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M Donowitz

Publications and source records attributed to M Donowitz.

At least 37 records · Page 2Linked to original sources
PubMed2000

NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization.

Teleost fishes, living in fresh water, engage in active ion uptake to maintain ion homeostasis. Current models for NaCl uptake involve Na(+) uptake via an apical amiloride-sensitive epithelial Na(+) channel (ENaC), energized by an apical vacuolar-type proton pump (V-ATPase) or alternatively by an amiloride-sensitive Na(+)/H(+) exchange (NHE) protein, and apical Cl(-) uptake mediated by an electroneutral, SITS-sensitive Cl(-)/HCO(3-) anion-exchange protein. Using non-homologous antibodies, we have determined the cellular distributions of these ion-transport proteins to test the predicted models. Na(+)/K(+)-ATPase was used as a cellular marker for differentiating branchial epithelium mitochondria-rich (MR) cells from pavement cells. In both the freshwater tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), V-ATPase and ENaC-like immunoreactivity co-localized to pavement cells, although apical labelling was also found in MR cells in the trout. In the freshwater tilapia, apical anion-exchanger-like immunoreactivity is found in the MR cells. Thus, a freshwater-type MR chloride cell exists in teleost fishes. The NHE-like immunoreactivity is associated with the accessory cell type and with a small population of pavement cells in tilapia.

Animals↗
PubMed2000

Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri).

The branchial epithelium of the mudskipper Periophthalmodon schlosseri is densely packed with mitochondria-rich (MR) cells. This species of mudskipper is also able to eliminate ammonia against large inward gradients and to tolerate extremely high environmental ammonia concentrations. To test whether these branchial MR cells are the sites of active ammonia elimination, we used an immunological approach to localize ion-transport proteins that have been shown pharmacologically to be involved in the elimination of NH(4)(+) (Na(+)/NH(4)(+) exchanger and Na(+)/NH(4)(+)-ATPase). We also investigated the role of carbonic anhydrase and boundary-layer pH effects in ammonia elimination by using the carbonic anhydrase inhibitor acetazolamide and by buffering the bath water with Hepes, respectively. In the branchial epithelium, Na(+)/H(+) exchangers (both NHE2- and NHE3-like isoforms), a cystic fibrosis transmembrane regulator (CFTR)-like anion channel, a vacuolar-type H(+)-ATPase (V-ATPase) and carbonic anhydrase immunoreactivity are associated with the apical crypt region of MR cells. Associated with the MR cell basolateral membrane and tubular system are the Na(+)/K(+)-ATPase and a Na(+)/K(+)/2Cl(-) cotransporter. A proportion of the ammonia eliminated by P. schlosseri involves carbonic anhydrase activity and is not dependent on boundary-layer pH effects. The apical CFTR-like anion channel may be serving as a HCO(3)(-) channel accounting for the acid-base neutral effects observed with net ammonia efflux inhibition.

Ammonia↗
PubMed1999

cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor.

The members of the regulatory factor (RF) gene family, Na(+)/H(+) exchanger (NHE)-RF and NHE3 kinase A regulatory factor (E3KARP) are necessary for cAMP to inhibit the epithelial brush border NHE isoform 3 (NHE3). The mechanism of their action was studied using PS120 fibroblasts stably transfected with rabbit NHE3 and wild type rabbit NHE-RF or wild type human E3KARP. 8-Bromo-cAMP (8-Br-cAMP) had no effect on Na(+)/H(+) exchange activity in cells expressing NHE3 alone. In contrast, in cells co-expressing NHE-RF, 8-Br-cAMP inhibited NHE3 by 39%. In vivo phosphorylation of NHE3 demonstrated that cAMP increased phosphorylation in two chymotrypsin-generated phosphopeptides of NHE3 in cells containing NHE-RF or E3KARP but not in cells lacking these proteins. The requirement for phosphorylation of NHE-RF in this cAMP-induced inhibition of NHE3 was examined by studying a mutant NHE-RF in which serines 287, 289, and 290 were mutated to alanines. Wild type NHE-RF was a phosphorylated protein under basal conditions, but treatment with 8-Br-cAMP did not alter its phosphorylation. Mutant NHE-RF was not phosphorylated either under basal conditions or after 8-Br-cAMP. 8-Br-cAMP inhibited NHE3 similarly in PS120/NHE3 cells containing wild type or mutant NHE-RF. NHE-RF and NHE3 co-precipitated and did so similarly with and without cAMP. Mutant NHE-RF also similarly immunoprecipitated NHE3 in the presence and absence of 8-Br-cAMP. This study shows that members of the regulatory factor gene family, NHE-RF and E3KARP, are necessary for cAMP inhibition of NHE3 by allowing NHE3 to be phosphorylated. This inhibition is not dependent on the phosphorylation of NHE-RF.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3.

Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 micrograms/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

Animals↗
PubMed1999

NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation.

The cloned epithelial cell-specific Na+/H+ exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH-terminal truncation mutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at the truncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/calmodulin (CaM) inhibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, was stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluorescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 in a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized into separate stimulatory and inhibitory growth factor/protein kinase regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two proteins.

Animals↗
PubMed1999

Na(+)/H(+) exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface.

NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na(+)/H(+) exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by (22)Na(+) uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 +/- 3.5, 64.6 +/- 3.3, 20.0 +/- 2.6, and 14.0 +/- 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 +/- 9.6, 92.1 +/- 8.6, and 99.2 +/- 9.1 s(-1), when V(max) was determined using (22)Na uptake at 22 degrees C and 742 +/- 47, 459 +/- 16, and 609 +/- 39 s(-1) when V(max) was determined using fluorometry at 37 degrees C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

Animals↗
PubMed1999

Development of an endogenous epithelial Na(+)/H(+) exchanger (NHE3) in three clones of caco-2 cells.

Expression of endogenous Na(+)/H(+) exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na(+)/H(+) exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 microM H(+)/s, whereas AP NHE1 activity decreased from 192 to 18 microM H(+)/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 microM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium.

Amiloride↗
PubMed1998

Membrane-limited expression and regulation of Na+-H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct.

1. Cell-specific reverse transcriptase-polymerase chain reaction (RT-PCR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+-H+ exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P2 receptors in the duct. 2. The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM). 3. Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct. 4. Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1-like activity in the BLM and NHE2-like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present. 5. Activation of P2 receptors resulted in activation of an NHE-independent, luminal H+ transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+. 6. Stimulation of P2U receptors expressed in the BLM activated largely NHE1-like activity, and stimulation of P2Z receptors expressed in the LM activated largely NHE2-like activity. 7. The interrelation between basolateral and luminal NHE activities and their respective regulation by P2U and P2Z receptors can be used to co-ordinate membrane transport events in the LM and BLM during active Na+ reabsorption by the SMG duct.

Adenosine Triphosphate↗
PubMed1998

Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vasculature.

The novel aminosterol, squalamine, inhibits angiogenesis and tumor growth in multiple animal models. This effect is mediated, at least in part, by blocking mitogen-induced proliferation and migration of endothelial cells, thus preventing neovascularization of the tumor. Squalamine has no observable effect on unstimulated endothelial cells, is not directly cytotoxic to tumor cells, does not alter mitogen production by tumor cells, and has no obvious effects on the growth of newborn vertebrates. Squalamine was also found to have remarkable effects on the primitive vascular bed of the chick chorioallantoic membrane, which has striking similarities to tumor capillaries. Squalamine may thus be well suited for treatment of tumors and other diseases characterized by neovascularization in humans.

Animals↗
PubMed1998

Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger.

Na+/H+ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na+/H+ exchanger gene family, plays an important role in reabsorption of Na+ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in Vmax, but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from approximately 4.3 to approximately 2.4. This translocation resulted in 10-15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes approximately 50% to the overall protein kinase C-induced inhibition of the exchanger.

Caco-2 Cells↗
PubMed1998

A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro.

There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell-cell contact areas. The Na+/K+-ATPase alpha1-subunit was detected predominantly in the basolateral membranes, while the Na+/H+ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (beta-galactosidase +/- a nuclear targeting signal, alpha1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line.

Adenoviridae↗
PubMed1998

Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange.

Intestinal neutral NaCl absorption, which is made up of brush-border (BB) Na+/H+ exchange linked to BB Cl-/HCO3- exchange, is up- and downregulated as part of digestion and diarrheal diseases. Glucocorticoids stimulate ileal NaCl absorption and BB Na+/H+ exchange. Intestinal BB contains two Na+/H+ exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbit ileal BB Na+/H+ exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BB Na+/H+ exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute approximately 50% to ileal Na+/H+ exchange. Glucocorticoids (methylprednisolone) increase BB Na+/H+ exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times), without an effect on NHE2 activity. Thus ileal BB Na+/H+ exchange in animals treated with glucocorticoids is 69% via NHE3. A quantitative Western analysis for NHE3 was developed, using as an internal standard a fusion protein of the COOH-terminal 85 amino acids of NHE3 and maltose binding protein. Glucocorticoid treatment increased the amount of BB NHE3. The quantitative Western analysis showed that NHE3 makes up 0.018% of ileal BB protein in control rabbits and 0.042% (2.3 times as much) in methylprednisolone-treated rabbits. Methylprednisolone treatment did not alter the amount of ileal BB NHE2 protein. NHE3 turnover number was estimated to be 458 cycles/s under basal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thus methylprednisolone stimulates ileal BB Na+/H+ exchange activity only by an effect on NHE3 and not on NHE2; it does so primarily by increasing the amount of BB NHE3, although it also increases the NHE3 turnover number.

Animals↗
PubMed1998

G protein-coupled receptors in gastrointestinal physiology. III. Asymmetry in plasma membrane signal transduction: lessons from brush-border Na+/H+ exchangers.

Signal transduction in epithelial cells adds another level of complexity to the signaling that occurs in symmetrical cells, in the form of the need to coordinate and keep separate signals at the apical and basolateral membranes. Regulation by protein kinases of ileal NaCl absorption and its component brush-border Na+/H+ exchanger are used as an example of how signaling in epithelial cells must deal with spatial localization of signals, protein-protein interactions, signaling molecules, and the involvement of the transport protein being regulated in collecting and focusing the signals generated at the receptor and beyond.

Animals↗
PubMed1998

In birds, NHE2 is major brush-border Na+/H+ exchanger in colon and is increased by a low-NaCl diet.

We previously reported that mammalian small intestinal and colonic brush borders (BBs) contained both epithelial Na+/H+ exchangers NHE2 and NHE3. We now show that, in the avian (chicken) colon, NHE2 is the major functional isoform under basal conditions and when stimulated by a low-NaCl diet. Hubbard chickens were maintained for 2 wk on a high- or low-NaCl diet. After the chickens were killed, the ileum and colon were removed, and BBs were prepared by Mg2+ precipitation and 22Na and D-[14C]glucose uptake determined in the BB vesicles. NHE2 and NHE3 were separated by differential sensitivity to HOE-694 (NHE2 defined as Na+/H+ exchange inhibited by 50 microM HOE-694). Chickens on a low-Na+ diet have increased plasma aldosterone (10 vs. 207 pg/ml). On the high-NaCl diet, both NHE2 and NHE3 contributed to ileal and colonic apical Na+/H+ exchange, contributing equally in ileum, but NHE2 being the major component in colon (86%). Low-NaCl diet significantly increased ileal and colonic BB Na+/H+ exchange; the increase in BB Na+/H+ exchange in both ileum and colon was entirely due to an increase in NHE2 with no change in NHE3 activity. In contrast, low-NaCl diet decreased ileal and colonic Na+-dependent D-glucose uptake. Western analysis showed that low-Na+ diet increased the amount of NHE2 in the ileal and colonic BB and decreased the amount of ileal Na+-dependent glucose transporter SGLT1. Both NHE2 and NHE3 were present in the apical but not basolateral membranes (BLM) of ileal and colonic epithelial cells. In summary, 1) NHE2 and NHE3 are both present in the BB and not BLM of chicken ileum and colon; 2) NHE2 is the major physiological colonic BB Na+/H+ exchanger under basal conditions; 3) low-NaCl diet, which increases plasma aldosterone, increases ileal and colonic BB Na+/H+ exchange and decreases Na+-dependent D-glucose uptake; 4) the stimulation of colonic BB Na+/H+ exchange is due to increased activity and amount of NHE2; and 5) the inhibition of ileal D-glucose uptake is associated with a decrease in SGLT1 amount. NHE2 is the major chicken colonic BB Na+/H+ exchanger.

Animals↗
PubMed1997

Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-gamma1. Role of cytoskeletal rearrangement in the carbachol-induced inhibition of ileal NaCl absorption.

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

Actins↗
PubMed1997

Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb.

Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat.

Amino Acid Sequence↗
PubMed1997

Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger.

The epithelial brush border Na+/H+ exchanger isoform 3 (NHE3) is regulated by growth factors and protein kinases. When stably expressed in PS120 fibroblasts, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by phorbol esters. To examine the role of phosphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicular stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/H+ exchanger null PS120 fibroblasts (PS120/NHE3V). NHE3V was regulated by serum, FGF, and phorbol ester in a manner identical to wild type non-VSVG-tagged NHE3. Phosphorylation of NHE3V was evaluated via immunoprecipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE3V cells with [32P]orthophosphate. NHE3V was phosphorylated under basal conditions. However, FGF and PMA, under conditions in which these agonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography nor two-dimensional phosphopeptide maps of tryptic digests of NHE3V. In contrast, while changes in NHE3V phosphorylation were not observed with serum exposure by one-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional studies showed increases in two phosphopeptides. Under all these conditions, phosphoamino acid analysis showed that NHE3V was phosphorylated only on serine residues. By cell surface protein biotinylation studies under basal conditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intracellular forms of NHE3V under basal conditions and determine whether the amount of phosphorylation of the surface form changes upon serum, FGF, and PMA regulation, the surface form of NHE3V was separated from intracellular form by biotinylation/avidin-agarose precipitation. Under basal conditions, both intracellular and surface forms of NHE3V were phosphorylated. However, the amount of phosphorylation of the surface form of NHE3V did not change upon stimulation by serum and FGF and inhibition by PMA based on one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, its regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphorylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.

Animals↗