Crescence Bookstein

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.

Aldosterone stimulates intestinal Na+ absorption in rats by increasing NHE3 expression of the proximal colon

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.

Expression of multiple Na+/H+exchanger isoforms in rat parotid acinar and ductal cells

Several members of the Na+/H+exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50 = 0.014 ± 0.005 μM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50 = 0.754 ± 0.104 μM) than cell lines expressing NHE1 or NHE2 (IC50 = 0.076 ± 0.013 or 0.055 ± 0.015 μM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50= 6.25 ± 1.89 μM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.

Localization of the Na+/H+ exchanger isoform NHE-3 in rabbit and canine kidney.

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.

Regulation of intestinal epithelial brush border Na(+)/H(+) exchanger isoforms, NHE2 and NHE3, in C2bbe cells.

Abstract. 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 pHi 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 KNa and Vmax. In contrast, the same two second messengers inhibit NHE3 by a decrease in Vmax exclusively. Phorbol ester activation of protein kinase C alters both Vmax and KNa 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.

INVERSE RELATIONSHIP BETWEEN MEMBRANE LIPID FLUIDITY AND ACTIVITY OF NA+/H+ EXCHANGERS, NHE1 AND NHE3, IN TRANSFECTED FIBROBLASTS

Abstract. 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 ≈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°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.

Differential regulation of Na+/H+ exchange and H+ -ATPase by pH and HCO3 - in kidney proximal tubules

This study examines the effects of acute in vitro acid-base disorders on Na+/H+ and H+-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na+/H+ exchange activity (assayed by 22Na+ influx) or abundance (using NHE-3 specific antibody) and H+-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na+/ H+ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO3−3 mm) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO3−90 mm). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H+-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na+/H+ exchanger activity increased by 29% in high HCO3−group (HCO3−96 mm) and decreased by 16% in the low HCO3−groups (HCO3−7mm. The NHE-3 abundance remained constant in high, normal, and low [HCO3−] tubules. The abundance of H+-ATPase, however, increased by 82% in high [HCO3−] and decreased by 77% in the low [HCO3−] tubules. In PT suspensions incubated in varying pCO2 and constant [HCO3−], Na+/H+ exchanger activity increased by 35% in high pCO2 (20% pCO2, respiratory acidosis) and decreased by 32% in low pCO2 (1.5% pCO2, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO2 tubules. However, the H+-ATPase abundance increased by 74% in high pCO2 and decreased by 69% in low pCO2 tubules.The results of these studies suggest that the luminal Na+/H+ exchanger is predominantly regulated by pH whereas H+-ATPase is mainly regulated by [HCO3−] and/ or pCO2. They further suggest that the adaptive changes in H+-ATPase transporter are likely mediated via endocytic/exocytic pathway whereas the adaptive changes in Na+/H+ exchanger are via the nonendocytic/exocytic pathway.The excellent technical assistance of Yollanda J. Hattabaugh, Gwen L. Bizal, and L. Yang is greatly appreciated. Portions of these studies were presented at the annual meeting of the American Society of Nephrology, Boston, MA, November 1993, and published in abstract form (J.Am.Soc.Neph. 4:840A, 1993)

Glycosylation of the Na+/H+ exchanger isoform NHE-3 is species specific

The glycosylation of Na+/H+ exchanger isoform NHE-3 was studied in brush border membrane (BBM) vesicles isolated from rabbit, dog, and rat kidney cortex. Western blot analyses were performed against BBM proteins by using polyclonal antibodies to an NHE-3 fusion protein. In rabbit kidney, NHE-3 antibody recognized a band with approximately 95 kd molecular mass. Treatment of rabbit cortical BBM with glycopeptidase F, at 16 U/ml, for 4 or 16 hours increased the apparent mobility of NHE-3 to 84 and 82 kd, respectively. Incubation of rabbit BBM proteins for 16 hours with endoglycosidase H, at 0.1 U/ml, did not alter the apparent mobility of NHE-3. Deglycosylation of NHE-3 with glycopeptidase F did not affect acid-stimulated, amiloride-sensitive sodium 22 influx in BBM vesicles as compared with that in controls (p > 0.05). Immunoblot analysis against BBM proteins from canine kidney cortex demonstrated the presence of an approximately 83 to 92 kd protein. Treatment of canine BBM with glycopeptidase F or endoglycosidase H or F for 16 hours did not alter the apparent mobility of NHE-3, suggesting that canine renal NHE-3 is not glycosylated. Treatment of canine kidney BBM with glycopeptidase F did not affect acid-stimulated 22Na+ influx as compared with that in controls (p > 0.05). Immunoblot analysis against BBM proteins from rat kidney cortex demonstrated the presence of a sharp band at 90 kd. Treatment of rat BBM with glycopeptidase F or endoglycosidase H or F for 16 hours did not alter the apparent mobility of NHE-3, suggesting that rat renal NHE-3 is not glycosylated. The above experiments suggest that NHE-3 glycosylation in mammalians is species specific and that glycosylation does not affect the exchanger activity.

Inhibition of glycosylation decreases Na+/H+ exchange activity, blocks NHE-3 transport to the membrane, and increases NHE-3 mRNA expression in LLC-PK1 cells

Recent studies have shown that NHE-3 is the luminal Na+/H+ exchanger isoform in cultured renal proximal tubule cells LLC-PK1 and OK (J Biol Chem 1994; 269:15613-8). The purpose of the current experiments was to study the role of NHE-3 glycosylation on antiporter activity in LLC-PK1 cells. Treatment of LLC-PK, cells with 1.5 microgram/ml tunicamycin for 24 hours, which blocks glycosylation in the endoplasmic reticulum, significantly decreased antiporter activity as asses sed by acid-stimulated sodium 22 uptake (9.52 +/- 1.0 nmol/mg protein in control cells vs 5.85 +/- 0.7 nmol/mg protein in tunicamycin-treated cells, p < 0.01, n = 4) and sodium-dependent pHi recovery from an acid load (0.46 +/- 0.05 pH/min in control cells vs 0.35 +/- 0.04 pH/min in tunicamycin-treated cells, p < 0.02, n = 6). Lactate dehydrogenase (LDH) concentration in the medium was the same in both groups (p > 0.05), indicating that the inhibitory effect of tunicamycin was not caused by cell toxicity. Northern hybridization of poly(A)+ RNA from LLC-PK1 cells illustrated that in tunicamycin-treated cells, NHE-3 mRNA expression increased threefold over control cells. Immunoblots of luminal membranes from control LLC-PK, cells with specific NHE-3 antiserum showed a doublet at 94 to 95 kd and a band at 90 kd. Luminal membranes from tunicamycin-treated cells showed only one strong band at 95 kd. NHE-3 immunoblots of whole cell extract from tunicamycin-treated cells showed that in addition to the 95 kd protein, an 87 kd band was also detected. These results are consistent with the possibility that the two bands in the 94 and 90 kd areas became deglycosylated and did not reach the membrane in the presence of tunicamycin. We conclude that glycosylation of the Na+/H+ exchanger isoform NHE-3 is essential for antiporter activity in LLC-PK, cells. The results further suggest that glycosylation of NHE-3 mediates the translocation and insertion of this exchanger in the plasma membrane.

A UNIQUE NA+/H+ EXCHANGER, ANALOGOUS TO NHE1, IN THE CHICKEN EMBRYONIC FIBROBLAST

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 μM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-α, 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.