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Phosphorylation of the band 3 anion exchange protein by the tyrosine kinases p72syk and p56lyn is thought to play a role in the pathway that regulates swelling-activated taurine efflux from the skate red blood cell. In this study, the protein tyrosine kinase (PTK) inhibitors piceatannol and tyrphostin A23 both inhibited taurine efflux and the activities of the tyrosine kinases p72syk and p56lyn in the skate erythrocyte. However, the PTK inhibitors genistein and tyrphostin A46 had only small effects on taurine efflux and PTK activities. In general, a strong correlation between the extent of inhibition of taurine efflux and of tyrosine kinase activity was observed. PTK inhibitors showed a similar pattern of inhibition of band 3 phosphorylation, with the greatest inhibition observed in cells treated with piceatannol. The protein kinase C inhibitors staurosporine and bisindolylmaleimide tested alone or in combination with piceatannol had little or no significant effect on swelling-activated taurine efflux. Overall the results support the hypothesis that phosphorylation of the skate band 3 protein by p72syk and p56lyn contributes to the regulation of volume-activated taurine efflux in skate red cells, and suggest that protein kinase C may not be involved in this regulation.

Until recently, studies to characterize the intestinal epithelial Na(+)/H(+) exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na(+)/H(+) exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH(i) only approximate Na(+)/H(+) exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used (22)[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca(++) involves changes to both K(Na) and V(max). In contrast, the same two second messengers inhibit NHE3 by a decrease in V(max) exclusively. Phorbol ester activation of protein kinase C alters both V(max) and K(Na) of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways.

Background & Aims: Barrier function of the inflamed intestinal mucosa can be compromised by reactive oxygen metabolites that increase mucosal permeability and disrupt the actin cytoskeleton, the integrity of which is important for maintaining tight epithelial junctions. Because heat-shock protein 72 (hsp72) protects intestinal epithelial cells against injury, we determined whether resistance of Caco2/bbe (C2) intestinal monolayer barrier function was related to their high endogenous hsp72 expression., Methods: hsp72 anti-sense (C2/AS) and vector-only transfected C2 (C2/CEP4) clones, lines that exhibit low and high hsp72 expression, respectively, were studied. Permeability was assessed by measuring electrical resistance and mannitol fluxes and actin organization by confocal fluorescein isothiocyanate-phalloidin analysis., Results: Basal transepithelial electrical resistance (TER) and mannitol fluxes were not significantly different between groups. However, the oxidant monochloramine rapidly decreased TER and increased mannitol permeability of C2/AS monolayers compared with C2/CEP4 (50% effective doses at 30 minutes were 0.53 +/- 0.11 and 2.06 +/- 0.34 mmol/L, respectively). Associated with these changes, decreased cell viability, dissociation and aggregation of perijunctional and stress actin filaments, loss of cell height, and increased intercellular separation were observed only in C2/AS cells treated with monochloramine., Conclusions: hsp72 protects intestinal epithelial barrier function against oxidant-induced stress, in part, by protecting the integrity of the actin cytoskeleton.

We report the characterization of an Na+/H+ exchanger (NHE) in embryonic fibroblasts (SL-29 cells) of the chicken, a terrestrial vertebrate, where Na+ conservation is important. This exchanger is electroneutral, has a single Na+ binding site, and is highly sensitive to amiloride (IC50 2 microM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-alpha, hypertonicity, and okadaic acid. Although these features make it resemble mammalian NHE1, other characteristics suggest distinct differences. First, in contrast to mammalian NHE1 it is inhibited by cAMP and shows a biphasic response to phorbol esters and a highly variable response to increased intracellular Ca2+ concentration. Second, whereas full-length human and rat NHE1 cDNA probes recognize a 4.8-kb transcript in rat tissues, they recognize only a 3.9-kb transcript in chicken tissues. An antibody against amino acids 631-746 of human NHE1 sequence fails to recognize a protein in SL-29 cells. Rat NHE2 and NHE3 probes do not recognize any transcript in chicken fibroblasts. The SL-29 exchanger differs markedly from the previously characterized chicken intestinal apical exchanger in its amiloride sensitivity and regulation by phorbol esters. These results suggest that a modified version of mammalian NHE1 is present in chicken tissues and imply that another functionally distinct Na+/H+ exchanger is expressed in aves.

We examined the regulation of the Na+/H+ exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane 22Na+ influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-(N,N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.

Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them.

Treatment of skate erythrocytes with FCCP, dinitrophenol, or sodium azide lowers ATP levels and inhibits Na+-independent taurine uptake after hypotonic volume expansion. Inside-out vesicles isolated from hypotonic volume-expanded cells demonstrate greater Na+-independent taurine uptake, and pretreatment of cells with FCCP abolishes this stimulation. Addition of ATP to the vesicles does not restore stimulated taurine uptake, suggesting that ATP does not act as a ligand modulator on the transporter. Therefore the role of protein phosphorylation was investigated. Because known protein kinase inhibitors have previously been found to have little effect on taurine fluxes in skate erythrocytes, we focused on the effects of protein phosphatase inhibition. When volume-expanded cells were returned to isotonic medium, taurine flux returned to basal values more slowly after treatment with the tyrosine phosphatase inhibitor pervanadate, suggesting that dephosphorylation may regulate inactivation. A similar effect of phosphatase inhibitors was observed in the inside-out vesicles from volume-expanded cells: the reversal of stimulated taurine uptake takes place more slowly in vesicles prepared from cells that had been incubated with pervanadate. Band 3, a major protein involved in the taurine transport pathway, shows increased tyrosine phosphorylation after hypotonic volume expansion. Pervanadate treatment of the cells potentiates and prolongs the increased phosphorylation. Therefore tyrosine phosphorylation of band 3 may play an important role in the activation of taurine fluxes after volume expansion.

Glutamine is a major metabolic fuel and trophic agent for intestinal epithelial cells that can enhance intestinal epithelial cell survival against oxidant and thermal stress, possibly through the induction of heat shock protein 72 (hsp72). In this study, we examined the possibility of a direct causal relationship and tissue specificity of glutamine-induced hsp72. Glutamine treatment of intestinal (epithelial) IEC18 cells, but not HeLa (epidermoid) cells, induces the production of hsp72 in a concentration-dependent fashion, without affecting the expression of the constitutively expressed heat shock protein, hsc73. To determine the specific role that hsp72 induction by glutamine plays in resistance of IEC18 cells to oxidant injury, stable transfected cells expressing an hsp72 antisense construct were established. Although thermal induction of hsp72 was blocked only partially by the antisense construct, glutamine induction of hsp72 was completely inhibited. This was associated with a loss of the protective effects of glutamine against oxidant-induced stress. Thus, glutamine specifically induces hsp72 in intestinal epithelial cells, which mediates cell protection against inflammation-induced stress such as oxidants.

Na+ retention by the colon in response to salt deprivation is mediated in part by the resulting secondary hyperaldosteronism. We show that experimental hyperaldosteronism, to levels seen with salt deprivation, causes an increase in the selective expression and activity of NHE3, an apically located isoform of the Na+/H+ exchange family that functions in transepithelial Na+ absorption. The effect of aldosterone on NHE3 expression is tissue specific, occurring in intestine and not in kidney. Within the intestine, these effects are regional, being observed only in proximal colon, and different in distribution from that observed with glucocorticoids, where the predominant effect occurs in ileum. Although glucocorticoids are well known to exert many effects via regulation of transcript levels, the present study demonstrates that aldosterone stimulates intestinal Na+ absorption by increasing cellular NHE3 expression, a response that is tissue and region specific.

Volume expansion of erythrocytes of little skate, Raja erinacea, triggers the opening of an osmolyte channel. We review this transport mechanism and further investigate the channel's physicochemical nature by probing the channel with a series of pyridoxine derivatives in skate RBC as well as in epithelial cells: MDCK and C6 glioma cells and in skate hepatocytes. The identity of the transport mechanism (band 3 vs. an anion channel) which mediates the swelling-activated efflux of osmolytes in fish RBC is controversial. Therefore, we compared taurine and Cl- effluxes in similar conditions. We found that there is significant Cl- loss from volume-expanded skate RBC. However, there was no effect of either hypotonicity or a number of taurine transport inhibitors on this loss. Utilizing changes in intracellular pH as a means of indirectly measuring H+/Cl- cotransport, we found that a rise in cell pH accompanied the loss of Cl-. This suggests that Cl- efflux could occur via a H+/Cl- cotransporter. To probe and compare the osmolyte channel (taurine efflux) of the skate RBC and three other cell types we used a family of pyridoxine inhibitors. The inhibitory patterns for the skate erythrocytes and hepatocytes differed from those for MDCK and C6 glioma cells and the two former cell types differed from each other. Therefore, the results show that the osmolyte channel in the skate differs from that in other epithelial cells with regard to pyridoxine derivative binding properties.

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

Background & Aims: Mesalamine has many effects and is commonly used for the treatment of inflammatory bowel diseases. Because sodium salicylate, a related compound, modulates the heat shock protein (hsp72) response in nonepithelial cells, the possibility that mesalamine confers cell protection by increasing intestinal epithelial hsp72 expression was examined., Methods: Rat intestinal IEC-18 cells were treated with 0.3-3 mmol/L mesalamine and thermally stressed (39 degrees C-42 degrees C) for 23 minutes. The effects of mesalamine on basal expression and the threshold and time course of hsp72 thermal induction were determined., Results: Although mesalamine had no effects on the basal hsp72 expression or its thermal activation threshold in IEC-18 cells, it accelerated and augmented thermal induction of hsp72 within the first 2 hours of exposure. This was associated with a transient increase in heat shock factor-heat shock element binding and enhanced cellular protection against oxidant-induced injury. In contrast, both mesalamine and sodium salicylate have been shown to lower the thermal induction threshold but not to enhance the hsp72 response in HeLa cells., Conclusions: Mesalamine augments thermal induction of the intestinal epithelial hsp72 expression in a manner that differs from that in nonintestinal epithelial cells. This effect is accompanied by increased cellular protection against oxidant injury.

Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conservation in land birds. In mammals, at least five isoforms of the exchanger, NHEs 1-5, have been cloned, with NHE-1 occurring in epithelial basolateral and nonepithelial cell membranes and NHE-3 being restricted to epithelial apical/brush border membranes. We had demonstrated earlier that chicken intestinal brush border membranes possess NHE activity that functionally resembles mammalian NHE-3. In this study, we used mammalian NHE-1 and NHE-3 probes to examine if chicken enterocytes possess these transporters. Antisera against rat NHE-3 recognized a 97 kDa protein in chicken intestinal brush border membrane, while a NHE-3 cDNA probe failed to recognize any transcript. A NHE-1 antibody failed to recognize any protein in brush border or basolateral membrane, while a NHE-1 cDNA probe recognized a 3.9 kb transcript. Thus, there is more than one NHE isoform in chicken intestine, and our results suggest a novel avian NHE family.

We present evidence that tissue distribution of two highly conserved Na+/H+ exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5' (noncoding and coding) and 3' coding regions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrast to earlier work that concluded that both NHE2 and NHE4 were expressed throughout the intestine and in the kidney, our data show that there is no NHE2 message in the kidney and NHE4 is not expressed in small or large intestine. Analyses of intestinal epithelial and kidney membrane proteins by an NHE2-specific antibody identified a doublet at < 90 kDa in intestine but not in kidney. NHE2 is highly expressed in the Na(+)-absorptive epithelium of jejunum, ileum, and ascending and descending colon. NHE4 mRNA message is found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994) and not in the intestine. From these data, we speculate that neither NHE2 nor NHE4 has a role in renal Na+ absorption. NHE2 is likely involved in gut Na+ absorption, whereas NHE4 may have a specialized role in cell volume rectification of inner medullary collecting duct cells. Knowledge of the correct tissue and cell-specific distribution of these two antiporters should help significantly in understanding their physiological roles.

Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the beta-amino acid taurine through an osmolyte channel. Previous studies have suggested that the osmolyte channel in epithelial cells is a swelling-activated anion channel (pICln). In skate heart, a 37-kDa protein is present which is recognized by a specific antibody to a protein characterized in MDCK cells as pICln. This protein is present predominantly in the cytosol (only 10% in the membrane fraction) of heart incubated under isotonic conditions. After transfer to hypotonic medium (one-half osmolarity), the distribution of this protein is markedly altered and significant amounts of the protein are found in the membrane fraction. After hypotonic exposure, the amount of the protein in the membrane fraction rises to 38 +/- 11% (range 18-53, n = 3). The translocation to the membrane fraction suggests that this protein may play a role in the taurine efflux in this tissue stimulated by hypotonic stress.

Erythrocytes of the skate (Raja erinacea) demonstrate a regulatory volume decrease response when exposed to hypotonic stress by releasing taurine and other organic osmolytes. We review the transport characteristics, physical changes and signaling pathways of a volume-activated osmolyte channel associated with band 3 in skate red blood cells that can be distinguished from anion channels and is separate from the anion exchanger function of band 3.

Volume expansion of little skate (Raja erinacea) erythrocytes increases the affinity of ankyrin binding without altering in the number of binding sites. Potassium iodide-stripped inside-out vesicles (KI-IOV) were used to assess ankyrin binding under volume-expanded conditions. Under isoosmotic conditions, ankyrin binds nearly exclusively to a single class of sites (Bmax, 52 +/- 12 microg/mg; Kd, 150 +/- 28 nM). KI-IOV from volume-expanded cells (either with one-half osmolarity medium or with inclusion of the permeant solute ethylene glycol) demonstrate two ankyrin-binding populations. A high affinity population occurs transiently under volume-expanded conditions. This population has a Bmax of 18 +/- 7 microg/mg and a Kd of 25 +/- 9 nM. Total binding of high and low affinity sites is 57 +/- 17 microg/mg. This change in ankyrin affinity is reversible on volume regulatory decrease. A major target protein in the KI-IOV was identified as the skate homolog of the mammalian red cell anion exchanger band 3. Inclusion of the purified cytoplasmic domain of band 3 competes away more than 80% of the ankyrin binding. To determine whether increased ankyrin affinity is due to band 3 tetramer formation that occurs in volume expansion, cells were treated with pyridoxal 5-phosphate or 4,4'-dinitrostilbene-2,2'-disulfonic acid, two agents that increase tetramer formation under isoosmotic conditions. Both treatments altered the binding affinity with a shift toward higher affinity binding without significant alteration in the number of binding sites.

Erythrocytes of the skate (Raja erinacea) exposed to hypotonic stress swell and then undergo a volume regulatory decrease by releasing taurine and other osmolytes. Previous studies showed that taurine release occurs via a volume-activated, Na(+)-independent, bi-directional transporter that has the properties of a size-limited channel. We now report on the transport characteristics of this channel and its regulation. Kinetic, competition and inhibitor studies indicate that polyols (myo-inositol) and trimethylamines (betaine) are transported by the same channel as taurine. Although the identity of the channel is still unknown a variety of evidence suggests that band 3 is involved in either channel formation or regulation. Hypotonicity causes phosphorylation and structural changes in band 3. Under isotonic conditions band 3 is predominantly in the dimeric form. Hypotonicity causes a shift to tetrameric band 3. We hypothesize that the band 3 tetramer either forms or regulates an osmolyte channel. The finding that expression of band 3 protein increases osmolyte channel activity in Xenopus oocytes supports this hypothesis.

Taurine, a beta amino acid, is a primary osmolyte in nucleated skate erythrocytes and is involved in the regulation of cell volume. Growth factors may be involved in the regulation of cell volume which occurs during cell division. Erythropoietin (EPO) is the primary growth factor controlling erythropoiesis. To investigate its mechanism of action, we used nucleated skate erythrocytes. EPO stimulates Na(+)-independent uptake of taurine in a concentration-dependent manner. The uptake was inhibited by the tyrosine kinase inhibitor genistein. Concomitantly, EPO stimulates tyrosine phosphorylation of a number of proteins, particularly ones of molecular masses 145, 120, 100, 80, 65, and 35 kDa. Using specific antibodies, the 145 kDa protein is identified as phospholipase C gamma-1 (PLC gamma-1) and the 100 kDa protein as the skate homolog of the anion exchanger band 3. Since PLC gamma-1 is activated, turnover of membrane lipids was determined. EPO increased 1,2-diacylglycerol formation from phosphatidylinositols (phosphatidylinositol-4-monophosphate and 4,5-biphosphate) during an early phase and later preferentially from phosphatidylcholine. The early hydrolysis of phosphoinositides was confirmed measuring generation of inositol-1,4,5-trisphosphate, demonstrating an activation of PLC gamma-1 activity. To determine if phospholipase D (PLD) stimulation also occurred, ethanol was included in the reactions. Phosphatidylethanol, synthesized by PLD-mediated transphosphatidylation, appeared at times longer than 5 min, suggesting delayed activation of PLD. These results demonstrate that EPO, via simulation of tyrosine phosphorylation, stimulates taurine transport in skate erythrocytes.

Activation and phosphorylation of the Na+/H+ exchanger occurs with a diverse group of mitogens such as phorbol myristate acetate (PMA), epidermal growth factor (EGF), thrombin and serum (Sardet et al., 1990, Science, 247:723-726). Some of these growth factors have been shown to differentially activate the Na+/H+ exchanger in various fibroblasts (Etscheid et al., 1990, Am. J. Physiol., 259:C549-C556; Muldoon, L.L., et al., 1987, Am. J. Physiol., 253:C219-C229). However, alterations in the expression and phosphorylation of NHE1 in various fibroblasts has not been examined with respect to a potential mechanism of differential activation of the exchanger. To pursue this question, a novel antibody, anti-XB17, directed to the cytoplasmic tail of NHE1 was characterized and then utilized to examine the expression of NHE1 protein and the level of phosphorylation of the serum stimulated exchanger in human embryonic lung fibroblasts (WI-38), SV40-transformed WI-38, and nontransformed human foreskin fibroblasts (HSWP) cells. The level of mRNA expressed in these cells was also examined. Results indicate that the parental cell lines and other nontransformed fibroblasts express NHE1. Although the transformed cell lines express NHE1 mRNA in approximately similar abundance to the parental lines, they contain decreased quantity of NHE1 exchanger/mg membrane protein as recognized by anti-XB17 Ab. The mechanism that results in the apparent decrease in NHE1 protein levels in the transformed cells is not known. Also, the SV40-transformed cells express an exchanger with a higher apparent molecular weight. The WI-38 cells demonstrate phosphorylation of NHE1 in response to mitogenic stimulation. ALthough the nontransformed HSWP cells have a high level of Na+/H+ exchanger protein, they do not show a significant increase in phosphorylation following serum stimulation, when examined by immunoprecipitation, and analysis on 1-D gels. However, subsequent studies of tryptic phosphopeptides from the immunoprecipitated exchanger reveal that serum-stimulated phosphorylation of one tryptic peptide does occur but may be masked in the first dimension by differential phosphorylation of other tryptic peptides that are more heavily phosphorylated in unstimulated cells.

The distribution and subcellular localization of Na+/H+ exchanger isoform NHE-3 was studied in rabbit and canine kidney using polyclonal antibodies to an NHE-3 fusion protein. Western blot analyses were performed against microsomal, brush-border, and basolateral membranes isolated from rabbit kidney cortex, outer medulla, and inner medulla. Immunoblots indicated that NHE-3 antibody recognized a strong band with 95-100 kDa molecular mass in cortical microsomes. Subcellular localization studies showed that NHE-3 was expressed in brush-border membranes of kidney cortex. Expression of NHE-3 in the medullary regions was studied by immunoblot analysis of NHE-3 antibody against the microsomal membranes from the outer and inner medulla. NHE-3 antibody specifically labelled a 95-100 kDa protein in outer but not inner medulla. Subcellular localization studies demonstrated that NHE-3 is localized to the brush-border membranes of the outer medulla. Immunoblot analysis against brush-border membranes from canine kidney cortex and outer medulla demonstrated the presence of an 83-90 kDa protein. The above experiments suggest that NHE-3 in rabbit kidney is a 95-100 kDa protein and is expressed in brush-border membranes of the cortex and outer medulla. The canine kidney NHE-3 is a 83-90 kDa protein and is expressed in brush-border membranes of the cortex and outer medulla. Based on its subcellular localization, we conclude that NHE-3 may be involved in vectorial Na+ and HCO3- transport and pHo regulation.

Volume expansion of little skate (Raja erinacea) erythrocytes leads to the formation of oligomeric forms of the band 3 protein. The oligomers are formed following incubation in hypotonic media or inclusion of a permeant solute such as ethylene glycol. Oligomers were detected by specifically labeling the skate erythrocyte band 3 homolog with the radiolabeled stilbene [3H]4,4'-diisothiocyano-1,2 diphenylethane-2,2'-disulfonic acid and cross-linking membrane proteins with the homobifunctional agent bis(sulfosuccinimidyl) suberate. Under isoosmotic conditions, the distribution of band 3 in the monomer, dimer, and tetramer forms was 38, 54, and 8%. Medium of 0.5 osmolarity caused the distribution to change to 22% monomer, 31% dimer, and 47% tetramer. Upon return to isoosmotic conditions, cell volume, as well as the distribution of band 3 in the monomer, dimer, and tetrameric forms, returned to control values. Return of hypotonically swollen cells to isoosmotic conditions also returned taurine efflux back to control values. Hyperosmolarity caused a 20% shrinkage of cell volume but did not have any effect on either taurine efflux or the distribution of band 3 in the oligomeric forms. Pyridoxal 5'-phosphate and dinitrostilbene disulfonate, two agents that interact with and cause the formation of the tetrameric form of human band 3, also caused a shift toward to tetrameric peak in skate band 3. To determine whether the cross-linked oligomers had proteins that closely associate with band 3 in the intact cell, Western blots were performed to detect ankyrin and band 4.1. Neither protein appeared with the dimeric or tetrameric forms of band 3, suggesting that the mobility shift was in fact due to association of band 3 monomers. This is the first demonstration that oligomerization of band 3 occurs during a physiologic effect that may be mediated by the band 3 protein.

Na+/H+ exchanger isoform and the effect of high osmolality on its function was studied in cultured renal epithelial cells (LLC-PK1 and OK). Using NHE-3-specific antibody, immunoblots of luminal membranes from LLC-PK1 and OK cells specifically labeled proteins with molecular masses 90 and 95 kDa, indicating that NHE-3 is the isoform expressed on the luminal membranes of these epithelia. Proximal tubular suspensions from rabbit kidney cortex were incubated in control (310 mosm/liter) or high osmolality (510 mosm/liter) medium for 45 min and utilized for brush border membrane vesicle preparation. Influx of amiloride-sensitive 22Na+ at 10 s (pHo 7.5, pHi 6.0) into brush border membrane vesicles was 37% lower in the high osmolality group (p < 0.03). LLC-PK1 or OK cells were grown to confluence and examined for Na+/H+ exchange activity. An increase in medium osmolality to 510 mosm following acid loading decreased the 5-min uptake of the amiloride-sensitive 22Na+ in LLC-PK1 and OK cells (p < 0.04 and < 0.03 for LLC-PK1 cell OK cells, respectively). An increase in medium osmolality to 510 mosm in vascular smooth muscle cells, which express NHE-1, produced 45 and 64% stimulation of the amiloride-sensitive 22Na+ influx at base-line pHi and acid-loaded condition, respectively (p < 0.03 and < 0.01). Down-regulation of protein kinase C by preincubation with phorbol 12-myristate 13-acetate or inhibition of Ca(2+)-calmodulin-dependent protein kinase (calmodulin-kinase II) by N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7) in LLC-PK1 cells did not block the inhibitory effect of high osmolality on Na+/H+ exchange activity. We conclude that renal proximal tubule epithelial cells express Na+/H+ exchange isoform NHE-3 on their luminal membranes and that hyperosmolality decreases transporter activity during cell acidification. This inhibitory effect might be unique to the NHE-3 isoform, since vascular smooth muscle cells which express NHE-1 exhibit an increase in Na+/H+ exchange activity in response to high osmolality.

We have demonstrated that phorbol esters such as phorbol dibutyrate (PhE) transiently inhibit Na/H exchange both in intact avian enterocytes and in brush border membrane (BBM) vesicles prepared from enterocytes treated with PhE (Chang et al., 1991, Am. J. Physiol. 260: C1264-C1272). Maximal inhibition occurs at 90 sec and values return to baseline by 15 min. In this study we examined if PhE causes changes in BBM protein phosphorylation by two methods: 1) in situ phosphorylation in which intact cells prelabeled with 32Pi were treated with PhE; 2) in vitro phosphorylation in which BBM, isolated from untreated and PhE-treated enterocytes, were exposed to gamma 32P-ATP. In situ phosphorylation studies showed that, at 90 sec, PhE increases the phosphorylation of BBM proteins of M(r) (pI): 150 (6.5), 89 (approximately 6.2), and 48 (approximately 6.1) kDa which declined to control values at 15 min, suggesting that these may be transport-related substrates. These labeled substrates were recovered in the detergent-insoluble fraction after extraction with 0.1% Triton X-100 overnight. Transient phosphorylation of a number of proteins was also observed when BBM prepared from control or PhE-treated cells were incubated with gamma 32P-ATP +/- 10 nM PhE, phosphatidyl serine, Ca2+, and/or exogenous protein kinase C (PKC). The in vitro phosphoproteins included both Triton-soluble and Triton-insoluble proteins. However, none of these proteins labeled in vitro coincided with those labeled in situ. The decline in phosphorylation with time can be accounted for by phosphatase action as these BBM possess a Ca-dependent phosphatase. In summary, we have demonstrated that the BBM possess PKC-specific substrates which can be visualized by in situ and in vitro phosphorylation. Treatment of intact enterocytes with PhE results in the phosphorylation of three detergent-insoluble proteins with a time course similar to that of PhE inhibition of Na/H transport.

1. Na/H exchange is the major pathway for Na uptake in brush border membrane vesicles from chicken small intestine. Hanes-Woolf analysis demonstrated that Na and H competed at the same extravesicular site. The KNa for Na+ at extravesicular pH 6.6 is 35 mM and at pH 7.4, 12 mM. 2. Similar to mammalian intestinal cells, the Na/H exchanger does not appear to have an internal proton modifier site. Varying intravesicular pH from 6.1 to 7.8 stimulates uptake, but a sigmoidal relationship is not observed. 3. The ability of several amiloride analogs to inhibit the exchanger was tested and the inhibitory profile was similar, but not identical to Na/H exchangers in mammalian tissues. The potency series (from most to least potent) is hexamethylamiloride approximately ethylisopropylamiloride > methylisobutylamiloride > dimethylamiloride >> amiloride.

Bethanechol, a muscarinic agonist, inhibits the initial rate of amiloride-sensitive Na uptake by intact mucosa of avian small intestine as well as by isolated chicken villus enterocytes, an effect that is maximal at 90 seconds and reverses by 6 minutes. Bethanechol similarly decreases intracellular pH in isolated cells suspended in bicarbonate-free buffer in a time course similar to inhibition of enterocyte Na uptake, suggesting inhibition of Na/H exchange. In brush border membrane vesicles rapidly prepared from cells stimulated with bethanechol, proton-dependent 22Na uptake is transiently inhibited in a time course similar to inhibition of cell Na uptake. Bethanechol also stimulates transient translocation of protein kinase C from the cytosol to the particulate fraction, a portion of this activity translocating to the brush border membrane. To determine the calcium dependence of bethanechol's action, enterocytes were loaded with varying concentrations of the calcium buffering agent quin-2. Inhibition of cell Na uptake by the calcium ionophore ionomycin could be completely reversed by quin-2 buffering in a concentration-dependent manner. In contrast, quin-2 buffering had little or no effect on the inhibition of Na uptake caused by the protein kinase C activators phorbol esters and oleoylacetylglycerol. Bethanechol's inhibitory effects were partially, but not completely reversed by quin-2 buffering. These data suggest that the effects of bethanechol on chicken villus enterocyte brush border Na/H exchange are mediated by calcium-dependent process(es) as well as by protein kinase C.

The relative abundance of cystic fibrosis transmembrane regulator (CFTR) message at various stages of postconfluence development was compared with evolving cellular ion-nutrient transport properties of CaCo-2 human intestinal cells. Initially these cells demonstrate electrogenic Cl secretion manifested by secretagogue-induced changes in short-circuit current. Over time, however, the secretory characteristics of CaCo-2 monolayers diminish, and brush border hydrolase activities and glucose-dependent and amiloride-sensitive Na transport increase. With a polymerase chain reaction-derived cDNA probe to CFTR exon 13, two distinct mRNA transcripts of 6.5 and 4.3 kb were found. No significant differences in their abundance were noted in cells at 6 and 28 days after confluence. These data suggest two possible interpretations for the role of CFTR protein. First, if CFTR is membrane Cl channel, its protein expression or activity could be differentially regulated during CaCo-2 cell development. Alternatively, CFTR may not be a Cl channel but may serve as an important regulatory membrane protein.

Phorbol 12,13-dibutyrate, an activator of protein kinase C, stimulates electrogenic anion secretion in chicken ileum. This effect can be inhibited by the cyclooxygenase inhibitor piroxicam, suggesting arachidonic acid metabolites as mediators. Phorbol 12,13-dibutyrate stimulates increases in prostaglandin E2 release and mucosal 3',5'-cyclic adenosine monophosphate content in intact mucosa. Stimulation of prostaglandin E2 release appears to come mainly from the subepithelial compartment and not from epithelial cells. Unlike its effect on anion secretion, phorbol 12,13-dibutyrate inhibition of sodium uptake by sodium-hydrogen exchange in isolated mature villus enterocytes is not inhibited by piroxicam. This study concludes that phorbol ester effects on anion secretion in chickens appear to be mediated by arachidonic acid metabolites most likely produced by cells of the lamina propria and submucosa, whereas phorbol ester effects on inhibiting upper villus cell sodium-hydrogen exchange may be a direct effect of phorbol esters.

Interleukin 1 or 3 added to the serosal side of chicken small intestine transiently increases short-circuit current. Replacement of bathing-medium Cl and HCO3 with gluconate and HEPES abolished the short-circuit current increase, consistent with these cytokines stimulating electrogenic anion secretion. Cytokine-stimulated short-circuit current changes were inhibited by preincubation with piroxicam (10(-5) M), an inhibitor of arachidonic acid cyclooxygenase, suggesting prostaglandin formation as an intermediate step for cytokine stimulation of short-circuit current. In intact mucosal strips, interleukin 1 and 3 stimulated prostaglandin E2 release and elevated tissue 3',5'-cyclic adenosine monophosphate concentration. When prostaglandin E2 release from epithelial and subepithelial fractions of the mucosa by interleukin 1 was determined, increases were found only from the subepithelium. The secretory actions of cytokines appear to be mediated by arachidonic acid metabolites most likely produced by cells of the lamina propria and submucosa and may play a role in inflammatory processes in which intestinal secretion is enhanced.

Effects of HgCl2 (100 microM) para-chloromercuribenzene sulfonate (PCMBS) (1 mM), and oxophenylarsine (OPA) (250 microM) were determined on (a) the rate of Na pump activity in intact winter flounder intestine; (b) activity of Na-K-ATPase in tissue homogenates; and (c) Na-dependent and Na-independent uptake of tyrosine in brush border membrane vesicles. Initial rate of uptake (influx) of 86Rb from the serosal solution of tissues mounted in Ussing chambers, a measure of Na-K-ATPase activity in the intact cell, was inhibited by all three agents with differing time courses. Rapidly permeating HgCl2 inhibited influx to the same degree as ouabain at 30 min, whereas the effects of PCMBS and OPA required 90 min. Cell potassium was also measured as an indirect indicator of ATPase activity and cell membrane permeability. All three agents decreased cell K, although effects on cell K lagged behind those for inhibition of the ATPase. At the concentrations used in the Ussing chamber (or at one-tenth concentration), all agents completely inhibited Na-K-ATPase activity in enzyme assays performed with tissue homogenates. In contrast, only HgCl2 decreased Na-dependent uptake of tyrosine by brush border membrane vesicles. These results suggest that mercurial and arsenical effects on tyrosine absorption are due to inhibition of the Na-K-ATPase thus decreasing the driving force for the cellular uptake by the Na-tyrosine cotransport system. Direct effects on Na-tyrosine cotransport may play a role in the inhibition observed with HgCl2, but not for PCMBS or OPA.

Carbachol (CCH), serotonin (5HT), divalent ionophore A23187, cAMP, and certain neuropeptides, i.e. substance P (SP), inhibit the initial rate of uptake (influx) of 22Na into isolated chicken villus enterocytes. All these agents also increase cytosolic Ca. However, the increases stimulated by CCH, 5HT, and cAMP are not blocked by chelation of extracellular Ca, whereas those of A23187 and SP are. Only CCH and 5HT stimulate hydrolysis of membrane phosphoinositides to form inositol phosphates. CCH and 5HT also stimulate incorporation of [32P]-PO4 into membrane polyphosphoinositides. These studies suggest that at least three mechanisms exist to increase cytosolic Ca in chicken enterocytes and thereby inhibit Na influx. Certain neurohumoral agents such as SP open a plasma membrane permeability for Ca, permitting extracellular Ca to enter the cell down its electrochemical gradient. These agents do not stimulate phosphatidylinositol breakdown. CCH and 5HT stimulate phosphatidylinositol breakdown and via the formation of inositol trisphosphate release Ca from intracellular stores. A third mechanism exists for cAMP which mobilizes Ca from intracellular stores, but does not involve the metabolism of membrane phosphatidylinositols.

The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1 microM), in the presence of calcium. These cells produce 12.94 +/- 1.8 ng/10(6) cells of LTC4 and 3.8 +/- 0.4 ng/10(6) cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1 microM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91 +/- 0.42 ng/10(6) cells of LTC4 and 0.41 ng +/- 0.06/10(6) cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5 +/- 0.2 ng of LTC4 and 1.45 +/- 0.2 ng of LTB4 per 10(6) cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.

A cloned murine mast cell MC9 expresses phospholipase and lipoxygenase activity when stimulated with IgE and hapten. Addition of DNP-BSA to sensitized MC9 cells causes release of 58% of the cell histamine and 127 pmoles LTC4/10(6) cells. Prelabelling studies with [1-14C]-arachidonic acid showed that LTC4 production was proceeded by the release of arachidonic acid from membrane phospholipids. Approximately 8.7% of the cell arachidonic acid was released and half of this was converted to LTC4. The remaining radioactivity was converted to diHETES including LTB4 (15%), 5-HETE (10%), free arachidonic acid (10%), reesterified 5-HETE and arachidonic acid (8%) and prostaglandins (7%). This stimulation was dependent on hapten (DNP-BSA) and extracellular Ca++. Under identical conditions the calcium ionophore A23187 stimulated the release of 10.3% of the total cell arachidonic acid, and 51% of this was metabolized to LTC4. In addition the ionophore stimulated the release of 61% of the total cellular histamine.

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.

MC9 mast cells stimulated by a soluble (calcium ionophore A23187) or by an Fc epsilon-receptor agonist (IgE plus hapten) produce platelet activating factor (PAF). MC9 cells incorporate either exogenous [3H]acetic acid or [3H]lyso-PAF into PAF. PAF was identified by mobility on thin layer chromatography, platelet aggregatory activity inhibitable by known PAF antagonists, and by enzymatic modification. Quantified by aggregation of rabbit platelets, MC9 cells produce 6 pmoles PAF/10(6) cells. MC9 cells express acetyltransferase activity of 0.19 nmole/5 min-mg protein. Analysis of MC9 phospholipids by HPLC showed that MC9 cells contain large amounts of phosphatidylcholine (82 nmoles/10(7) cells) but contain little ether-linked phosphatidylcholine (4 nmoles/10(7) cells).

Intestine (both small and large) secretes chloride in response to prostaglandins and agents, such as bradykinin, that increase prostaglandin synthesis. The colon, but not the small intestine, can also be stimulated to secrete chloride by certain lipoxygenase products, such as 5-hydroperoxy-eicosatetraenoic acid and 5-hydroxy-eicosatetraenoic acid, but not by leukotrienes. Intestine generates these compounds and leukotriene B4 (but not C, D, and E) and their synthesis is increased by melittin, calcium ionophore A23187, and bradykinin.

The E prostaglandins (and to a lesser extent PGE2 alpha) stimulate active electrolyte secretion in mammalian small intestine and colon. They do so by stimulating intestinal mucosal adenylate cyclase and thereby increasing cAMP concentration. The diarrheagenic action of the prostaglandins is seen as a side effect of their therapeutic use, in certain hormone-secreting tumors, and in inflammatory lesions of the bowel in which leukocyte infiltrates are the probable sources of prostaglandin excess. Prostaglandins are also normally synthesized by intestinal epithelial cells and appear to play an important role in the physiologic regulation of intestinal fluid transport. In recent in vitro studies, we have shown that addition of arachidonic acid (K 1/2 congruent to 10(-6) M) also stimulates secretion, cAMP accumulation, and PGE2 production in rabbit ileal mucosa. In the continued presence of arachidonate, tachyphylaxis develops: both secretory and cAMP responses have a half-life of about twenty minutes and subsequent additions of arachidonate produce little or no further response. In contrast, PGE2 production continues undiminished. Similar tachyphylaxis develops when PGE2 itself is added. Resensitization following removal of PGE2 is rapid, 50% of the initial sensitivity being restored in 6-7 min. Prostaglandin desensitization has been noted in other cell systems and appears to be exerted on adenylate cyclase.

Both 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) increased the short-circuit current (Isc) in rabbit colonic mucosa mounted in vitro in Ussing chambers. Measurements of chlorine-36 fluxes indicated that the Isc response to 5-HPETE is due to stimulation of active chlorine secretion. 9-, 11-, and 12-HPETE's and leukotrienes C4 and B4 produced either very small increases in Isc or no increase. In contrast to results in rabbit colon, no HPETE, HETE, or leukotriene was effective in rabbit ileal mucosa. The effects of 5-HPETE in the rabbit colon were unaffected by mepacrine, but could be partially blocked by indomethacin. These results suggest that drugs which block both cyclooxygenase and lipoxygenase may be effective antidiarrheals in patients with colitis.