Ira Kurtz

Molecular Cloning, Chromosomal Localization, Tissue Distribution, and Functional Expression of the Human Pancreatic Sodium Bicarbonate Cotransporter

We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem. 272, 19111–19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an ∼7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5′ region of kNBC detected an ∼7.6-kilobase transcript only in the kidney. In situhybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescentin situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney.

Toxic Alcohol Ingestions: Clinical Features, Diagnosis, and Management

Alcohol-related intoxications, including methanol, ethylene glycol, diethylene glycol, and propylene glycol, and alcoholic ketoacidosis can present with a high anion gap metabolic acidosis and increased serum osmolal gap, whereas isopropanol intoxication presents with hyperosmolality alone. The effects of these substances, except for isopropanol and possibly alcoholic ketoacidosis, are due to their metabolites, which can cause metabolic acidosis and cellular dysfunction. Accumulation of the alcohols in the blood can cause an increment in the osmolality, and accumulation of their metabolites can cause an increase in the anion gap and a decrease in serum bicarbonate concentration. The presence of both laboratory abnormalities concurrently is an important diagnostic clue, although either can be absent, depending on the time after exposure when blood is sampled. In addition to metabolic acidosis, acute renal failure and neurologic disease can occur in some of the intoxications. Dialysis to remove the unmetabolized alcohol and possibly the organic acid anion can be helpful in treatment of several of the alcohol-related intoxications. Administration of fomepizole or ethanol to inhibit alcohol dehydrogenase, a critical enzyme in metabolism of the alcohols, is beneficial in treatment of ethylene glycol and methanol intoxication and possibly diethylene glycol and propylene glycol intoxication. Given the potentially high morbidity and mortality of these intoxications, it is important for the clinician to have a high degree of suspicion for these disorders in cases of high anion gap metabolic acidosis, acute renal failure, or unexplained neurologic disease so that treatment can be initiated early.

Cloning, Tissue Distribution, Genomic Organization, and Functional Characterization of NBC3, a New Member of the Sodium Bicarbonate Cotransporter Family

Previous functional studies have demonstrated that muscle intracellular pH regulation is mediated by sodium-coupled bicarbonate transport, Na+/H+ exchange, and Cl−/bicarbonate exchange. We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3 encodes a 1214-residue polypeptide with 12 putative membrane-spanning domains. The ∼ 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans ∼80 kb and is composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA inXenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.

Effect of Pistachio Nuts on Serum Lipid Levels in Patients with Moderate Hypercholesterolemia

BACKGROUND Elevated serum cholesterol levels play an important role in the development of coronary artery disease. Previous studies have suggested that nut consumption benefits lipid profile. Pistachio nuts are widely available, inexpensive and frequently consumed by the general population. OBJECTIVE To determine whether substituting 20% of the daily caloric intake in the form of pistachio nuts will improve the lipid profiles of humans with primary, moderate hypercholesterolemia. DESIGN Controlled, randomized crossover design. SETTING Outpatient dietary modification, counseling and blood analysis. PATIENTS Ten patients with moderate hypercholesterolemia. INTERVENTION Three weeks of dietary modification with 20% caloric intake from pistachio nuts. MEASUREMENTS Body weight, blood pressure, total cholesterol, LDL, HDL, and triglycerides were monitored. Lipid profiles were analyzed prior to, during and after dietary modification. RESULTS After three weeks, there was a decrease in total cholesterol (p<0.04), an increase in HDL (p<0.09), a decrease in the total cholesterol/HDL ratio (p<0.01) and a decrease in the LDL/HDL ratio (p<0.02). Triglycerides and LDL levels decreased, but not significantly. Body weight and blood pressure remained constant throughout the study. CONCLUSIONS Results suggest that eating pistachio nuts instead of other dietary fat calories can improve lipid profiles, thereby decreasing coronary risk. Further studies will be required to confirm these results and to determine the mechanism of this effect.

Blindness and auditory impairment caused by loss of the sodium bicarbonate cotransporter NBC3

Normal sensory transduction requires the efficient disposal of acid (H+) generated by neuronal and sensory receptor activity. Multiple highly sensitive transport mechanisms have evolved in prokaryotic and eukaryotic organisms to maintain acidity within strict limits. It is currently assumed that the multiplicity of these processes provides a biological robustness. Here we report that the visual and auditory systems have a specific requirement for H+ disposal mediated by the sodium bicarbonate cotransporter NBC3 (refs. 7,8). Mice lacking NBC3 develop blindness and auditory impairment because of degeneration of sensory receptors in the eye and inner ear as in Usher syndrome. Our results indicate that in certain sensory organs, in which the requirement to transduce specific environmental signals with speed, sensitivity and reliability is paramount, the choice of the H+ disposal mechanism used is limited.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell‐type dependent

1 The pancreatic variant of the sodium bicarbonate cotransporter, pNBC1, mediates basolateral bicarbonate influx in the exocrine pancreas by coupling the transport of bicarbonate to that of sodium, with a 2 HCO3−:1 Na+ stoichiometry. The kidney variant, kNBC1, mediates basolateral bicarbonate efflux in the proximal tubule by coupling the transport of 3 HCO3− to 1 Na+. The molecular basis underlying the different stoichiometries is not known. 2 pNBC1 and kNBC1 are 93 % identical to each other with 41 N‐terminal amino acids of kNBC1 replaced by 85 distinct amino acids in pNBC1. In this study we tested the hypothesis that the differences in stoichiometry are related to the difference between the N‐termini of the two proteins. 3 Mouse renal proximal tubule and collecting duct cells, deficient in both pNBC1‐ and kNBC1‐mediated electrogenic sodium bicarbonate cotransport function were transfected with either pNBC1 or kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The stoichiometry was calculated from the reversal potential by measuring the current‐voltage relationships of the cotransporter at different Na+ concentration gradients. 4 Our data indicate that both kNBC1 and pNBC1 can exhibit either a 2:1 or 3:1 stoichiometry depending on the cell type in which each is expressed. In proximal tubule cells, both pNBC1 and kNBC1 exhibit a 3 HCO3−:1 Na+ stoichiometry, whereas in collecting duct cells, they have a 2:1 stoichiometry. These data argue against the hypothesis that the stoichiometric differences are related to the difference between the N‐termini of the two proteins. Moreover, the results suggest that as yet unidentified cellular factor(s) may modify the stoichiometry of these cotransporters.

Acid-base analysis : a critique of the Stewart and bicarbonate-centered approaches

When approaching the analysis of disorders of acid-base balance, physical chemists, physiologists, and clinicians, tend to focus on different aspects of the relevant phenomenology. The physical chemist focuses on a quantitative understanding of proton hydration and aqueous proton transfer reactions that alter the acidity of a given solution. The physiologist focuses on molecular, cellular, and whole organ transport processes that modulate the acidity of a given body fluid compartment. The clinician emphasizes the diagnosis, clinical causes, and most appropriate treatment of acid-base disturbances. Historically, two different conceptual frameworks have evolved among clinicians and physiologists for interpreting acid-base phenomena. The traditional or bicarbonate-centered framework relies quantitatively on the Henderson-Hasselbalch equation, whereas the Stewart or strong ion approach utilizes either the original Stewart equation or its simplified version derived by Constable. In this review, the concepts underlying the bicarbonate-centered and Stewart formulations are analyzed in detail, emphasizing the differences in how each approach characterizes acid-base phenomenology at the molecular level, tissue level, and in the clinical realm. A quantitative comparison of the equations that are currently used in the literature to calculate H(+) concentration ([H(+)]) is included to clear up some of the misconceptions that currently exist in this area. Our analysis demonstrates that while the principle of electroneutrality plays a central role in the strong ion formulation, electroneutrality mechanistically does not dictate a specific [H(+)], and the strong ion and bicarbonate-centered approaches are quantitatively identical even in the presence of nonbicarbonate buffers. Finally, our analysis indicates that the bicarbonate-centered approach utilizing the Henderson-Hasselbalch equation is a mechanistic formulation that reflects the underlying acid-base phenomenology.

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.

Regulation of pH During Amelogenesis

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation.

Regulation of the sodium bicarbonate cotransporter knbc1 function: role of Asp986, Asp988 and kNBC1‐carbonic anhydrase II binding

The HCO3− : Na+ cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (Erev) and thus the net direction of transport of these ions through the cotransporter. Previously, we showed that phosphorylation of kNBC1‐Ser982 in the carboxy‐terminus of kNBC1 (kNBC1‐Ct), by cAMP‐protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the cotransporter is electrostaticaly modulated. These results raise the possibility that phosphorylated kNBC1‐Ser982, or other nearby negatively charged residues shift the stoichiometry by blocking a bicarbonate‐binding site. In the current study, we examined the role of the negative charge on Ser982‐phosphate and three aspartate residues in a D986NDD custer in altering the stoichiometry of kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in an Ussing chamber for electrophysiological studies. Enhanced green fluorescence protein (EGFP)‐tagged mutant constructs expressed in the same cells were used to determine the phosphorylation status of kNBC1‐Ser982. The data indicate that both kNBC1‐Asp986 and kNBC1‐Asp988, but not kNBC1‐Asp989, are required for the phosphorylation‐induced shift in stoichiometry. A homologous motif (D887ADD) in the carboxy‐terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). In isothermal titration calorimetry experiments, CAII was found to bind to kNBC1‐Ct with a KD of 160 ± 10 nm. Acetazolamide inhibited the short‐circuit current through the cotransporter by 65 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. Acetazolamide did not affect the cotransport stoichiometry or the ability of 8‐Br‐cAMP to shift the stoichiometry. Although CAII does not affect the transport stoichiometry, it may play an important role in enhancing the flux through the transporter when kNBC1‐Ser982 is unphosphorylated.