na greater than choline. The Ba-sensitive Rb flux is dependent on Ca within the vesicles, with a very high affinity estimated as K0.5 10 to 100 nM. The vesicles appear to be right-side-out. The Ba-sensitive 86Rb uptake is also inhibited by quinine K0.5 30 microM but is insensitive to tetraethyl ammonium ions and apamin. These isotope flux experiments complement electrophysiological experiments in providing independent evidence for the existence of K channels in the luminal surface of cells of this ascending limb of the loop of Henle. The very high Ca affinity suggests that cytoplasmic Ca could play an important role in regulation of transepithelial salt flux in this region of the nephron.

Animals↗
PubMed1985

Identification and reconstitution of a Na+/K+/Cl- cotransporter and K+ channel from luminal membranes of renal red outer medulla.

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na+/K+/Cl- cotransport system and a K+ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na+/K+/Cl- cotransporter and K+ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of 86Rb+ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two 86Rb+ fluxes. (A) A furosemide-inhibited 86Rb+ flux in the absence of Na+ (K+-K+ exchange). This flux is stimulated by an inward Na+ gradient (Na+/K+ cotransport) and is inhibited also by bumetanide. (B) A Ba2+-inhibited 86Rb+ flux, through the K+ channel. Luminal membranes containing the Na+/K+/Cl- cotransporter and K+ channels, and basolateral membranes containing the Na+/K+ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na+/K+/Cl- cotransporter and K+ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.

Animals↗
PubMed1985

Electrolyte transport across the basolateral membrane of the parietal cells.

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.

Amiloride↗
PubMed1982

Ion pathways in renal brush border membranes.

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na+/H+ or Li+/H+ exchange was demonstrated by diluting Na2SO4 or Li2SO4 loaded vesicles into Na+-or Li+-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K+ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na+ conductance smaller than electroneutral Na+/H+ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na+ conductance greater than the H+ conductance was suggested. K+ gradients also induced changes of pH, at about 10% of the Na+ or Li+ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K+ was a low affinity substrate for the Na+/H+ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K+ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles has a low K+ conductance. Inward Cl- gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl- conductance greater than the H+ conductance and a Cl-/OH- exchange were present. The rate of Na+/H+ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent Km for Na+ or Li+ of about 10 mM and an EA of 14.3 kcal per mol. A 61-fold Na2SO4 gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower delta pH even in the presence of gramicidin showing that the H+ and Cl- pathways could alter the effect of the Na+/H+ exchange.

Acridine Orange↗
PubMed1976

Determinants of regional distribution of a bolus inhaled from residual volume.

The volume of lung at residual volume (RV) which fails to receive an inhaled tracer bolus (EXV) was quantitated in 13 normals by comparison of a scintigram of the distribution of a tracer bolus inhaled from RV (BORV) with a scintigram at RV of lung equilibrated with the tracer (EQRV). EXV was found in the dependent lung in the erect position in all subjects but also occurred to a lesser degree at the apex in 11 of 13 subjects. Basal EXV ranged from 1 to 7% of TLC, and unlike apical EXV increased with age (r= 0.91, P less than 0.01). EXV in the decubitus position shifted largely to the dependent lung with none remaining in the original erect apical and basal locations, demonstrating that gravity determined EXV location. Nitrous oxide, which is highly diffusible, failed in four subjects to carry the tracer to basal EXV even though perfusion was documented to persist to this area, implying basal EXV airways were closed, not narrowed. In one of the four subjects apical EXV was readily definable. Nitrous oxide carried tracer into this region, implying patent apical EXV airways.

Adolescent↗