The distribution of Na'-independent Cl --HCO3 exchange was studied in individual intercalated cells from in vitro perfused rabbit outer CCDs using dual excitation laser scanning confocal microscopy by measuring the pH1 response to sequential removal of Cl-from both sides of the tubule. Three patterns of intracellular pH (pH1) response were observed. 39% of intercalated cells had only apical Cl--HCO3 exchange (,B cell), 4%had only basolateral Cl--HCO-exchange (a cell), and 57% had both apical and basolateral Cl --HCO3 exchange (ay cell). Valinomycin-high K+ voltage clamping had no effect on the pH1 response of intercalated cells with bilateral Cl --HCO3 exchange. Although the mean rates of dpH1/dt following apical Cl-removal were similar in ,8 cells compared to y cells, a wide range ofapical rates was seen among individual iB and y intercalated cells. Neither the apical nor the basolateral CI--HCO3 exchanger in y cells was inhibited by 0.5 mM H2DIDS. Binding ofapical peanut lectin was seen both in ft cells and in y cells.In 41% of CCDs with four to seven intercalated cells studied, all intercalated cells were of the same subtype. We conclude that the majority of intercalated cells from the rabbit outer CCD have both apical and basolateral Na'-independent Cl--HCO3 exchangers (y cells), which are stilbene-insensitive. Intercalated cells with only basolateral CI--HCO3 exchange are very uncommon in the rabbit outer CCD. There is a tendency for all intercalated cells in a given rabbit outer CCD to be of the same subtype (either all 6 cells or all y cells), suggesting the presence of CCD intertubule heterogeneity at the same cortical level. This finding may account for intertubule differences in transepithelial H+-base transport. (J. Clin. Invest. 1994. 93:417423.)

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na '-independent Cl -/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na '-independent Cl-/ base exchanger on both membranes. HCO3 fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3 production does not alter the rate of apical Cl-/base exchange in Hepesbuffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is -6 ,uM and in vitro tubule formate production is < 0.6 pmol/min per mm.Formate in the micromolar range stimulates Jv in a dosedependent manner in the absence of a transepithelial Na+ and Cl-gradient and without a measurable effect on Cl--induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH-exchanger and that the secreted H+ and OH -ions form H20 in the tubule lumen. (J. Clin. Invest. 1994. 94:173-183.) Key words: Na+/H+ exchange-

To functionally characterize transport properties of the apical anion exchanger of rabbit β-intercalated cells, the mean change in anion exchange activity, dpHi/d t(where pHi is intracellular pH), was measured in response to lumen Cl− replacement with gluconate in perfused cortical collecting ducts (CCDs). β-Cell apical anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS in the presence of 115 mM Cl−. In contrast, apical anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the absence of Cl−. β-Cell apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However, β-cell apical anion exchange was inhibited by α-cyano-4-hydroxycinnamic acid, with an IC50 of 2.4 mM. Substitution of either sulfate or gluconate for lumen Cl− resulted in a similar rate of alkalinization. Conversely, pHi was unchanged by substitution of sulfate for lumen gluconate, confirming the lack of transport of sulfate on the β-cell apical anion exchanger. Taken together, the results demonstrate a distinct “fingerprint” of the rabbit CCD β-cell apical anion exchanger that is unlike that of other known anion exchangers.

In order to investigate the reproducibility of maximum progressive exercise we studied 6 healthy volunteers twice a day for 3 nonconsecutive days. The average within-subject coefficients of variation for the maximal ventilatory and heart rate (HR) responses ranged from 3.8% for HR to 12% for minute ventilation (Ve) and anaerobic threshold. Three-way analysis of variance revealed no significant variations in exercise parameters for the group as a whole except for the carbon dioxide output (VCO₂) and respiratory exchange ratio (R) which was significantly greater in the afternoon (p < 0.05). This was attributed to dietary effects of carbohydrate loading upon VCO₂ and was associated in 1 subject with a significant increase in Ve (p < 0.01). In another subject, morning values of Ve were consistently and significantly (p < 0.05) greater than the afternoon values which could only be attributed to increased effort as indicated by an increased oxygen uptake. Tests of resting pulmonary mechanics (FEV1, FVC, FRC, RAW) measured before each exercise procedure showed no significant diurnal or day-to-day variations. Results indicate that while the maximal responses to progressive exercise are generally reproducible and the first exercise procedure can usually be considered representative, diurnal variations in R, VCO₂ and Ve may occur which can be best avoided on repeated testing by exercising subjects at a standardized time of day.

To investigate halide selectivity patterns of cortical collecting duct (CCD) intercalated cells (ICs), intracellular pH (pHi) was measured in perfused rabbit outer CCD ICs in Na(+)-free, HCO-3-buffered, 115 mM Cl solutions. Apical anion exchange activity was measured as the dpH/dt after lumen Cl- replacement with gluconate. The perfusate was randomly changed to equimolar Br-, F-, or I-. Addition of Br- to the lumen in place of gluconate caused a brisk acidification similar to that with Cl- return. The acidification rate with I- replacement was only approximately 25% of that seen with Cl- readdition, and F- substitution resulted in a small alkalinization. In a separate protocol, each halide was substituted for lumen Cl-. Replacement of lumen Cl- with Br- resulted in intracellular acidification, whereas F- substitution caused an increase in pHi similar to that with gluconate. A slower rate of alkalinization was seen with I-. Separate tubules were perfused and bathed in Cl(-)-free gluconate solutions for 10 min, and then either Cl- or Br- was returned to the lumen. Similar rates of acidification were found with either Cl- or Br- return. Taken together, the results show that, at a halide concentration of 115 mM, the halide selectivity transport pattern of the apical anion exchanger of CCD ICs is Cl- = Br- > I- > F-.