Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.     

The effect of dexamethasone on urinary acidification.

Although it is well recognized that mineralocorticoids enhance renal acid excretion, the effect of glucocorticoids on renal acidification is unclear. Oral administration of dexamethasone to six healthy volunteers for 1 week at a daily dose of 4.5 mg was associated with mild respiratory alkalosis and a small but statistically significant increase in baseline urine pH. However, neither the ability to lower urine pH nor to excrete titratable acid and ammonium after NH4Cl acid-loading was altered. Administration of a single intravenous dose of dexamethasone sodium phosphate (7.5 mg) was associated with a significant rise in urine pH and potassium excretion and decreased titratable acid, ammonium , and phosphorus excretion in the absence of changes in blood acid-base status, creatinine clearance, or urine flow.     

Effect of NH4Cl acidosis on the function of renin-angiotensin-aldosterone system in newborn infants.

The present study was undertaken to assess the influence of acute metabolic acidosis on the activity of renin-angiotensin-aldosterone system and renal function in a group of seven one-week-old neonates with mean birth weight of 2164 g (range: 1300-3750 g) and mean gestational age of 34 weeks (range: 28-40 weeks) undergoing oral NH4Cl load. NH4Cl was given in a dose of 2.8 mEq/kg to evaluate renal acidification. Prior to and following NH4Cl administration blood acid-base parameters, plasma urinary electrolytes, creatinine and aldosterone concentration as well as plasma renin activity, glomerular filtration rate, urine flow rate and net acid secretion were measured. NH4Cl administration significantly depressed blood pH (P < 0.05), total CO2 content (P < 0.01) and base excess (P < 0.01) and resulted in a significant elevation of plasma potassium concentration (P < 0.05). Furthermore, NH4Cl ingestion significantly increased urine flow rate, sodium, chloride and net acid excretion. In response to NH4Cl acidosis no consistent change in plasma renin activity and plasma aldosterone concentration could be detected. There was, however, an about 50% increase in urinary aldosterone excretion from the control value of 4.1 +/- 1.2 micrograms/day to 6.8 +/- 2.3 micrograms/day (P < 0.05) after NH4Cl administration. These data suggest that the responsiveness of neonatal adrenals to stimulation by metabolic acidosis is blunted, acidosis therefore, may play a minor role in the neonatal hyperfunction of renin-angiotensin-aldosterone system.     

Cutaneous and renal responses to intravascular infusions of HCl and NH4Cl in the bullfrog (Rana catesbiana)

This study examined the ability of bullfrogs to correct a non-respiratory acidosis by renal and cutaneous mechanisms. Acidosis was induced by intravascular infusions of HCl (3 mmole/kg) or NH4Cl (4 mmole/kg). The acid load was removed primarily by increased renal excretion of NH4+, while urine pH and titratable buffer acid excretion changed little. Acid loading resulted in an increase in cutaneous permeability, shown by large ion losses and elevated water uptake across the skin. It is concluded that infused mineral acids were immediately buffered by the extracellular fluids, moved rapidly into the intracellular fluid compartment, and only later were slowly cleared.     

Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR.

The aim of this study was to investigate the effect of NH4+ on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH4+ transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pHi) under standard conditions was 7.24 +/- 0.04 (n = 46). Exposure to NH4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pHi value. The NH4(+)-induced acidification followed saturation kinetics up to 20 mM NH4Cl (delta pHmax = 0.2 pHunits). Half-maximal acidification was observed at 0.6 mmol/l. The intracellular acidification due to NH4Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter. The effect of bumetanide was dose-dependent and a Ki value of 8.10(-7) M was calculated. NH4+ influx via K+ channels or the (Na+ + K+)ATPase could not be detected. pHi recovery to the initial value was caused mainly by amiloride-sensitive Na+/H+ exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (beta i) in TALH SVE.1 cells of 26 +/- 3 mM x pH-1 (pHi = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal 'functional buffer capability' between pHout 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na+/K+/2Cl- cotransporter is the main site of NH4+ entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH4+ influx into the cell.     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Role of intracellular pH in secretion from adrenal medulla chromaffin cells

The role of intracellular pH in stimulus-secretion coupling was investigated in cultured bovine adrenal medullary chromaffin cells. NH4Cl (1-25 mM) did not affect basal catecholamine or ATP release but markedly inhibited nicotine- or high K+-induced release by up to 60%. The inhibition had a rapid onset (less than 1 min) and was maximal at about 5 mM NH4Cl. The effect of NH4Cl was largely sustained over 20 min and was reversed upon NH4Cl removal. Sodium propionate did not affect secretion but partially reversed the inhibition by NH4Cl in a concentration-dependent manner. Methylamine (10 mM) produced a similar, but slower, inhibition than NH4Cl. Monensin (1-10 microM) inhibited catecholamine secretion by 30-60%, and its effect was reduced in the presence of NH4Cl. Using the fluorescent Ca2+ probe Fura-2, we found that the increase of [Ca2+]i following stimulation was not altered by concentrations of NH4Cl which inhibited secretion maximally. Measurement of cytosolic pH (pHi) with the fluorescent probe 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) revealed an alkalinization by NH4Cl (2.5-25 mM) of 0.1-0.23 pH units and acidification by sodium propionate (10-20 mM) of 0.2-0.25 pH units, with intermediate combined effects. Monensin (1 microM) caused a cytosolic acidification of 0.26 pH units. All pHi changes were partly recovered in 15 min. Fluorescence quenching measurements using the weakly basic fluorescent probe acridine orange indicated the accumulation of the probe into acidic compartments, presumably the chromaffin granules, which was strongly reduced by both NH4Cl and monensin. From these findings we conclude that the pH of the chromaffin granule modulates secretion by affecting some step in the secretory process unrelated to the rise in [Ca2+]i.     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.     

The role of the adrenal gland in control of H+ and NH4+ excretion by toad urinary bladder

The urinary bladder of Bufo marinus has been shown to excrete H+ and NH4+ and this excretion is increased by metabolic acidosis. The involvement of the adrenal gland and its steroid secretions in the adaptation for increased acid and ammonia excretion by the bladder was tested during the course of this study. Groups of toads were adrenalectomized and maintained in chronic NH4Cl-induced acidosis. Three other groups of toads were adrenalectomized and put in acidosis but repleted with 2.5 mg/day of either cortisol (CT), dexamethasone (Dexa), or deoxycorticosterone acetate (DOCA). All control groups were sham-operated. The bladders were excised after 3 days and mounted between 2-ml Lucite chambers. Net H+ and NH4+ fluxes into the mucosal media were measured and reported in units of nanomoles per 100 mg bladder per minute. In control acidotic toads H+ excretion was 20.1 +/- 2.0 and the adrenalectomized nonreplete group H+ excretion was 14.2 +/- 1.87 (P less than 0.04). For the same groups NH4+ excretion was 2.90 +/- 0.26 for the controls and 1.38 +/- 0.19 for the adrenalectomized (P less than 0.001). The H+ excretion in CT-, Dexa-, and DOCA-repleted toads was not significantly different from the control group. NH4+ excretion, however, showed a 55% decrease (P less than 0.001) in the CT group, and a 45% decrease (P less than 0.05) in the Dexa group. The NH4+ excretion in the DOCA repleted group was significantly different from the control group. Therefore, we conclude that the adrenal gland plays a role in the adaptive increase of H+ and NH4+ excretion by the urinary bladder in acidosis through the secretion of steroid hormones. The increase in NH4+ excretion appears to be a mineralocorticoid-stimulated process. We were not able to determine in this study if the steroid hormones had an exacting regulatory role or one of a permissive role over H+ and NH4+ excretion in the toad urinary bladder.     

Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.     

Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Ca2+ influx and stimulation of protein synthesis in sea urchin eggs

Acid release, Ca2+ influx and stimulation of protein synthesis were investigated with sea urchin eggs submitted to an excess of KCl, to NH4Cl, and to a combination of both. KCl, though unable to promote any acid release, triggers a large 45Ca uptake by eggs and slightly stimulates protein synthesis, provided that external Ca2+ is present. NH4Cl, which induces an intracellular pH increase, triggers a late and small 45Ca uptake but highly stimulates protein synthesis. The combined use of NH4Cl + KCl allows a large 45Ca uptake to occur but the level of protein synthesis is similar to that obtained with NH4Cl alone and is identical whether external Ca2+ is present or not. In contrast to previous works, our results show that the large stimulation of protein synthesis triggered by an intracellular pH increase, as after NH4Cl activation, cannot be enhanced by a Ca2+ influx. This suggests that the Ca2+ influx occurring after fertilization has only a minimal effect on the overall stimulation of protein synthesis.     

The effect of the anions, phosphate and sulphate, on normal rates of excretion of potassium in dogs.

Small doses of (NH4)2HPO4 or KH2PO4 by stomach tube caused increase in plasma PO4 and PO4 excretion. Above a threshold of 0-8 mmol. 1(-1), increase of plasma PO4 by 0-5 mmol. 1(-1) caused PO4 excretion to increase by about 35 mumol. min.-1 After KH2PO4 this relationship was not altered by the concurrent increases in plasma K and K excretion. After doses of (NH4)2SO4 or K2SO4, excretion of SO4 was similarly related to plasma SO4 and was independent of plasma K and K excretion. An effect of PO4 on K excretion was observed after doses of (NH4)2HPO4, when increased excretion of PO4 was accompanied by increased excretion of K without change in plasma K. There was also increased excretion of NH4 and a small increase in Na excretion. The changes were similar to those produced by (NH4)2SO4 [O'Connor and Summerill, 1976]. KH2PO4 and K2SO4 produced increase in plasma K and increased excretion of K not significantly different from the changes produced by KCl or KHCO3 [Baylis and O'Connor, 1976]. After KH2PO2 or K2SO4, the urinary anion was PO4 or SO4, instead of Cl and HCO3. Any effect of anions on K excretion was much less than the effect of increase in plasma K. At low rates of excretion of K, increased urinary excretion of impermeant anion can determine increased excretion of K. However, the effect of anion is small in comparison with the effect of increase in plasma K.     

The relationship between glutamate deamination and gluconeogenesis in kidney.

The effect of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], was tested on NH3 formation via the purine nucleotide cycle and glutamate dehydrogenase (EC 1.4.1.2). NH3 excretion in rats increased 70-fold after 48 h of NH4Cl feeding, from 12.2 +/- 4.5 to 862 +/- 190 mumol/mg of creatinine. At 4 h after a single intraperitoneal injection of 3-mercaptopicolinate into NH4Cl-fed rats, NH3 excretion was inhibited by 93%. Kidneys of NH4Cl-fed plus 3-mercaptopicolinate-treated rats, compared with those of NH4Cl-fed rats, showed a 3.5-fold increase in the content of IMP, 5-fold increase in adenylosuccinate, 4-fold increase in aspartate, and a 30% increase in AMP. 3-Mercaptopicolinate completely inhibited NH3 and glucose formation from glutamate in tubules from acidotic rats and NH3 formation from aspartate in kidney perfusion experiments. When transamination in tubules was prevented by 2-amino-4-methoxy-trans-but-3-enoic acid, formation of glucose, but not of NH3, from glutamate was inhibited. 3-Mercaptopicolinate completely inhibited NH3 formation from aspartate in the presence of the aminotransferase inhibitor in kidney tubules. The data show that NH3 can be formed via glutamate dehydrogenase and the purine nucleotide cycle at significant and approximately equal rates. 3-Mercaptopicolinate has no direct effect on NH3 formation via glutamate dehydrogenase, but inhibits that via the purine nucleotide cycle. We conclude that gluconeogenesis is not regulatory for NH3 formation in kidney.     

African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].