pH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells.

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.

Role of NBCe1 and AE2 in secretory ameloblasts.

The H(+)/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

Basolateral membrane Na+/H+ antiport, Na+/base cotransport, and Na+-independent Cl-/base exchange in the rabbit S3 proximal tubule.

The basolateral membrane Na+ and Cl(-)-dependent acid-base transport processes were studied in the isolated perfused rabbit S3 proximal straight tubule. Intracellular pH (pHi) was measured with 2'7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a microfluorometer coupled to the tubule perfusion apparatus. Reduction of basolateral HCO3- from 25 to 5 mM caused pHi to decrease at a rate of 0.81 pH/min. Approximately 50% of this rate was Na+-dependent, 30% Cl(-)-dependent and 20% Na+ and Cl(-)-independent. Two basolateral Na+-dependent acid base transport pathways were detected: (a) an amiloride-sensitive Na+/H+ antiporter and (b) a stilbene-sensitive Na+/base cotransporter. No evidence was found for a Na+-dependent Cl-/base exchanger. The Cl(-)-dependent component of basolateral base efflux was mediated by a stilbene-sensitive Na+-independent Cl-/base exchange pathway. The results suggest that the acid base transport pathways of the basolateral membrane of the S3 proximal tubule differ from more proximal nephron segments.

Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.

Functional characterization of three intercalated cell subtypes in the rabbit outer cortical collecting duct.

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

Induction of hypertrophy in cultured proximal tubule cells by extracellular NH4Cl.

Ammonia production increases in several models of renal hypertrophy in vivo. The present study was designed to determine whether ammonia can directly modulate the growth of renal cells in the absence of a change in extracellular acidity. In serum-free media NH4Cl (0-20 mM) caused JTC cells and a primary culture of rabbit proximal tubule cells to hypertrophy (increase in cell protein content) in a dose-dependent fashion without a change in DNA synthesis. Studies in JTC cells revealed that the cell protein content increased as a result of both an increase in protein synthesis and a decrease in protein degradation. Total cell RNA content and ribosome number increased after NH4Cl exposure and the cell content of the lysosomal enzymes cathepsin B and L decreased. Inhibition of the Na+/H+ antiporter with amiloride did not prevent the hypertrophic response induced by NH4Cl. The results indicate that ammonia is an important modulator of renal cell growth and that hypertrophy can occur in the absence of functioning Na+/H+ antiport activity.

Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.

Spontaneous luminal disequilibrium pH in S3 proximal tubules. Role in ammonia and bicarbonate transport.

We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.

Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family.

We report the cloning, characterization and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4, from human heart. NBC4 maps to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. NBC4 encodes a 1074-residue polypeptide with 12 putative membrane-spanning domains. Unlike other members of the NBC family, NBC4 has a unique glycine-rich region (amino acids 438-485). In addition, NBC4 lacks the lysine-rich C-terminus of NBC1 with which it is most homologous. The first of two putative stilbene binding motifs (K(M/L)(X)K) is lacking in NBC4 (amino acids 655-658). The approximately 6 kb NBC4 transcript is moderately expressed in heart, with the highest expression in liver, testes and spleen.

Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3.

Several electrogenic sodium bicarbonate cotransporters have been cloned from different human organs. In the renal proximal tubule, the electrogenic sodium bicarbonate cotransporter kNBC1 (1035aa) mediates the majority of basolateral sodium bicarbonate absorption. In pancreatic ducts, the electrogenic sodium bicarbonate cotransporter pNBC1 (1079aa) mediates basolateral sodium bicarbonate influx. hNBC1 (hhNBC), cloned from human heart, is identical to pNBC1 at the amino acid level. We have demonstrated that kNBC1 and pNBC1 are highly homologous proteins that have different N-termini. In kNBC1, 41 amino acids replace the initial 85 amino acids of pNBC1. Whether these proteins are coded by one or more genes is unknown. In order to determine the genetic basis for these transcripts, we first characterized the genomic organization of the NBC1 gene (SLC4A4). NBC1 spans approximately 450 kilobases containing 26 exons that are flanked by typical splice donor and acceptor sequences at the intron-exon boundaries. Exon 1 is specific for the pNBC1 transcript. The first alternative exon of the hNBC1 transcript, containing the 5'-untranslated region, is derived from the last 43 nucleotides of intron 1 in the NBC1 gene coupled to exon 2. kNBC1 is transcribed from an alternative promoter in intron 3. In the first alternative exon of kNBC1, the last 313 nucleotides of intron 3 are coupled to exon 4, which is common to pNBC1 and hNBC1. The major transcription initiation site in kNBC1 is located 192 nucleotides upstream from the translation initiation codon. A minor start site is located 182 nucleotides upstream from the translation initiation codon. Structural analysis of the proximal kNBC1 promoter revealed an atypical TATA sequence (-33) and several potentially important transcription factor binding sites. Functional studies showed that the 5'-flanking region of the alternative kNBC1 promoter (-159 to+43) is sufficient for promoter activity. This work is the first demonstration that the three N-terminal transcripts of the human electrogenic sodium bicarbonate cotransporter NBC1 are encoded by the SLC4A4 gene. Furthermore, knowledge of the genomic organization and alternative promoter usage in the NBC1 gene provides a molecular basis for understanding disorders involving electrogenic sodium bicarbonate cotransporters and facilitates the elucidation of transcriptional control of NBC1 expression.

Disorders of distal nephron function.

In this review, the distal nephron is considered to be that portion of the renal tubule commencing with the thick ascending limb of the loop of Henle and ending with the papillary collecting duct. The collecting duct, including its subdivisions in the cortex and medulla, originates from a different embryologic anlage than more proximal nephron segments, which may explain its morphologic and functional dissimilarities from the thick ascending limb and the distal convoluted tubule. This review summarizes selected aspects of the physiology of the distal nephron, with particular emphasis on the physiology of distal nephron transport of sodium, potassium, chloride and hydrogen ion. The pathophysiologic features of the following disorders of distal nephron function are reviewed: (1) pseudohypoaldosteronism, a heterogenous group of disorders in which the signs and symptoms are suggestive of aldosterone deficiency, but in which aldosterone levels are supernormal and administration of exogenous mineralocorticoid is not ameliorative; (2) pseudohyperaldosteronism (Liddle syndrome), a familial disorder in which the clinical manifestations closely resemble those resulting from an aldosterone-producing adenoma of the adrenal gland (primary aldosteronism), but in which the measured rate of aldosterone secretion and excretion is greatly subnormal; (3) Bartter syndrome and related syndromes of renal potassium wasting; (4) type 1 renal tubular acidosis (classic, distal); (5) type 4 renal tubular acidosis (hyperkalemic). Reference citations are generally to articles reporting recent advances in these areas and to review articles that contain comprehensive bibliographies.

Use of base in the treatment of severe acidemic states.

Severe acidemia (blood pH < 7.1 to 7.2) suppresses myocardial contractility, predisposes to cardiac arrhythmias, causes venoconstriction, and can decrease total peripheral vascular resistance and blood pressure, reduce hepatic blood flow, and impair oxygen delivery. These alterations in organ function can contribute to increased morbidity and mortality. Although it seemed logical to administer sodium bicarbonate to attenuate acidemia and therefore lessen the impact on cardiac function, the routine use of bicarbonate in the treatment of the most common causes of severe acidemia, diabetic ketoacidosis, lactic acidosis, and cardiac arrest, has been an issue of great controversy. Studies of animals and patients with these disorders have reported conflicting data on the benefits of bicarbonate, showing both beneficial and detrimental effects. Alternative alkalinizing agents, tris-hydroxymethyl aminomethane and Carbicarb, have shown some promise in studies of animals and humans, and reevaluation of these buffers in the treatment of severe acidemic states seems warranted. The potential value of base therapy in the treatment of severe acidemia remains an important issue, and further studies are required to determine which patients should be administered base therapy and what base should be used.

Freedom through a single switch: coping and communicating with artificial ventilation.

Challenging clients often stretch the limits of the imagination in terms of developing creative technologies to allow access to computers, communication devices and environmental controls. This paper provides a description of an innovative system that enables clients with motor neurone disease/amyotrophic lateral sclerosis (MND/ALS), on full ventilatory support and virtually no movement, to communicate and manipulate their environments, through the use of a myoelectrically controlled switch.

Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase.

In the male reproductive tract, the epididymis plays an important role in mediating transepithelial bicarbonate transport and luminal acidification. In the proximal vas deferens, a significant component of luminal acidification is Na+-independent, and mediated by specific cells that possess apical vacuolar proton pumps. In contrast, luminal acidification in the cauda epididymidis is an Na+-dependent process. The specific apical Na+-dependent H+/base transport process(es) responsible for luminal acidification have not been identified. A potential clue as to the identity of these apical Na+-dependent H+/base transporter(s) is provided by similarities between the transport properties of the epididymis and the mammalian nephron. Specifically, the H+/base transport properties of caput epididymidis resemble the mammalian renal proximal tubule, whereas the distal epididymis and vas deferens have characteristics in common with renal collecting duct intercalated cells. Given the known expression of the Na+/H+ antiporter, NHE3, in the proximal tubule, and of the electroneutral sodium bicarbonate cotransporter, NBC3, in renal intercalated cells, we determined the localization of NHE3 and NBC3 in various regions of rat epididymis. NBC3 was highly expressed on the apical membrane of apical (narrow) cells in caput epididymidis, and light (clear) cells in corpus and cauda epididymidis. The number of cells expressing apical NBC3 was highest in cauda epididymidis. The localization of NBC3 in the epididymis was identical to the vacuolar H+-ATPase. The results indicate that colocalization of NBC3 and the vacuolar H+-ATPase is not restricted to kidney intercalated cells. Moreover, the close association of the two transporters appears to be a more generalized phenomenon in cells that express high levels of vacuolar H+-ATPase. Unlike NBC3, NHE3 was most highly expressed on the apical membrane of all epithelial cells in caput epididymidis, with less expression in the corpus, and no expression in the cauda. These results suggest that apical NBC3 and NHE3 potentially play an important role in mediating luminal H+/base transport in epididymis.

Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.

New paradigms on the transport functions of maturation-stage ameloblasts.

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

Role of ammonia in the induction of renal hypertrophy.

Recent experiments from our laboratory have documented the importance of ammonia as a modulator of renal cell growth in vitro. Ammonia induces renal hypertrophy by increasing the rate of protein synthesis and decreasing the rate of protein degradation. These results have led to the hypothesis that an increase in renal ammoniagenesis contributes to renal growth in several seemingly unrelated clinical disorders. In chronic hypokalemia and metabolic acidosis, mitochondrial ammoniagenesis is stimulated directly. During protein loading, uninephrectomy, and diabetes mellitus, renal ammoniagenesis may be stimulated by an increase in single-nephron glomerular filtration rate (SNGFR).