Na+/H+ exchangers in the brush-border (luminal, apical) membrane of renal proximal tubules are responsible for active, transcellular reabsorption of NaHCO3 and NaCl. Although well characterized kinetically, the protein that mediates Na+/H+ exchange in the renal brush border has not been identified. Several Na+/H+ exchanger genes, including NHE1, NHE2, NHE3, and NHE4, are expressed in the kidney. To identify the NHE3 gene product and to determine its cellular and subcellular localization in the rabbit kidney, an NHE3-isoform-specific antibody was prepared. Guinea pigs were immunized with purified fusion protein containing the carboxy-terminal 40 amino acids of NHE3 (fpNHE3-C40). After affinity purification, immune sera demonstrated specific reactivity to the NHE3 sequence within the fusion protein as well as to an 80-kDa polypeptide expressed in NHE3-transfected LAP1 cells. Western blot analysis showed that anti-fpNHE3-C40 specifically reacted with an 80-kDa protein that is relatively enriched in renal brush-border membrane compared with basolateral membrane. Immunocytochemical studies confirmed that the Na+/H+ exchanger isoform NHE3 is expressed along the microvillar membrane of the brush border of proximal tubule cells in the rabbit kidney.

Previous immunochemical studies have shown that NHE3 is an apical Na+/H+ exchanger in some renal epithelia. The purpose of the present study was to develop high-affinity, isoform-specific monoclonal antibodies (MAbs) that would be useful for carrying out high-resolution immunocytochemical studies of NHE3 in the adult and neonatal mammalian kidney. Three MAbs were developed to a fusion protein containing amino acids 702-832 of rabbit NHE3. Specificity was established by immunoblotting membranes from NHE-deficient LAP cells that had been transfected with either NHE1,-2, -3, or -4. With the use of high-resolution immunocytochemical techniques, NHE3 was found in vesicles in the apical cytoplasm of proximal tubule cells, as well as in the apical plasma membrane of the proximal tubule, and in both the thin and thick limbs of the loop of Henle. When localized in the 1-day-old rat kidney, NHE3 was first detected in the late stages of the S-shaped body. In later stages of nephron development, the pattern of NHE3 staining was similar to that seen in the adult. This study demonstrates 1) the specificity of three MAbs for Na+/H+ exchanger isoform NHE3; 2) NHE3 is present in an intracellular vesicular compartment in cells of the proximal tubule, consistent with possible regulation by membrane recycling; and 3) NHE3 is expressed on the apical membrane in early stages of the developing nephron.

1. The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch-clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo. 2. Neurones were exposed to changes in COµ from 5% to 3 or 9% while maintaining a constant [NaHCO×]. Fifty-one per cent of neurones (n = 210) did not change their firing rate by more than 20% in response to hypercapnic acidosis. However, 22% of neurones responded to 9% COµ with an increase in firing rate ('stimulated'), and 27% of neurones responded with a decrease in firing rate ('inhibited'). 3. Chemosensitivity has often been considered an all-or-none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 ± 215 % of control when pH decreased from 7·40 to 7·19. Inhibited neurones had a mean increase in firing rate to 232 ± 265 % of control when pH increased from 7·38 to 7·57. 4. Neurones were also exposed to isocapnic acidosis. All COµ-stimulated neurones tested (n = 15) were also stimulated by isocapnic acidosis, and all COµ-inhibited neurones tested (n = 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n = 12). Thus, the effects of COµ on these neurones were mediated in part via changes in pH. 5. In stimulated neurones, acidosis induced a small increase in the after-hyperpolarization level of 1·38 ± 1·15 mV per −0·2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole-cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential. 6. The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo. 7. Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.

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Diverse physiological and therapeutic insults that increase the amount of unfolded or misfolded proteins in the endoplasmic reticulum (ER) induce the unfolded protein response, an evolutionarily conserved protective mechanism that manages ER stress. Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP) is an ER-resident protein that plays a central role in the ER stress response and is the only known substrate of the proteolytic A subunit (SubA) of a novel bacterial AB(5) toxin. Here, we report that an engineered fusion protein, epidermal growth factor (EGF)-SubA, combining EGF and SubA, is highly toxic to growing and confluent epidermal growth factor receptor-expressing cancer cells, and its cytotoxicity is mediated by a remarkably rapid cleavage of GRP78/BiP. Systemic delivery of EGF-SubA results in a significant inhibition of human breast and prostate tumor xenografts in mouse models. Furthermore, EGF-SubA dramatically increases the sensitivity of cancer cells to the ER stress-inducing drug thapsigargin, and vice versa, demonstrating the first example of mechanism-based synergism in the action of a cytotoxin and an ER-targeting drug.