Impaired urinary acidification in the hypothyroid rat

Summary Since abnormalities in the renal handling of sodium and water in both the proximal and distal tubule have been described in primary hypothyroidism, this study was undertaken to examine renal tubular hydrogen secretion in this disorder. Metabolic acidosis was induced in hypothyroid rats (H) and their age matched controls (C) by the administration of an oral ammonium chloride load of 0.15 g/24 h/kg for three days. On day 3 animals were prepared for clearance and acid-base studies, receiving an infusion of Ringer's solution of 0.6 ml/hr/100 g during surgery and the experimental procedure. A 26% decrease in GFR (P<0.005) and a doubling in fractional excretion of sodium (P<0.02) were observed in H rats. The lowest blood pH and average bicarbonate concentration and the excretion of chloride were similar in the two groups, indicating that the acid load was reabsorbed and led to similar degrees of systemic acidification. Urine flow also was comparable in the two groups. Minimal urine pH after NH₄Cl was 6.21±0.06 in H and 5.68±0.09 in C (P<0.001). Ammonium excretion was 28% (P<0.05) lower in H than in C. The defect in urine acidification in H was only partially corrected after 5 days on a low sodium diet and DOCA administration for 2 days. Fractional bicarbonate excretion at normal blood pH and bicarbonate concentration was not different in the two groups. These data indicate that hypothyroid rats have a mild defect in urine acidification and that it is localized predominantly in the distal tubule.     

Effect of NH4 +/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K⁺. In excised membrane patches the luminal K⁺ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (V _m) in slow-whole-cell (SWC) recordings and the open probability (P _o) of K⁺ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of V _m can be recorded. The pipette solution contained K⁺ 125 mmol/l and Cl^– 32 mmol/l. Intracellular pH was determined by 2,7 bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH₄ ⁺/NH₃ to the bath. In the presence of NH₄ ⁺/NH₃ intracellular pH acidified by 0.53±0.11 units (n=7). Inhibition of the Na⁺2Cl^– K⁺ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62±0.14 units (n=7). In SWC experiments V _m of TAL cells was -72±1 mV (n=70). NH₄ ⁺/NH₃ depolarised V _m by 22±2 mV (n=25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH₄ ⁺ from 24±3 mV to 7±3 mV. Under control conditions the single-channel conductance of TAL K⁺ channels in CAN experiments was 66±5 pS and the reversal voltage for K⁺ currents was 70±2 mV (n=35). The P _o of K⁺ channels in CAN patches was reduced by NH₄ ⁺/NH₃ from 0.45±0.15 to 0.09±0.07 (n=7). NH₄ ⁺/NH₃ exposure depolarised the zero current voltage of the permeabilised patches by-9.7±3.6 mV (n=5). The results show that TAL K⁺ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH₄ ⁺/NH₃ exposure at concentrations which are physiologically relevant because Na⁺2Cl^–K⁺(NH₄ ⁺) cotransporter-mediated import of NH₄ ⁺ exceeds the rate of NH₃ diffusion into the TAL. K⁺ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH₄ ⁺ uptake occurs by the Na⁺2Cl^–K⁺ cotransporter. The findings that, in the presence of NH₄ ⁺/NH₃ and furosemide, V _m is not completely repolarised and that K⁺ channels are not activated suggest that the respective K⁺ channels may in addition to their pH regulation be inhibited directly by NH₄ ⁺/NH₃.     

pH dependence of K+ conductances of rat cortical collecting duct principal cells

The K⁺ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K⁺ secretion is decreased with increased H⁺ secretion during acidosis. We examined whether the pH sensitivity of these K⁺ channels is present also in the intact cell and thus could explain the coupling between K⁺ and H⁺ secretion. Membrane voltages (V _m), whole-cell conductances (g _c), and single-channel currents of K⁺ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pH_i) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V _m, g _c, or the activity of the K⁺ channels in these cells. Acetate, however, acidified pH_i slightly by 0.17±0.04 pH units (n=19). V _m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g _c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO₂, respectively. The same CO₂ concentrations decreased pH_i by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P _o) of all four K⁺ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO₂. pH_i increased with the addition of 20 mmol/l NH₄ ⁺/NH₃ by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH₄ ⁺/NH₃ removal. In the presence of NH₄ ⁺/NH₃ V _m depolarized by 16±2 mV (n=66) and g _c decreased by 26±7% (n=16). The activity of all four K⁺ channels was also strongly inhibited in the presence of NH₄ ⁺/NH₃. The effect of NH₄ ⁺/NH₃ on V _m and g _c was markedly increased when the pH of the NH₄ ⁺/NH₃-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K⁺ conductances, thus reduces K⁺ secretion by direct inhibition of K⁺ channel activity. This pH dependence is present in all four K⁺ channels of the rat CCD. The inhibition of K⁺ channels by NH₄ ⁺/NH₃ is independent of changes in pH_i and rather involves an effect of NH₃.     

The effects of chronic papillary necrosis on acid excretion

Complete papillary necrosis in rats can be induced within 1 month following a single injection of 2-bromoethylamine hydrobromide (BEA) (50 mg, i.v.). Utilizing a combination of clearance and balance techniques the effects of complete absence of the papilla was examined as regards urinary acidification, whole kidney glomerular filtration rate (GFR), single nephron GFR, and morphology. Whole kidney GFR was not different from control, however, the percent filtering juxtamedullary nephrons was markedly diminished (87.2±2.1 vs. 31.5±3.6% filtering, control vs. BEA, respectively,P<0.001) and significantly reduced in the superficial nephrons (80.6±3.6 vs. 62.2±6.1% filtering, control vs. BEA, respectively,P<0.05). There was a significant decrease in juxtamedullary single nephron GFR and an increase in the superficial single nephron GFR as assessed by the quantitative Hanssen's technique in the animals with chronic papillary necrosis. Complete papillary necrosis was associated with normal arterial bicarbonate concentration, pH, and plasma electrolyte concentrations. At the same degree of acidemia (induced by NH₄Cl administration) minimal urinary pH, ammonium excretion, and titratable acid excretion were not different than seen in age matched controls. The response to Na₂SO₄ infusion and phosphate infusion was the same in both groups of animals. The urineblood (U-B)pCO₂, an index of urinary acidification, was identical in BEA and control animals. Scanning electron microscopy showed scarring of the juxtamedullary glomeruli one month after BEA. The papilla was sloughed and lying free in the renal pelvis in every experimental animal. These data demonstrate that complete papillary necrosis is not associated with acidosis nor a defect in urinary acidification.     

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid     

H+ ion secretion in proximal tubule of low-CO2/HCO 3 − perfused isolated rat kidney

Acidification in proximal tubule of the isolated rat kidney, perfused in vitro, was studied by stopped-flow microperfusion techniques, using Sb microelectrodes to measure luminal pH. The kidney was perfused with mammalian Ringer's solution at pH 7.4 buffered by 20 mmol/l phosphate and containing 7.5 g/100 ml bovine albumin, equilibrated with air. Final urine pH was 6.88±0.5. Steady-state pH in proximal segments was 6.81±0.03 (n=80), and acidification half-time (t/2) 7.25±0.33 (80) s, giving a net secretory H⁺ ion flux of 0.51±0.05 nmol·cm^–2·s^–1. This flux was about 70% of in vivo (blood perfused kidneys). During luminal perfusion with solutions at pH 6.2, back-flux of H⁺ was 0.82 ±0.08 nmol·cm^–2·s^–1, with an alkalinizationt/2 of 6.33 ±0.34 (34) s. The difference between acidification and alkalinizationt/2 was not significant. This is compatible with a pump-leak system of H⁺ transport. The back flux of H from the lumen was markedly reduced in low Na⁺ perfused kidneys in the presence of 10^–4 mol/l amiloride in the lumen, indicating that this process is mediated by the luminal Na/H exchanger. Observations in the presence of high K levels suggest that it may have also a charged component. 10^–4 mol/l acetazolamide added to the kidney perfusate reduced acidification to 0.5% of control, and 10^–6 mol/l SITS to 25% of control. Thus, despite the lowpCO₂ (0.1–0.4 kPa, or 1–3 mm Hg), the CO₂/Hco ₃ ^– buffer system still plays an important role in tubular acidification in this preparation.     

Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas

Intracellular pH (pH_i) has been measured in intact, perfused rat mandibular salivary glands loaded with the fluorescent pH indicator BCECF [2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectrofluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was measured ratiometrically at 6-s intervals. The mean value of pH_i in glands perfused with a HCO ₃ ^– -free, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES)-buffered solution at 37°C was 7.36±0.01 (n=52) which is comparable with values obtained by ³¹P nuclear magnetic resonance (NMR) spectroscopy. NMR data confirmed that the BCECF loading period was accompanied by a transient acidification of the cells, but there was no significant change in the content of the major phosphorus metabolites. Changes in pH in response to NH₄Cl pulses and acetylcholine stimulation were comparable with results reported previously for isolated acini. Additional, preliminary experiments show that the method can also be used to monitor intracellular Ca²⁺ (using fura-2) in perfused salivary glands, and can be adapted for studies of the isolated, perfused pancreas.     

Proximal tubular HCO 3 − , H+ and fluid transport during maleate-induced acidification defect

The mechanism of tubular acidification was studied in proximal tubular acidification defect induced in rats by acute parenteral infusion of maleate (200 mg/kg), which causes diuresis and bicarbonaturia. Proximal tubular bicarbonate reabsorption and H⁺ ion secretion were determined by stopped-flow microperfusion and measurement of luminal pH by Sb microelectrodes. Stationary pH increased in proximal tubule from 6.78 to 7.25 and bicarbonate reabsorption decreased from 1.32 to 0.51 nmol/cm² ·. In these segments, mean cell PD fell from −66.6 to −20.2 mV, whileJ _v as estimated by the Gertz technique fell to 15% of controls. A similar impairment of acidification was observed during luminal and capillary perfusion with phosphate Ringer's. Since H⁺-ion efflux from the lumen was not significantly increased and both acidification and alkalinization half-times (t/2) were increased, no evidence for an increase in passive permeability for H⁺/HCO ₃ ⁻ was obtained. The increasedt/2 found during luminal perfusion with acid phosphate indicates, according to an electrical analog model, a reduction in pump series conductance. These results show that maleate affects both proximal Na⁺ and H⁺ transport; this effect may be ascribed to impairment of sodium-dependent transport systems in the brush-border membrane.     

Changes of pH affect calcium currents but not outward potassium currents in rat myometrial cells

Spontaneous contraction of uterine smooth muscle is enhanced by alkalinization and depressed by acidification. We have investigated the ionic currents responsible for this in single myometrial cells. Intracellular acidification (20 mM butyrate) at constant external pH depressed the magnitude of the calcium current to 58±6% of control, but had little effect on outward currents. Similar but slower effects were also observed when the extracellular pH was lowered to 6.9 (56±9% of control). Correspondingly, when the intracellular or extracelluar pH was elevated (20 mM NH₄Cl or pH 7.9 respectively) the calcium current magnitude increased (165±15 % in NH₄Cl; 136±2 % at pH 7.9) and there was, again, no effect on the outward currents. These observations are consistent with the effects of pH on spontaneous contractile activity being due to an effect on the membrane calcium current.     

NH(4)(+) conductance in Xenopus laevis oocytes. III. Effect of NH(3)

Exposure of Xenopus laevis oocytes to NH₄Cl caused intracellular acidification, cell membrane depolarization and the generation of an inward current. To determine the contribution of uncharged NH₃ and positively charged NH₄⁺, the NH₄Cl-induced inward current was measured in the presence of increasing [NH₃] at constant [NH₄Cl] (10 mM) or increasing [NH₄Cl] at constant [NH₃] (0.045 mM) with pH varying in both cases. At –70 mV, the NH₄Cl-induced current was barely detectable at pH 6.5, 0.01 mM NH₃, but increased successively at pH 7.5, 0.1 mM NH₃ and pH 8.5, 1 mM NH₃. In contrast, NH₄Cl-associated currents were independent of changes of the [NH₄Cl] at constant [NH₃] and variable pH. Similar results with respect to acidification, depolarization and inward current in response to concentration and pH changes were obtained with trimethylamine HCl. Increasing concentrations of the weak acid propionate led to a reduction of the NH₄Cl-induced current. These data suggest that NH₃ entry may induce local alkalinization that, in turn, may trigger the opening of a conductance for NH₄⁺ or trimethylamine-H⁺ entry.     

The effect of ammonium chloride and sodium bicarbonate ingestion on the physical working capacity at the fatigue threshold

Summary The purpose of this investigation was to examine the effect of ammonium chloride (NH₄Cl) and sodium bicarbonate (NaHCO₃) ingestion on the physical working capacity at the fatigue threshold (PWC_FT). Eighteen adult males (mean age, SD=23, 2 years) volunteered for two experiments (experiment 1,n=9 ; experiment 2,n=9). In both experiments, the subjects orally ingested 0.3 g · kg^–1 body weight of NH₄Cl and NaHCO₃ over a 3-h period in random order on days separated by 72 h or more. In experiment 1, following ingestion of the substance, the subjects performed a discontinuous incremental cycle ergometer test to the onset of PWC_FT which was estimated from integrated electromyography voltages at the vastus lateralis muscle. In experiment 2, the subjects performed a continuous PWC_FT test. The results of these experiments indicated that NH₄Cl and NaHCO₃ ingestion had no significant (P>0.05) effect on PWC_FT (experiment 1: NH₄Cl=257, SD 26 W; NaHCO₃=256, SD 22 W;t=0.06;r=0.866; experiment 2: NH₄Cl=231, 14 W; NaHCO₃=216, 16 W;t=1.78;r=0.857).     

Proton transport mechanism in the cell membrane of Xenopus laevis oocytes

Mechanisms of H⁺ transport across the plasma cell membrane of prophase-arrested oocytes of Xenopus laevis were investigated by testing the effect of ion substitutions and inhibitors on cytoplasmic pH (pH_i), membrane potential (V _m) and membrane resistance (R _m). During superfusion with control solution of pH=7.4, pH_i was 7.49±0.12 (n=15), V _m was –61.9±7.8 mV (n=34) (cytoplasm negative), and R _m was 2.9±1.5 M (n=19). These data confirm that H⁺ ions are not distributed at electrochemical equilibrium. By following pH_i during recovery of the oocytes from an acid load (20 mmol/l NH₄Cl) in the presence and absence of extracellular Na⁺ or amiloride (1 mmol/l), a Na/H exchanger was identified. On the basis of the known Na⁺ gradient across the cell membrane, this transporter could suffice to generate the observed H⁺ disequilibrium distribution. Utilizing blockers or ion-concentration-step experiments no evidence was obtained for an ATP-driven H⁺ pump or for passive acid/base transporters such as H⁺ conductances or Na⁺ (HCO ₃ ^– )₃ cotransport. The membrane depolarization observed in response to extracellular acidification appeared to result from a pH-dependent, Ba²⁺-inhibitable K⁺ 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.     

Effects of vasopressin on water and NaCl transport across the in vitro perfused medullary thick ascending limb of Henle's loop of mouse,rat, and rabbit kidneys

Direct tubular effects of arginine vasopressin (AVP) on water and NaCl transport across the medullary thick ascending limb of Henle (MAL) were examined by the in vitro perfusion of isolated nephron fragments of mice, rats, and rabbits. Osmotic water permeability of the MAL of mice and rats was low and remained unchanged with 2 mU/ml AVP added to the bath. A dose-dependent increase in transepithelial electrical potential difference (PD) with AVP was observed in the mouse MAL when the ambient medium was isotonic. A similar result was also obtained when 2×10^–4 mol/l dibutyryl adenosine 3,5-cyclic-monophosphate was added to the bath. In this preparation, AVP also caused an increase in the unidirectional Cl efflux from 323±45 to 398±61 pmoles·mm^–1 ·min^–1 (n=6,P<0.05). In contrast, under similar condition, we could not demonstrate any effect of AVP on PD, Cl efflux, or net Na flux in the rat MAL and on PD and Cl efflux in the rabbit MAL. Both PD and Cl efflux in the rat MAL were unaffected by AVP when the perfusate was made hypotonic. However, when the ambient medium was made hypertonic by adding NaCl and urea, a significant increase in PD was observed. In addition, we confirmed that AVP stimulated adenylate cyclase activity in the MAL as well as in the collecting tubule of mice and rats. We conclude that AVP stimulates Cl transport across the MAL of mice and rats by activating adenylate cyclase-cyclic AMP system. However, this effect of AVP may quantitatively vary among species.     

Intracellular pH regulation of human colonic crypt cells

In order to investigate the regulation of intracellular pH (pH_i) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37°C). In N-2-hydroxyethylpiperazine — N — 2 — ethanesulphonic — acid — (HEPES)-buffered Ringer solution (HBS) a baseline pH_i of 7.35±0.03 (mean ± SD; n=42) was found for human colonocytes and in HBS, NH₄Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na⁺/H⁺ exchanger. Switching from HBS to HCO ₃ ^– buffered solution (BBS) led to a transient intracellular acidification (7.29±0.09), followed by a recovery to a final resting pH_i of 7.43±0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (H₂DIDS)-sensitive HCO ₃ ^– -dependent mechanisms. The functional activity of an acid-extruding Na⁺/HCO ₃ ^– cotransporter in human colonocytes was observed in response to the reintroduction of Na⁺ into amiloride-containing Na⁺/Cl^–-free BBS. In addition, the mechanism leading to alkalinization (7.56±0.05) in Cl^–-free BBS was identified as Na⁺-dependent Cl^–/HCO ₃ ^– exchange, by its H₂DIDS sensitivity and the specific requirement for Cl^– and Na⁺. The intrinsic buffering capacity ( _i) of the human colonocytes was calculated from pH changes induced by sequential NH₄Cl-loading steps during blockage of acid/base transporters. With _i=80 mmol · l^–1 · pH unit^–1 for the pH interval ranging from 6.9 to 7.1 (n=8) the colonocytes exhibited a relatively high intrinsic buffering in comparison with other cell types. In conclusion, the freshly isolated human colonocytes express a Na⁺/H⁺ exchanger, a Na⁺/HCO ₃ ^– cotransporter and a Na⁺-dependent Cl^–/HCO ₃ ^– exchanger, all of them likely to be involved in the regulation of pH homeostasis in vivo in the presence of widely varying extracellular conditions. Maintenance of a stable pH_i of human colonocytes seems to be facilitated by a comparatively high _i at physiological pH values.     

Water handling in the rat jejunum: effects of acidification of the medium

The minute-by-minute net water movement (J _w) in the rat jejunum was studied in relation to the diffusive water (P _w) and mannitol (P _s) permeabilities with the following results, (a) J _w was a linear function of the applied hydrostatic and osmotic transepithelial gradients (hydrostatic permeability coefficient, P _hydr=0.052±0.011 cm s^–1; osmotic permeability coefficient, P _osm= 0.0069±0.0014 cm s^–1. (b) A fraction of this absorptive J _w (transport-associated J _w, J _wt=0.086±0.024 l min^–1 cm^–2) was independent of the presence of any osmotic, hydrostatic or chemical gradient. (c) In the absence of Na⁺, J _wt was not significantly different from zero and there was an increase in P _hydr but no change in P _osm. (d) In the presence of a hydrostatic gradient (10 cm H₂O, mucosal side), acidification of the medium (95% CO₂ bubbling, pH 6.2) simultaneously and reversibly increased J _w and decreased P _w. (e) When an osmotic gradient was present (40 mM polyethyleneglycol on the serosal side) a net increase in J _w was observed. CO₂ bubbling in these conditions reversibly reduced J _w while increasing P _s. (f) These effects were not observed when the serosal or mucosal pH was reduced in the presence of a nonpermeant buffer (HEPES/TRIS; MES/TRIS). If we accept that P _s is a good marker of paracellular movements and that P _w mainly reflects transcellular water movements, we may conclude that acidification of the medium, in the presence of bicarbonate, modifies both paracellular and transcellular routes. The experimental evidence indicates that an increase in proton concentration opens the paracellular pathway and probably has a blocking effect on a transcellular route.     

Acid base status in unanesthetized,unrestrained guinea pigs

Acid-base status of arterial blood was measured in chronically cannulated, unanesthetized, unrestrained guinea pigs. Normal values were: pH=7.444±0.032,PaCO₂=35.7±4.4; HCO ₃ ^– =24.4±2.8; BE=+0.4±2.1 (n=69) andPaO₂=91.9±7.3 (n=25) (Values are mean±S.D.).Induction of light anesthesia with thiopentone caused a respiratory depression (decrease inPaO₂) accompanied by respiratory acidosis (increase inPaCO₂ and decrease in pH) and a development of slight metabolic acidosis (decrease in base excess and standard bicarbonate). Acid base parameters of guinea pigs are compared to those obtained from rats under identical experimental conditions.     

Electrical properties of cultured renal tubular cells (OK) grown in confluent monolayers

OK cells grown to confluent monolayers were investigated by microelectrode techniques and microinjection. Cell membrane potential difference (PD_m) in bi-carbonate-free solution is –61.8±0.6 mV (n=208), cell membrane resistance (R_m) amounts to 1.4±0.2k · cm² (n=8). The apparent transference number for potassium (t_K ⁺) is 71±3% (n=28) and can be reduced by 3 mmol/l BaCl₂ to 7.5±4.0%; (n=8). In the presence of extracellular CO₂ and HCO ₃ ^– (pH 7.4) the cells acidify by 0.34±0.05 pH units (n=12). This leads to a depolarization of PD_m by 8.4±1.8 mV (n=8), an increase in R_m by 49±10% (n= 10), and a reduction of K⁺-conductance to 63±5% (n= 13). Intracellular acidification by the NH₄Cl-prepulse technique also inhibits K⁺-conductance and depolarizes the membrane. Recovery from an intracellular acid load is reflected by cell membrane repolarization. This recovery can be inhibited by amiloride (10^–3 mol/l). Na⁺- and Cl^–-conductances could not be detected.The transepithelial resistance (R _te) of OK cell monolayers 1 day after plating is 41±6 ·cm² and decreases with time after plating. Intercellular communication (electrical or dye coupling) was not observed.Conclusions: 1. The membrane potential of OK cells is largely determined by a pH-sensitive, barium-blockable K⁺-conductance. 2. Amiloride-blockable Na⁺/H⁺-exchange is reflected by membrane potential changes via this K⁺-conductance. 3. Monolayers of OK cells are electrically leaky.Parts of this study were presented at the 66th meeting of the Deutsche Physiologische Gesellschaft, Würzburg, September 1988 [Pflügers Arch 412 (Suppl 1):R55].     

Changes in “Active” and “Inactive” renin in the juxtaglomerular apparatuses of rat nephrons and plasma induced by different salt intake

The juxtaglomerular apparatuses (JGA) of deep and superficial nephrons were isolated by microbiopsy or by microdissection. Inactive renin content was determined by acidification of JGA or plasma to pH 3.0.In rats with low salt intake the renin content of superficial JGA was 13.4±3.0 ng AI/JGA/h before and 20.4±3.4 ng AI/JGA/h (n=9,P<0.05) after acidification. The corresponding values for deep JGA were 9.1±1.2 ng AI/JGA/h and 12.7±2.7 ng AI/JGA/h (n=9,P<0.01). The plasma renin concentration was 54.1±15.0 ng AI/ml/h before and 56.0±10.6 ng AI/JGA/h (n=7, N.S.) after acidification.In rats with a normal salt intake the superficial renin JGA renin content was 11.6±2.3 ng AI/JGA/h before and 11.0 ±2.7 ng AI/JGA/h (n=9, N.S.) after acidification. The renin content of deep JGA was 4.6±0.6 ng AI/JGA/h before and 8.6±3.1 ng AI/JGA/h (n=9,P<0.005) after acidification. Plasma renin concentration was 34.5±4.7 ng AI/ml/h and did not change after acidification.In rats with a high salt intake superficial JGA content was 6.8±1.7 ng AI/JGA/h before and 8.4±2.1 ng AI/JGA/h (n=9, N.S.) after acidification. The corresponding values for deep JGA were 5.7±1.6 ng AI/JGA/h and 6.9±1.6 ng AI/JGA/h (n=9, N.S.) respectively. Plasma renin concentration was 13.1±1.1 ng AI/ml/h and this to 21.8±2.9 ng AI/ml/h (n=8,P<0.01) after acidification.These results suggest that although the synthesis of active and inactive renin is linked, the secretion of the two forms may be independent.     

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca²⁺-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3±0.3 pS (n=15) under symmetrical inger conditions. The channel excluded anions (P _Na/P _Cl=18 whereP _Na/P _Cl denotes the relative permeability of Na to Cl); it was equally permeant to the Na⁺ and K⁺ ions and exhibited a low permeability toN-methyl-D-glucamine and Ba²⁺ or Ca²⁺. Channel opening required a free Ca²⁺ concentration of about 10^–6 mol/l on the internal side of the membrane and the open probability (P _o) was maximal at 10^–3 mol/l (P _o=0.72±0.06,n=12). Adenosine 5mono-, tri- and di-phosphate reducedP _o to 29±14 (n=5), 42±10 (n=8) and 51±12 (n=5) % of controlP _o, respectively, when they were added at a concentration of 10^–3 mol/l to the internal side. The channel was partially blocked by flufenamic acid (10^–4 mol/l) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10^–5 mol/l). This type of channel, together with Ca²⁺-activated K⁺ channels, might participate in the control of membrane potential and modulate the motility of OHCs.