Flow dependence of transtubular potential difference in isolated perfused segments of rabbit proximal convoluted tubule.

Transmembrane potential difference (pd) was studied in isolated perfused segments of rabbit proximal convoluted tubules. At perfusion flow rates above 10 nl/min the pd was -5.80 +/-0.3 mv (lumen negative) when perfusing with isosmolal ultrafiltrate of same rabbit serum as the bath. That this pd is generated by transport activity of the tubule is supported by three separate observations: (a) pd reversibly decreased with cooling from 37 degrees C to 25 degrees C; (b) pd decreased when 10(-5) M ouabain was added to the bath and reversed to control levels when ouabain was removed; and (c) heating to 47 degrees C irreversibly decreased pd to zero. The magnitude of the pd was related to perfusion flow rate at slower rates than 10 nl/min. A decrease in flow rate was associated with a decrease in pd. The tubular geometry and transmembrane hydrostatic pressure were ruled out as the mediating factors governing the magnitude of observed pd.

Mechanism of NaCl and water reabsorption in the proximal convoluted tubule of rat kidney.

The role of chloride concentration gradients in proximal NaCl and water reabsorption was examined in superficial proximal tubules of the rat by using perfusion and collection techniques. Reabsorptive rates (Jv), chloride concentrations, and transtubular potential difference were measured during perfusion with solutions (A) simulating an ultrafiltrate of plasma; (B) similar to (A) except that 20 meq/liter bicarbonate was replaced with acetate; (C) resembling late proximal fluid (glucose, amino acid, acetate-free, low bicarbonate, and high chloride); and (D) in which glucose and amino acids were replaced with raffinose and bicarbonate was partially replaced by poorly reabsorbable anions (cyclamate,sulfate, and methyl sulfate). In tubules perfused with solutions A and B, Jv were 2.17 and 2.7 nl mm-1 min-1 and chloride concentrations were 131.5 +/- 3.1 and 135 +/- 395 meq/liter, respectively, indicating that reabsorption is qualitatively similar to free-flow conditions and that acetate adequately replaces bicarbonate. With solution C, Jv was 2.10 nl mm-1 min-1 and potential difference was +1.5 +/- 0.2 mV, indicating that the combined presence of glucose, alanine, acetate, and bicarbonate per se is not an absolute requirement. Fluid reabsorption was virtually abolished when tubules were perfused with D solutions; Jv was not significantly different from zero despite sodium and chloride concentrations similar to plasma; chloride concentration was 110.8 +/- 0.2 meq/liter and potential difference was -0.98 mV indicating that chloride was close to electrochemical equilibrium. These results suggest the importance of the chloride gradient to proximal tubule reabsorption in regions where actively reabsorbable solutes (glucose, alanine, acetate, and bicarbonate) are lacking and provide further evidence for a passive model of NaCl and water transport.

Proton transport and cell function.

The past five years have witnessed an explosion of information on the many and varied roles of H+ transport in cell function. H+ transport is involved in three broad areas of cell function: (a) maintenance and alteration of intracellular pH for initiation of specific cellular events, (b) generation of pH gradients in localized regions of the cell, including gradients involved in energy transduction, and (c) transepithelial ion transport. These processes each involve one or more of several H+ translocating mechanisms. The first section of this review will discuss these H+ translocating mechanisms and the second part will deal with the cellular functions controlled by H+ transport.

The mechanisms of sodium absorption in the human small intestine.

The present studies were designed to characterize sodium transport in the jejunum and ileum of humans with respect to the effects of water flow, sodium concentration, addition of glucose and galactose, and variations in aniomic composition of luminal fluid. In the ileum, sodium absorption occurred against very steep electrochemical gradients (110 mEq/liter, 5-15 mv), was unaffected by the rate or direction of water flow, and was not stimulated by addition of glucose, galactose, or bicarbonate. These findings led to the conclusion that there is an efficiently active sodium transport across a membrane that is relatively impermeable to sodium. In contrast, jejunal sodium (chloride) absorption can take place against only the modest concentration gradient of 13 mEq/liter, was dramatically influenced by water movement, and was stimulated by addition of glucose, galactose, and bicarbonate. The stimulatory effect of glucose and galactose was evident even when net water movement was inhibited to zero by mannitol. These observations led to the conclusion that a small fraction of jejunal sodium absorption was mediated by active transport coupled either to active absorption of bicarbonate or active secretion of hydrogen ions. The major part of sodium absorption, i.e. sodium chloride absorption, appeared to be mediated by a process of bulk flow of solution along osmotic pressure gradients. The stimulatory effect of glucose and galactose, even at zero water flow, was explained by a model in which the active transport of monosaccharide generates a local osmotic force for the absorption of solution (NaCl and water) from the jejunal lumen, which, in the presence of mannitol, is counterbalanced by a reverse flow of pure solvent (H(2)O) through a parallel set of channels which are impermeable to sodium. Support for the model was obtained by the demonstration that glucose and bicarbonate stimulated the absorption of the nonactively transported solute urea even when net water flow was maintained at zero by addition of mannitol to luminal contents.

The role of intraluminal sodium in glucose absorption in vivo.

Active glucose absorption is thought to depend on a gradient of sodium ion concentration across the brush border membrane of intestinal epithelial cells. This concept is generally accepted, although its validity has never been adequately evaluated in the human small intestine in vivo. According to this hypothesis, the rate of glucose absorption should decrease markedly if the luminal sodium concentration is markedly reduced, and glucose absorption against a concentration gradient should cease entirely if luminal sodium is lower than intracellular sodium concentration. In the present series of experiments we were not able to show an important role of intraluminal sodium concentration in the active absorption of glucose from the human, rat, and dog ileum in vivo. Specifically, glucose absorption was minimally reduced or not reduced at all when intraluminal sodium concentration was reduced from 140 to as low as 2.5 mEq/liter. The discrepancy between our results and those of previous workers whose data suggest that removal of intraluminal sodium should markedly inhibit active glucose absorption is not entirely clear, but there are a number of differences in experimental design between most previous studies and our own. Although our data show that active glucose absorption proceeds at a near normal rate even when lumen sodium concentration is reduced below 3 mEq/liter, our results do not disprove the sodium gradient theory because of the theoretic possibility that the microclimate adjacent to the brush border has a high concentration of sodium even when luminal sodium concentration is markedly reduced. The validity of the sodium gradient hypothesis would appear to be critically dependent on such a microclimate.

Angiotensin II in arterial and renal venous plasma and renal lymph in the dog.

Angiotensin II was determined by radioimmunoassay in systemic arterial, pulmonary arterial, and renal venous plasma and in renal hilar lymph in dogs. Levels of the peptide were determined prior to and during progressive graded hemorrhage or reduction in renal perfusion pressure. Levels of angiotensin II in plasma consistently rose during transit through the lung indicating pulmonary conversion of angiotensin I to angiotensin II. On the other hand, angiotensin II in the renal vein plasma was less than that in arterial plasma indicating renal extraction of the peptide from plasma. When renal hilar lymph was sampled under similar conditions, angiotensin II in lymph was consistently higher than that in arterial or renal venous plasma. Furthermore, in some experiments angiotensin II in lymph increased at a time when the concentration in plasma was undetectable. No evidence was found to indicate that angiotensin II in plasma entered renal lymph. It was concluded that angiotensin II levels in lymph reflected the concentration of angiotensin II in renal tissue. The data further suggested that angiotensin II is partially removed from arterial plasma by hydrolysis during transit through the kidney.

Effect of luminal and peritubular HCO3(-) concentrations and PCO2 on HCO3(-) reabsorption in rabbit proximal convoluted tubules perfused in vitro.

The effect of luminal and peritubular HCO3(-) concentrations and PCO2 on HCO3(-) reabsorption was examined in rabbit proximal convoluted tubules perfused in vitro. Increasing luminal HCO3(-) concentration from 25 to 40 mM without changing either peritubular HCO3(-) concentration or PCO2, stimulated HCO3(-) reabsorption by 41%. When luminal HCO3(-) concentration was constant at 40 mM and peritubular HCO3(-) concentration was increased from 25 to 40 mM without changing peritubular PCO2, a 45% reduction in HCO3(-) reabsorption was observed. This inhibitory effect of increasing peritubular HCO3(-) concentration was reversed when peritubular pH was normalized by increasing PCO2. Passive permeability for HCO3(-) was also measured and found to be 1.09 +/- 0.17 X 10(-7) cm2 s-1. Using this value, the passive flux of HCO3(-) could be calculated. Only a small portion (less than 23%) of the observed changes in net HCO3(-) reabsorption can be explained by the passive HCO3(-) flux. We conclude that luminal and peritubular HCO3(-) concentrations after HCO3(-) reabsorption by changing the active H+ secretion rate. Analysis of these data suggest that both luminal and peritubular pH are major determinants of HCO3(-) reabsorption.

Effects of extracellular fluid volume and plasma bicarbonate concentration on proximal acidification in the rat.

The effects of systemic bicarbonate concentration and extracellular fluid volume status on proximal tubular bicarbonate absorption, independent of changes in luminal composition and flow rate, were examined with in vivo luminal microperfusion of rat superficial proximal convoluted tubules. Net bicarbonate absorption and bicarbonate permeability were measured using microcalorimetry. From these data, net bicarbonate absorption was divided into two parallel components: proton secretion and passive bicarbonate diffusion. The rate of net bicarbonate absorption was similar in hydropenic and volume-expanded rats when tubules were perfused with 24 mM bicarbonate, but was inhibited in volume-expanded rats when tubules were perfused with 5 mM bicarbonate. Volume expansion caused a 50% increase in bicarbonate permeability, which totally accounted for the above inhibition. The rate of proton secretion was unaffected by volume expansion in both studies. The rate of net bicarbonate absorption was markedly inhibited in alkalotic expansion as compared with isohydric expansion. Bicarbonate permeabilities were not different in these two conditions, and the calculated rates of proton secretion were decreased by greater than 50% in alkalosis. Net bicarbonate absorption was stimulated in acidotic rats compared to hydropenic rats. This stimulation was attributable to a 25% increase in the rate of proton secretion. We conclude that (a) proton secretion is stimulated in acidosis, inhibited in alkalosis, and is not altered by volume status; (b) bicarbonate permeability is increased by volume expansion but is not altered by increases in plasma bicarbonate concentration; (c) when luminal bicarbonate concentrations are similar to those of plasma, net bicarbonate absorption is dominated by proton secretion and is thus sensitive to peritubular bicarbonate concentrations, and insensitive to extracellular fluid volume; (d) when luminal bicarbonate concentrations are low and proton secretion is slowed, bicarbonate permeability and thus extracellular fluid volume have a greater influence on net bicarbonate absorption.

Control of proximal bicarbonate reabsorption in normal and acidotic rats.

This free-flow micropuncture study examined the dependence of bicarbonate reabsorption in the rat superficial proximal convoluted tubule to changes in filtered bicarbonate load, and thereby the contribution of the proximal tubule to the whole kidney's response to such changes. The independent effects of extracellular fluid (ECF) volume expansion and of acidosis on proximal bicarbonate reabsorption were also examined. When the plasma volume contraction incurred by the micropuncture preparatory surgery was corrected by isoncotic plasma infusion ( congruent with1.3% body wt), single nephron glomerular filtration rate (SNGFR), and the filtered total CO(2) load increased by 50%. Absolute proximal reabsorption of total CO(2) (measured by microcalorimetry) increased by 30%, from 808+/-47 during volume contraction to 1,081+/-57 pmol/min.g kidney wt after plasma repletion, as fractional total CO(2) reabsorption decreased from 0.90 to 0.77. Aortic constriction in these plasma-repleted rats returned the filtered load and reabsorption of total CO(2) to the previous volume contracted levels. In other animals isohydric ECF expansion with plasma (5% body wt) or Ringer's solution (10% body wt), or both, produced no further diminution in fractional proximal total CO(2) reabsorption (0.76-0.81). Metabolic acidosis was associated with very high fractional proximal total CO(2) reabsorptive rates of 0.82 to 0.91 over a wide range of SNGFR and ECF volumes. At a single level of SNGFR, end-proximal total CO(2) concentration progressively decreased from 5.6+/-0.5 to 1.6 +/-0.2 mM as arterial pH fell from 7.4 to 7.1. Expansion of ECF volume in the acidotic rats did not inhibit the ability of the proximal tubule to lower end-proximal total CO(2) concentrations to minimal levels. In conclusion, bicarbonate reabsorption in the superficial proximal convoluted tubule is highly load-dependent (75-90%) in normal and acidotic rats. No inhibitory effect of ECF volume per se on proximal bicarbonate reabsorption, independent of altering the filtered bicarbonate load, could be discerned. Acidosis enabled the end-proximal luminal bicarbonate concentration to fall below normal values and reduced distal bicarbonate delivery.

Effect of saline infusions on intrarenal distribution of glomerular filtrate and proximal reabsorption in the dog.

The effect of acute extracellular volume expansion with saline on the intrarenal distribution of glomerular filtrate, was studied in dogs utilizing micropuncture techniques in which samples were obtained by both recollection and from new tubules. Recollection was examined in seven dogs during continuous hydropenia and in five dogs during continuous saline diuresis. Recollection was associated with an increase in nephron flow rate of 8% during hydropenia and 27% during saline diuresis. In addition, during continuous saline diuresis, shortened transit times and lowered intratubular pressures were recorded in previously punctured tubules. Despite increased tubular flow, fractional reabsorption was unchanged. Nephron glomerular filtration rates (gfr) were measured during hydropenia and then after acute volume expansion in 10 dogs. In the repunctured tubules gfr rose 38% more than total glomerular filtration rate (GFR). In contrast, when new tubules were punctured during volume expansion, nephron gfr and total GFR changed proportionately. The disproportionate rise in nephron gfr after volume expansion noted with the recollection technique appears to be artifactual when contrasted to micropuncture of new tubules. With acute volume expansion, fractional reabsorption decreased 15% in recollected samples and 16% in newly sampled tubules. Increased nephron gfr cannot account for the fall in fractional reabsorption. It is concluded that in dogs, saline diuresis is not associated with redistribution of filtrate from deep to superficial nephrons, and that the fall in proximal fractional reabsorption is caused by diminished absolute reabsorption.

Effective glomerular filtration pressure and single nephron filtration rate during hydropenia, elevated ureteral pressure, and acute volume expansion with isotonic saline.

Free-flow and stop-flow intratubular pressures were measured in rats with an improved Gertz technique using Landis micropipets or a Kulite microtransducer. In hydropenia, average single nephron glomerular filtration rate was 29.3 nl/min, glomerular hydrostatic pressure (stop-flow pressure + plasma colloid osmotic pressure) was 70 cm H(2)O and mean glomerular effective filtration pressure was 12.7-14.3 cm H(2)O, approaching zero at the efferent end of the glomerulus. Thus, the glomerulus is extremely permeable, having a filtration coefficient four to five times greater than previously estimated. Mean effective filtration pressure and single nephron glomerular filtartion rate fell with elevated ureteral pressure and rose with volume expansion, more or less proportionately. Changes in effective filtration pressure were due primarily to increased intratubular pressure in ureteral obstruction and to reduced plasma colloid osmotic pressure in volume expansion; glomerular hydrostatic pressure remained constant in both conditions and thus played no role in regulation of filtration rate.

Resting transmembrane potential difference of skeletal muscle in normal subjects and severely ill patients.

The resting membrane potential difference (Em) of skeletal muscle was measured in 26 normal human subjects, 7 patients with mild illness, and 21 patients with severe, debilitating medical disorders. A closed transcutaneous approach to the muscle was made by needle puncture and the Em was measured utilizing standard Ling electrodes. Measurements revealed an Em of -88 +/-3.8 mv in healthy subjects and -89 +/-2.1 mv in patients hospitalized for minor medical problems. The mean Em in 21 in-hospital patients, judged to be severely ill clinically from a variety of causes, was -66.3 +/-9.0 mv. Open deltoid muscle biopsies were performed in 7 of the healthy subjects and in 13 of the severely ill group. Estimation of the intra-extracellular water partition was made by calculating the chloride space from the previously measured Em. Analysis of the muscle samples revealed no significant difference in the intra-extracellular potassium ratios of the two groups biopsied. Intracellular Na(+) concentrations were uniformly increased in the muscle samples of the severely ill subjects and averaged 42.3% higher than those of the normal subjects. The mechanisms which might account for the elevation of intracellular Na(+) and a depression of Em independent of changes in intra-extracellular K(+) ratios are discussed and it is suggested that this defect may be a generalized cellular abnormality which is a common quality of serious illnesses.

Mechanism of bicarbonate absorption and its relationship to sodium transport in the human jejunum.

Using a constant perfusion technique, sodium and bicarbonate absorption was studied in human subjects. The following observations were made on sodium absorption from saline solution: (a) the rate of sodium absorption is markedly influenced by bulk water flow, (b) when net water flow is zero, sodium absorption is zero if there are no concentration gradients between plasma and lumen that favor net NaCl diffusion; and (c) the PD between abraded skin and jejunal lumen is near zero when saline is perfused and does not change with partial substitution of sulfate or bicarbonate for chloride. Based on these observations, we conclude that sodium absorption from saline is entirely passive in the human jejunum. On the other hand, in the presence of bicarbonate sodium is absorbed actively against electrochemical gradients. The mechanism of the link between bicarbonate and sodium absorption was studied in normal subjects and in 11 patients with pernicious anemia; the latter were chosen because they do not secrete gastric acid which can react with bicarbonate in the jejunal lumen. We observed that bicarbonate absorption (a) occurs against steep electrochemical gradients, (b) does not generate a potential difference between abraded skin and jejunal lumen, (c) is inhibited by acetazolamide, and (d) generates a high CO2 tension in jejunal fluid. These observations suggest that bicarbonate absorption is mediated by active hydrogen secretion, rather than by bicarbonate ion transport per se, and that the link between sodium and bicarbonate transport is best explained by a sodium-hydrogen exchange process.

PIP: In this study of bicarbonate and sodium absorption in the intestine, absorption in a 30-cm segment of intestine was studied by the Ingelfinger triple-lumen perfusion system, which involves perfusion of test solutions into the intestine and sampling of gut contents 10 and 40 cm beyond the infusion marker. Human subjects were used. Observations made from these experiments on the mechanism of bicarbonate absorption and its relationship to sodium transport in the jejunum from saline solutions include: 1) the rate of sodium absorption is influenced greatly by bulk water flow; 2) when net water flow is zero, sodium absorption is zero in the absence of concentration gradients betwee plasma and lumen; and 3) the potential difference between abraded skin and jejunal lumen is near zero when saline is perfused and does not change when sulfate or bicarbonate is partially substituted for the chloride. It is concluded that sodium absorption from saline is entirely passive in the human jejunum; in the presence of bicarbonate, sodium is actively absorbed against electrochemical gradients. This study also looked at the mechanism of the link between bicarbonate and sodium absorption. Normal subjects and 11 patients with pernicious anemia were studied. Bicarbonate absorption was found to 1) occur against steep electrochemical gradients; 2) not generate a potential difference between abraded skin and jejunal lumen; 3) be inhibited by acetazolamide; and 4) generate a high carbon dioxide tension in jejunal. These observations led to the conclusion that bicarbonate absorption is mediated by active hydrogen secretion rather than by bicarbonate ion transport per se, making the best explanation for the link between sodium and bicarbonate transport a sodium-hydrogen exchange process.

Factors governing the transepithelial potential difference across the proximal tubule of the rat kidney.

Previous measurements of the transepithelial potential difference (PD) of the proximal tubule have yielded widely conflicting values (range -20 to +3 mV). In a recent study, Kokko has demonstrated that the PD of the in vitro perfused isolated proximal tubule of the rabbit varies in a predictable way from -6 to +3 mV, depending on the concentration of chloride, bicarbonate, glucose, and amino acids in the perfusing solution. The present micropuncture study examines the effect of tubular fluid composition on the PD profile along the proximal tubule of the in vivo rat kidney. Low resistance measuring electrodes with large tips (3-5 microns OD) filled with 3 M KCl, were used to provide stable PD recordings. Experiments were performed to validate the use of these electrodes. Transepithelial PD measurements were made in immediate postglomerular segments identified by injection of dye into Bowman's space of accessible surface glomeruli and in randomly selected more distal segments of the proximal tubule. In the control state, the first loop was found to have a small but consistently negative PD which could be obliterated by an infusion of phloridzin. In contrast, the PD in later segments was consistently positive. Infusion of acetazolamide abolished the positive PD in the later segments. Acetazolamide and glucose infusion resulted in a negative PD which was abolished by the additional infusion of phloridzin. These data provide evidence that glucose reabsorption is electrogenic and can account for the small negative PD normally present in the early proximal tubule. The positive PD in later segments appears to be a passive chloride diffusion potential. This positive potential is discussed as an important electrochemical driving force for significant passive reabsorption of sodium in the proximal tubule.

Effect of variation in dietary NaCl intake on the intrarenal distribution of plasma flow in the rat.

The effect of dietary variation in sodium chloride intake on the intrarenal distribution of plasma flow was investigated in rats using the antiglomerular basement membrane antibody technique. Rats were placed on a liquid diet containing either 9.86 (n = 9) or 0 (n = 9) mEq NaCl/daily portion for 2 wk. Labeled antibody was injected and the diets were reversed. After an additional 2 wk period, antibody labeled with a different radionuclide was injected and the animals were sacrificed. Fractional plasma flow distribution was then calculated for each dietary period. No change in flow to any cortical region could be detected. In six additional awake rats on identical dietary regimen, total plasma flow was estimated by the clearance of hippuran-(131)I. No change in this parameter occurred with changes in NaCl intake. We conclude, therefore, that no change in either total renal plasma flow or intracortical distribution of plasma flow occurs with wide variations in dietary sodium chloride intake in the rat. The implications of this constancy of regional plasma flow are discussed with reference to presumed concomitant alterations in the intrarenal distribution of nephron filtration rate.

The mechanism of suppression of proximal tubular reabsorption by saline infusions.

The mechanism by which expansion of extracellular fluid volume with isotonic saline suppresses reabsorption in the proximal tubule was studied in rats by examining the relations among glomerular filtration rate (GFR), absolute and fractional reabsorption of filtrate, intrinsic reabsorptive capacity (rate of reabsorption per unit tubular volume), transit time, and tubular volume. Saline infusions reduced the per cent of the glomerular filtrate reabsorbed in the proximal tubule from 50% during antidiuresis to 25% during saline diuresis. The suppression of proximal reabsorption was the result of two factors: 1) a 30% reduction of intrinsic reabsorptive capacity, and 2) a 26% reduction of tubular volume per unit GFR.GFR invariably rose during saline diuresis. However, prevention of the rise in GFR by aortic clamping had no effect on either the inhibition of intrinsic reabsorptive capacity or the reduction in tubular volume per unit GFR produced by saline infusions. Expansion of extracellular fluid volume with isotonic saline, therefore, depressed intrinsic reabsorptive capacity and tubular volume per unit GFR by some mechanism completely independent of GFR. The effects of furosemide administration were contrasted with those of saline infusions. Furosemide inhibited intrinsic reabsorptive capacity by 40% but had no significant effect on proximal fractional reabsorption. The failure to suppress fractional reabsorption was the consequence of a disproportionate rise in tubular volume (relative to GFR) that was sufficient to completely overcome the inhibition of intrinsic reabsorptive capacity. Inhibition of intrinsic reabsorptive capacity alone, therefore, will not result in a net suppression of reabsorption of filtrate in the proximal tubule. We concluded that, although intrinsic reabsorptive capacity was inhibited during saline diuresis, the critical factor responsible for translating this inhibition into effective net suppression of proximal reabsorption was the observed reduction in tubular volume per unit GFR.

Water and solute movement in the small intestine of patients with sprue.

Water and electrolyte movement in the jejunum of normal subjects and patients with sprue was measured during perfusion with isotonic electrolyte solutions. Normal subjects absorbed water, sodium, and potassium. By contrast, in patients with sprue (seven with adult celiac sprue and one with tropical sprue) who had diarrhea and steatorrhea, these substances were secreted into the intestinal lumen. This indicates that the jejunal mucosa of these patients was in a secretory state with respect to water and electrolytes.A method is presented for detecting abnormalities in the effective pore size in disease states. The method is based on the principle of restrictive diffusion and involves measuring the simultaneous diffusion rates of solutes of different molecular size. Since the method does not depend on measurement of water flow in response to osmotic pressure gradients, it can be used in disease states in which absorption and secretory processes involving water may be abnormal.The ratio of urea to tritiated water diffusion in the jejunum of normal subjects averaged 0.8, compared to 0.2 in patients with sprue. This indicates a marked decrease in the effective pore size of the jejunal mucosa in sprue. This conclusion was strengthened by the finding that erythritol and L-xylose, which are somewhat larger solutes than urea, are essentially non-absorbable in small bowel involved with sprue.

Measurement of intracellular pH of skeletal muscle with pH-sensitive glass microelectrodes.

We used three methods to examine the relationship among intracellular pH, transmembrane potential, and extracellular pH. Single-barreled electrodes permitted the determination of resting potential and intracellular pH with a minimum of cellular injury. Double-barreled electrodes, which incorporated a reference as well as a pH-sensitive electrode in a single tip, facilitated the direct measurement of intracellular pH without the interposition of the transmembrane potential. Triple-barreled electrodes permitted measurement of intracellular pH during the controlled hyperpolarization or depolarization of the cell membrane. The results of all three methods were in close agreement and disclosed that the H(+) activity of intracellular and extracellular fluid is in electrochemical equilibrium at any given transmembrane potential. This implies that the determinants of intracellular pH are the transmembrane potential and the blood pH. The actual pH of the normal resting muscle cell is 5.99, as estimated from the normal transmembrane potential and blood pH, or as determined by direct measurements of intracellular pH.

Functional characteristics of the diluting segment of the dog nephron and the effect of extracellular volume expansion on its reabsorptive capacity.

The functional characteristics of the ascending limb of Henle's loop were examined during hypotonic saline infusion by measuring solutefree water clearance (C(H2O)) at varying rates of solute delivery. The influence of expansion of extracellular volume was studied by comparing C(H2O) during hypotonic saline diuresis in normal dogs with dogs whose extracellular volume had been expanded acutely by saline infusions or chronically by the administration of deoxycorticosterone acetate and salt. In normal animals hypotonic saline infusions greatly increased urine flow (V) and C(H2O) without appreciably augmenting osmolar clearance (C(osm)). C(H2O) was, therefore, analyzed as a function of V, rather than C(osm), since V was the best estimate of delivery of filtrate to the diluting segment. C(H2O) increased as a linear function of V without any evidence of saturation.The validity of interpreting increases in C(H2O) and V as indications of increased sodium reabsorption and delivery was reinforced by tissue studies that disclosed a rise in papillary osmolality with rising urine flows. The observed increase in C(H2O) and V could not, therefore, be due to a decrease in back diffusion of solute-free water as a result of a diminished osmotic driving force, but probably represented increased formation consequent to augmented delivery as a result of decreased fractional reabsorption in the proximal tubule. In animals whose extracellular volume was acutely or chronically overexpanded before the infusion of hypotonic saline, sodium excretion was greater, and C(H2O) less, at any given V. Although the curve relating C(H2O) to V was flatter than in the control group, no tubular maximum was observed. The diminished C(H2O) in this group was interpreted to mean that massive expansion of extracellular volume inhibits sodium reabsorption in the ascending limb of Henle's loop.

Regulation of cell pH by ambient bicarbonate, carbon dioxide tension, and pH in the rabbit proximal convoluted tubule.

UNLABELLED: To study the regulation of cell pH by ambient pH, carbon dioxide tension (PCO2), and bicarbonate (HCO3), cell pH was measured in the isolated, in vitro microperfused rabbit proximal convoluted tubule using the fluorescent dye (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein. For the same changes in external pH, changes in [HCO3] and PCO2 affected cell pH similarly ([HCO3]: pHi/pHe = 0.67, PCO2: pHi/pHe = 0.64, NS). Isohydric changes in extracellular [HCO3] and PCO2 did not change cell pH significantly. Changes in peritubular [HCO3] elicited larger changes in cell pH than changes in luminal [HCO3], which were enhanced by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). The cell pH defense against acute increases and decreases in PCO2 was inhibited by sodium, but not by chloride removal. Peritubular SITS inhibited the cell pH defense against increases and decreases of PCO2, whereas luminal amiloride inhibited cell pH defense against increases in PCO2. CONCLUSIONS: (a) Steady-state cell pH changes in response to changes in extracellular [HCO3] and PCO2 are quantitatively similar for a given change in extracellular pH; (b) the rate of the basolateral Na/(HCO3)3 cotransporter is a more important determinant of cell pH than the rate of the apical membrane mechanism(s); (c) cell pH defense against acute changes in PCO2 depends on the basolateral Na/(HCO3)3 cotransporter (acid and alkaline loads) and the luminal Na/H antiporter (acid loads).