Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Acetazolamide inhibition of basolateral base exit in rabbit renal proximal tubule S2 segment

The influence of the carbonic anhydrase inhibitor acetazolamide (ACZ) was investigated on HCO ₃ ^– transport mechanisms in the basolateral cell membrane of rabbit renal proximal tubule. Experiments were performed on isolated S2 segments using double-barrelled microelectrodes to measure cell membrane potential (V _b) and cell pH (pH_i) during step changes in bath perfusate ion concentrations. Peritubular application of ACZ (1 mmol/l) reduced the initial V _b response to 101 reduction of bath HCO ₃ ^– concentration only slightly, from +53.8±4.2 mV to+49.1±0.3 mV (n=5), but caused an intermittent overshooting repolarization in the secondary V _b response. In conjunction with these effects it left the initial pH_i response virtually unchanged but induced a secondary slow acidification. These observation indicate that — under the present experimental conditions — ACZ does not block the Na⁺-HCO ₃ ^– cotransporter but acts via inhibition of cytosolic carbonic anhydrase. This was confirmed by studying the effect of elevated intracellular HCO ₃ ^– concentrations under reduced flux conditions and by comparing the concentration dependence of the V _b response with the inhibition kinetics of cytosolic carbonic anhydrase. In contrast, peritubular ACZ inhibited Na⁺-independent Cl^–/HCO ₃ ^– exchange in the basolateral cell membrane of S2 segments directly in a similar way to that described in the preceding publication for S3 segments.     

The chloride/base exchanger in the basolateral cell membrane of rabbit renal proximal tubule S3 segment requires bicarbonate to operate

Isolated microperfused S3 segments of rabbit renal proximal tubule were investigated with pH-sensitive double-barrelled intracellular microelectrodes to determine whether the Cl^–/base exchanger, which we have previously identified in the basolateral cell membrane of this segment requires HCO₃ ^– or can also work in CO₂/HCO₃ ^– free conditions. Cell pH (pH_i) was measured in response to sudden substitution of bath Cl^– by gluconate. In control solutions containing 25 mmol/l HCO₃ pH_i increased initially by 5.0±0.3 × 10^–3 unit/s but after perfusion with CO₂/HCO₃ ^–-free solutions pH_i of the same cells increased only by 1.3±0.2 × 10^–3 unit/s in response to Cl^– substitution. From measurements of the cellular buffering power it was calculated that the control base flux had fallen drastically from 3.7±0.3 to 0.3±0.1 × 10^–12 mols/s·cm tubule length. To test whether the remaining flux might have resulted from metabolic CO₂, oxidative metabolism was poisoned with cyanide (5 mmol/l). This abolished the pH change (pH_i) in CO₂/HCO₃ ^–-free solutions, but did not affect the pH shift in the presence of HCO₃ ^–. The data indicate that basolateral Cl^–/base exchange in S3 segment requires HCO₃ ^– to operate. A model in which HCO₃ ^– absorption proceeds in form of OH^– and CO₂ can be largely excluded.     

Contraluminal bicarbonate transport in the proximal tubule of the rat kidney

In order to measure the contraluminal bicarbonate flux in situ we applied the stopped flow capillary microperfusion technique and measured the influx of¹⁴C-bicarbonate buffer into cortical tubular cells at pH 8. It was found that the influx in percent of the starting concentration is larger at 20 mmol/l bicarbonate than at 1 mmol/l, indicating a sigmoidal type influx curve. At 20 mmol/l bicarbonate the influx was inhibited by 44%, when Na⁺ was replaced by choline. Replacement of gluconate by chloride or sulfate did not change H¹⁴CO ₃ ^– influx. At this bicarbonate concentration, influx is inhibited by 10 mmol/l 4,4-diisothiocyanato-2,2-stilbenedisulfonate (DIDS) (22%), 5 mmol/l of the carbonic anhydrase blocker ethoxyzolamide (40%) as well as by 5 mmol/l of the arginine reagent 4-nitrophenylglyoxal (31%). At 1 mmol/l bicarbonate starting concentration, bicarbonate influx was inhibited when chloride in the perfusate was present or when sulphate was added. Replacement of sodium by choline did not change bicarbonate influx. Addition of DIDS and 8-anilino-naphthalene-1-sulfonate (5 mmol/l each) inhibited 1 mmol/l bicarbonate influx 39 and 49%, respectively. The para-aminohippurate transport blocker dipropylsulfamoyl-benzoate (probenecid), the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the SH group blocker 2-(3-hydroxymercuri-2-methoxypropyl)-carbamoyl-phenoxyacetate (mersalyl), and formate did not inhibit bicarbonate influx, at 20 and at 1 mmol/l H¹⁴CO ₃ ^– starting concentration. The data are compatible with the assumption of 1. a contraluminal (HCO ₃ ^– )₃/Na⁺ cotransporter inhibitable by DIDS, carbonic anhydrase inhibitors and 4-nitrophenylglyoxal, 2. a HCO ₃ ^– /anion exchange system, which accepts sulfate and chloride and is inhibitable by the anion exchange blockers DIDS and 8-anilino-naphthalene-1-sulfonate, and 3. a HCO ₃ ^– influx component which could not be influenced by Na⁺, Cl^–, nor by the inhibitors applied.     

Evidence for OH−/H+ permeation across the peritubular cell membrane of rat renal proximal tubule in HCO 3 − -free solutions

Membrane potentials and intracellular pH were measured on rat renal proximal tubular cells in vivo to test whether sodium-bicarbonate cotransport across the peritubular cell membrane accepts OH^– (or H⁺ in opposite direction) or whether it requires the CO₂, HCO ₃ ^– , CO ₃ ⁼ buffer to operate. It was found that step changes of peritubular pH in nominally HCO ₃ ^– -free and CO₂-free solutions produced qualitively similar initial potential responses and cell pH responses as changes in peritubular HCO ₃ ^– concentrations. These responses, however, were considerably smaller and they were neither reduced in Na⁺-free solutions nor inhibited by the stilbene derivative SITS which is known to block Na⁺ (HCO ₃ ^– ) _n cotransport completely. We conclude that the cotransporter requires the CO₂, HCO ₃ ^– , CO ₃ ⁼ buffer for its physiological operation but that high rates of OH^– or H⁺ can also be transferred across the peritubular cell membrane in HCO ₃ ^– -free solutions, probably through a separate transport system.     

The mechanism of action of harmaline on renal solute transport

Summary The effect of the hallucinogenic drug harmaline was tested on rat kidney proximal tubular solute and water transport, using in vivo micropuncture and electrophysiological techniques as well as in vitro biochemical techniques. During peritubular application harmaline (5 mmol/l) was found to block net tubular volume absorption reversibly (by 85%) through inhibition of active Na⁺ transport and possibly active HCO ₃ ^– transport. The inhibition was accompanied by a rapid strong depolarization of the tubular cell membranes. As a biochemical equivalent harmaline inhibited the Na⁺–K⁺-ATPase and the Mg²⁺-ATPase of peritubular cell membrane fractions as well as the HCO ₃ ^– -stimulated ATPase of a brush border membrane fraction with similar kinetics. By studying glucose tracer efflux and by measuring cell membrane potential and conductance changes in response to glucose perfusions, no evidence for a direct effect of harmaline on Na⁺-glucose (or amino acid) cotransport mechanisms in the brush border could be obtained. The data suggest that harmaline does not specifically compete with Na⁺ for transport sites. Neither are the cotransport systems in the brush border membrane specifically inhibited, nor could the inhibition of the Na⁺ pump in the peritubular cell membrane simply result from a competition between harmaline and Na⁺.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

The purpose of this study was to investigate intracytoplasmic pH (pH_i) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pH_i was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pH_i values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pH_i by means of the classic Na⁺/H⁺ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pH_i recovery was mediated by a Na⁺/H⁺ exchange present in the basolateral side. Moreover, at physiological initial pH_i values, chloride removal from the apical solution caused the pH_i to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pH_i recovery was induced in sodium-free media by 15 mM HCO ₃ ^– in the presence of an outward chloride gradient. This pH_i change was completely abolished by 4,4-diisothiocyanostilbene 2,2-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na⁺-independent Cl^–/HCO ₃ ^– exchanger.     

Coupling between proximal tubular transport processes

Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (-methyl-d-glycoside), H⁺ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (c) of these substances. In the case of calcium the electrochemical potential differencec +zFc_i /RT) was the criterion employed. The rate of isotonic Na⁺-absorption (J_Na) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4isothiocyanatostilbene 2,2-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited J_Na (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, -methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H⁺ (glycodiazine) transport and PAH transport was only slightly affected.When SITS (1 mM) was applied from both sides of the cell it inhibited H⁺ (glycodiazine) transport by 72% and reduced J_Na by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H⁺ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied.When the HCO ₃ ^– buffer or physically related buffers were omitted from the perfusate the absorption of Na⁺ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected.The data are consistent with the hypothesis that the active transport processes of histidine, -methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na⁺-Ca²⁺ countertransport located at the contraluminal cell side. The residual Na⁺ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H⁺ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO ₃ ^– from the cell and secondary to that, the luminal H⁺-Na⁺ exchange and consequently the Na⁺ reabsorption. In the absence of HCO ₃ ^– buffer in the perfusates the luminal H⁺-Na⁺ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP _i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.     

Mechanism of Cl− transport in eel intestinal brush-border membrane vesicles

Cl^– transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. ³⁶Cl^– uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K⁺ diffusion potential obtained by imposing a K⁺ concentration gradient (C _out>C_in) in the presence of valinomycin, (c) an inwardly directed H⁺ ion concentration gradient. The membrane-potential-driven Cl^– transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropylamino)-benzoic acid. Arachidonic acid also inhibited Cl^– uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na⁺ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl^– influx was the same in the presence of inwardly directed gradients of Li⁺, Na⁺ or K⁺, arguing against the presence of Na⁺-Cl^–, as well as K⁺-Cl^– cotransport. The absence of a significant contribution of the Na⁺-K⁺-2Cl^– cotransport mechanism to the Cl^– uptake in seawater eel intestinal BBMV was indicated from the observations that Cl^– uptake was not stimulated by the simultaneous presence of inwardly directed Na⁺ and K⁺ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na⁺ and K⁺. Furthermore, no evidence for the dependence of Cl^– uptake on the Na⁺ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K⁺ and valinomycin. The finding that the Cl^– uptake in the presence of a H⁺ gradient was not inhibited by 1 mM SITS and was significantly reduced in the presence of [K⁺]_in=[K⁺]_out and valinomycin, suggests that no anion exchanger is present in our experimental system. We conclude that Cl^– uptake in eel intestinal BBMV does not occur via an electroneutral Na⁺-dependent Cl^– transport mechanism (either cotransport or double exchange) and is realized by a Cl^– conductance.     

Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO 3 − (OH−) exit

Intracellular pH (pH_c) was measured on surface loops of rat kidney proximal tubules under free-flow conditions in vivo using fine tip double-barrelled pH microelectrodes based on a neutral H⁺ ligand. The microelectrodes had Nernstian slopes and a resistance of the order of 10¹² . By using a driven shield feed back circuit the response time to pH jumps was lowered to around 1 s. At a peritubular pH of 7.42 and a luminal pH of 6.68 ± 0.13 (n=27), pH_c was 7.17 ± 0.08 (n=19). Perfusing the peritubular capillaries suddenly with bicarbonate Ringer solutions of plasma-like composition which were equilibrated with high or low CO₂ pressures, acidified or respectively alkalinized the cells rapidly as expected from the high CO₂ permeability of the cell membranes. Such data allowed us to calculate the cytoplasmic buffering power of the tubular cells. Sudden peritubular perfusion with Ringer solution containing only 3 mmol/l of HCO ₃ ^– at constant physiological CO₂ pressure led to a similar fast cell acidification which indicated that the peritubular cell membrane is also highly permeable for bicarbonate or OH^– (H⁺). The latter response was completely blocked by the stilbene derivative SITS at the concentration of 10^–3 mol/l. The observations indicate first that pH_c of rat proximal tubule is more acidic than was previously thought on the basis of distribution studies of weak acids, second that intracellular bicarbonate concentration is around 13 mmol/l and third that bicarbonate exit across the peritubular cell membrane is a passive rheogenic process via a conductive pathway which can be inhibited by SITS. The latter point confirms the conclusion which we had derived previously from membrane potential measurements in response to changing peritubular bicarbonate concentrations.     

A stretch-activated K+ channel in the basolateral membrane ofXenopus kidney proximal tubule cells

The present study examined whether a basolateral potassium ion (K⁺) channel is activated by membrane-stretching in the cell-attached patch. A K⁺ channel of conductance of 27.5 pS was most commonly observed in the basolateral membrane ofXenopus kidney proximal tubule cells. Channel activity increased with hyperpolarizing membrane potentials [at more positive pipette potentials (V _p)]. Open probability (P _o) was 0.03, 0.13, and 0.21 atV _p values of 0, 40, and 80 mV, respectively. Barium (0.1 mM) in the pipette reducedP _o by 79% at aV _p of 40 mV. Application of negative hydraulic pressure (−16 to −32 cm H₂O) to the pipette markedly activated outward currents (fromP _o=0.01 to 0.75) at aV _p of −80 mV, but not inward currents at aV _p of 80 mV. The size of the activated outward currents (from cell to pipette) did not change by replacing chloride with gluconate in the pipette. These results indicate that a stretch-activated K⁺ channel exists in the basolateral membrane of proximal tubule cells. It may play an important role as a K⁺ exit pathway when the cell membrane is stretched (for example, by cell swelling).     

Active NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse does not require the presence of bicarbonate

The aim of the present study was to investigate whether bicarbonate buffer (CO₂ + HCO ₃ ^– ) is required to sustain maximal NaCl transport in the cortical thick ascending limb of Henle's loop (cTAL) of the mouse. Transepithelial Na⁺ and Cl^– net fluxes (J _Na, J _Cl, pmol min^–1 mm^–1), measured by electron microprobe analysis, were similar irrespective of the presence or absence of CO₂ + HCO ₃ ^– in luminal and bathing solutions J _NaCl with CO₂ + HCO ₃ ^– =203±25 pmol min^–1 mm^–1; J NaCl without CO₂ + HCO ₃ ^– =213±13 pmol min^–1 mm^–1, n=14). Furthermore the transepithelial potential difference, V _te, the transepithelial resistance, R _te, and the basolateral membrane potential, V _bl, were unaffected by CO₂ + HCO ₃ ^– . In the absence of CO₂ + HCO ₃ ^– , V _te was +17.0±1.7 mV(n=9) (lumen positive), R _te was 28±2 cm² (n=9) and V _bl was –76±4 mV (n=6). In the presence of CO₂ + HCO ₃ ^– , V _te, R _te and V _bl were +15.9±1.5 mV, 29±1 cm² and –73±5 mV, respectively. 4-Acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS; 0.1 mmol l^–1) and amiloride (1 mmol l^–1) added to the (CO₂ + HCO ₃ ^– )-containing lumen perfusate were without effect on V _te and R _te. Finally, the effect of furosemide (0.1 mmol l^–1) on V _te and V _bl in the presence of CO₂ + HCO ₃ ^– was investigated. Furosemide reversibly decreased V _te from +13.7±1.1 mV to +1.7±0.7 mV (n=6) and hyperpolarized V_bl from –70±1 to –89±3 mV (n=5), suggesting passive distribution of Cl^– across the basolateral membrane. In conclusion, these data suggest that active NaCl transport in the cTAL of the mouse does not require the presence of CO₂ + HCO ₃ ^– .     

Energy source for transepithelial sodium transport in rat renal proximal tubules

Summary The effects of various metabolic inhibitors on isotonic fluid absorption (J _V ) in rat proximal tubules and on the Na⁺–K⁺-ATPase of isolated cell membranes of rat kidney cortex were investigated by the shrinking split oil droplet technique and biochemical methods respectively.Both Oligomycin (5×10^–5 M, 10^–4 M) and Antimycin A (10^–5 M, 10^–4 M) inhibited isotonic fluid absorption by 80% when applied intratubularly but only in conjunction with bovine serum albumin. At these concentrations they inhibited a Na⁺–K⁺ activated adenosine triphosphate phosphohydrolase (Na⁺–K⁺ ATPase E.C. 3.6.1.3.) of cell membranes isolated from rat kidney cortex by 77%, 82% and 55%, 95%, respectively.Sodium phosphoenolpyruvate (PEP) 5×10^–3 M could partially reverse the inhibition of the isotonic fluid absorption but only with 10^–5 M Antimycin A when the Na⁺–K⁺ ATPase inhibition was apparently small.The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (10^–3 M), as well as sodium cyanide (5×10^–3 M) inhibitedJ _V 100%, but only when applied through peritubular blood capillary perfusion.From these findings it was concluded thatall proximal tubular isotonic fluid absorption is supported by energy fromoxidative processes, and that in a least 80% of this sodium reabsorption, ATP from oxidative phosphorylation is directly involved, while, for the remaining 20% non ATP energy is responsible.C. J. Martin Fellow of the National Health and Medical Research Council of Australia.     

Electrophysiological analysis of rat renal sugar and amino acid transport

Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion ofd-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na⁺ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brushborder membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition ofd-glucose transport by various inhibitors, such as phlorizin, harmaline and oubain. The latter experiments suggest that the active Na⁺ pump in the peritubular cell membrane, which establishes the Na⁺ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane.     

Ion transport and electrophysiology of the early proximal colon of rabbit

The ion transport properties of the mammalian descending colon have been the subject of numerous investigations during the last decade. In contrast, relatively few studies have investigated proximal segments of this organ. In the present study, we assessed transepithelial transport of Na⁺, K⁺ and Cl^– in the isolated initial segment (P1) of rabbit colon in vitro using radioisotopic tracer fluxes and electrophysiological techniques. Like the rabbit descending colon, the proximal colon actively absorbs sodium and chloride, howeveer, its transport systems are markedly different. In vivo, this segment absorbs potassium, however in vitro active potassium secretion was observed. Unlike the descending colon, Na⁺ absorption is relatively insensitive to amiloride and only a slight inhibition was obtained even at 1 mM concentrations of this drug. Na⁺ and Cl^– absorption appeared to be coupled (directly or indicrectly) since the absorption of each ion was inhibited by the removal of the other. Serosal ouabain also inhibited Na⁺ and Cl^– absorption and net K⁺ secretion. Unlike the descending colon, the proximal P1 segment did not have a net absorptive K⁺ transport system that was detectable in the presence of ouabain. Electrically, the early proximal colon has a low transepithelial resistance compared to descending colon (R _T=133±7 cm²) but a larger short-circuit current (l _sc=178±12 A/cm²). The transepithelial potential averaged –21±1 mV, in excellent agreement with values measured in vivo. The apical and basolateral membrane potentials averaged –21±1 mV and –42±1 mV and intracellular potassium activity was 70±2 mM. The findings indicate active K⁺ uptake across the basolateral membrane and passive exit across the apical membrane. The basolateral membrane conductance may be a potassium conductance that is blockable by barium. It is likely that K⁺ transport normally occurs by both cellular and paracellular routes in this epithelium. Because of the numerous differences between this segment and the descending colon, we conclude that the P1 segment of proximal colon has a distinct function in colonic electrolyte transport     

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pH_i) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pH_i measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na⁺/H⁺ exchanger and an apical Cl^–/HCO ₃ ^– exchanger [1]. The pH_i of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na⁺/H⁺ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pH_i is the Cl^–/HCO ₃ ^– exchanger. This study confirms the importance of this transporter in pH_i regulation within the physiological pH_i range and the influence of calcitonin in the regulation of DCTb cell function.     

Bidirectional active transport of thiosulfate in the proximal convolution of the rat kidney

Using the standing droplet method in the late proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of thiosulfate at 45 s was determined, the latter being taken as a measure of active thiosulfate transport. Under control conditions, in the presence of Na⁺, near zero c values were observed. When 1 mmol/l carinamide or paraaminohippurate (PAH) were added to the perfusates significant reabsorptive c arose. However, when 7.5 mmol/l sulfate was added to the Na⁺-free secretory c values were observed. Tested under Na⁺-free conditions, the secretory c was not influenced by simultaneously present 5 mmol/l of SO ₄ ^2– but was diminished by 50 mmol/l SO ₄ ^2– . PAH (1 mmol/l), carinamide (0.2 mmol/l) and probenecid (1 mmol/l) decreased the secretory c by 48, 65 and 48%, respectively. The PAH secretion was not influenced, when thiosulfate or sulfate up to 50 mmol/l was added to both perfusates. Under Na⁺-free conditions the c of thiosulfate in early loops of the proximal convolution is higher than in late loops, while for PAH this pattern is reversed. Taken together with the previously published inhibition of sulfate reabsorption by thiosulfate the data indicate 1. thiosulfate is reabsorved by the Na⁺-dependent sulfate transport system and 2. thiosulfate is simultaneously secreted by a carinamide-, probenecid-and PAH-sensitive secretory system. The secretory system might also be shared by sulfate. The thiosulfate net flux is the result of the difference in the activity of the counteracting transporters, located at the luminal and contraluminal cell side. Is is possible that the higher activity of the transporter at one cell side leads to a reversal of the flux through the transporter at the other cell side.     

Use of the pH-sensitive dye BCECF to study pH regulation in cultured human kidney proximal tubule cells

Summary This protocol describes the use of the pH-sensitive, intracellularly trapped dye 2′,7′-bis (2-carboxyethyl), 5 (and -6) carboxyfluorescein (BCECF), to characterize the pH regulating mechanisms in cultured human kidney proximal tubule cells. This is a reliable method for intracellular pH measurements and is applicable to single cells, cell suspensions, and confluent cultures. This study was supported in part by a Rapid Advisory Group grant from the Veterans Administration, Washington, DC.     

The effect of hyperosmotic challenge upon ion transport in cultured renal epithelial layers (MDCK)

Exposure of the basal-lateral surfaces of MDCK epithelia, mounted in Ussing chambers, to medium made hyperosmotic by the non-electrolyte mannitol, resulted in a marked inhibition of the adrenalinestimulated inward short-circuit current (Cl^– secretion). This inhibition was unaccompanied by a reversal of the adrenaline-stimulated increment in tissue conductance, indicating that the inhibition was due to modulation of ion transport at the basal-lateral membranes. Loop-diuretic-sensitive ⁸⁶Rb(K⁺) efflux mediated by the Na⁺-K⁺ — 2 Cl^– cotransporter at the basal-lateral membranes was markedly stimulated by hypertonic exposure. A diuretic-sensitive K⁺ (Cl^–) loss was observed in shrunken cells upon prolonged exposure (20 min), showing that the net direction of cotransport flux was outward. ⁸⁶Rb(K⁺) efflux stimulated by adrenaline (100 M), exogenous ATP (100 M) and A23187 (10 M) was attenuated in shrunken cells, suggesting that basal-lateral K⁺ conductance is reduced in hyperosmotic media. Cotransport stimulation by hyperosmotic medium was asymmetric, apical bathing hypertonicity being ineffective. These data are consistent with a low hydraulic permeability of the apical membranes.