Ursula Seidler

Intestinal Secretory Defects and Dwarfism in Mice Lacking cGMP-Dependent Protein Kinase II

Cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP. Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates. These results indicate that cGKII plays a central role in diverse physiological processes.

A FUNCTIONAL CFTR PROTEIN IS REQUIRED FOR MOUSE INTESTINAL CAMP-, CGMP- AND CA2+-DEPENDENT HCO3- SECRETION

1 Most segments of the gastrointestinal tract secrete HCO3−, but the molecular nature of the secretory mechanisms has not been identified. We had previously speculated that the regulator for intestinal electrogenic HCO3− secretion is the cystic fibrosis transmembrane regulator (CFTR) channel. To prove this hypothesis, we have now measured HCO3− secretion by pH‐stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein (‘CF‐mice’) and their normal littermates. 2 Basal HCO3− secretory rates were reduced in all small intestinal segments of CF mice. Forskolin, PGE2, 8‐bromo‐cAMP and VIP (cAMP‐dependent agonists), heat‐stable enterotoxin of Escherichia coli (STa), guanylin and 8‐bromo‐cGMP (cGMP‐dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short‐circuit current (ISC) and the HCO3− secretory rate (JHCO3‐) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3‐ in the intestine of CF mice. 3 To investigate whether Cl−–HCO3− exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied CF gradient‐driven 36Cl− uptake into brush‐border membrane (BBM) vesicles isolated from normal and CF mouse small intestine. Both the time course and the peak value for 4,4’‐diisothiocyanostilbene‐2’,2‐disulphonic acid (DIDS)‐inhibited 36Cl− uptake was similar in normal and CF mice BBM vesicles. 4 In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist‐induced stimulation of electrogenic HCO3− secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3− secretion exclusively via activation of the CFTR channel.

Slc2a5 (Glut5) Is Essential for the Absorption of Fructose in the Intestine and Generation of Fructose-induced Hypertension

The identity of the transporter responsible for fructose absorption in the intestine in vivo and its potential role in fructose-induced hypertension remain speculative. Here we demonstrate that Glut5 (Slc2a5) deletion reduced fructose absorption by ∼75% in the jejunum and decreased the concentration of serum fructose by ∼90% relative to wild-type mice on increased dietary fructose. When fed a control (60% starch) diet, Glut5-/- mice had normal blood pressure and displayed normal weight gain. However, whereas Glut5+/+ mice showed enhanced salt absorption in their jejuna in response to luminal fructose and developed systemic hypertension when fed a high fructose (60% fructose) diet for 14 weeks, Glut5-/- mice did not display fructose-stimulated salt absorption in their jejuna, and they experienced a significant impairment of nutrient absorption in their intestine with accompanying hypotension as early as 3–5 days after the start of a high fructose diet. Examination of the intestinal tract of Glut5-/- mice fed a high fructose diet revealed massive dilatation of the caecum and colon, consistent with severe malabsorption, along with a unique adaptive up-regulation of ion transporters. In contrast to the malabsorption of fructose, Glut5-/- mice did not exhibit an absorption defect when fed a high glucose (60% glucose) diet. We conclude that Glut5 is essential for the absorption of fructose in the intestine and plays a fundamental role in the generation of fructose-induced hypertension. Deletion of Glut5 results in a serious nutrient-absorptive defect and volume depletion only when the animals are fed a high fructose diet and is associated with compensatory adaptive up-regulation of ion-absorbing transporters in the colon.

Expression of AE2 anion exchanger in mouse intestine

We have characterized expression of anion exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA abundance was higher in colon than in more proximal segments. AE2a mRNA was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2 polypeptide abundance as detected by immunoblot qualitatively paralleled that of mRNA, whereas AE2 immunostaining exhibited a more continuous decrease in intensity from colon to duodenum. AE2 polypeptide was more abundant in colonic surface cells than in crypts, whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2 epitopes was restricted to the basolateral membranes of epithelial cells throughout the intestine with three exceptions. Under mild fixation conditions, anti-AE2 amino acids (aa) 109-122 detected nonpolarized immunostaining of ileal enterocytes and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224-1237 was also localized to subapical regions of Brunners gland ducts of duodenum and upper jejunum. These localization studies will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.

Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9−/−) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9−/− mice, in contrast to the predominant cytoplasmic localization in Slc26a9+/+ mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9−/− stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl−/HCO3− exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.

Effect of inhibitors of Na+/H+‐exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N‐formyl‐Met‐Leu‐Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP‐induced cell swelling. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP‐induced increase in CV. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP‐stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. Western analysis with a monoclonal anti gastric H+/K+ATPase β‐subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+‐dependent H+‐extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

Tissue-specific Regulation of Sodium/Proton Exchanger Isoform 3 Activity in Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Null Mice cAMP INHIBITION IS DIFFERENTIALLY DEPENDENT ON NHERF1 AND EXCHANGE PROTEIN DIRECTLY ACTIVATED BY cAMP IN ILEUM VERSUS PROXIMAL TUBULE

The multi-PDZ domain containing protein Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) binds to Na+/H+ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2′O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

Na+/HCO3− cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells

BACKGROUND & AIMS The gastric epithelium protects itself against luminal acid by secreting HCO3--rich fluid into the mucous layer and by HCO3--dependent intracellular pH regulation, but the basolateral HCO3- uptake mechanisms are incompletely characterized. This study examined the expression and functional significance of the Na+/HCO3- cotransporters NBC1 and NBC2 in rabbit gastric epithelial cells. METHODS Rabbit NBC1 and NBC2 complementary DNA fragments were cloned and sequenced, and cellular expression levels were assessed by semiquantitative polymerase chain reaction. Na+/HCO3- cotransport activity was measured fluorometrically in cultured rabbit parietal and mucous cells. RESULTS NBC1 expression was 4.5-fold lower in the stomach than kidney cortex and 5.5-fold higher in mucous than parietal cells. NBC2 expression in the stomach was much lower than in the eye, approximately 4-fold lower than NBC1 expression in the stomach, and 2.5-fold higher in mucous than parietal cells. The Na+- and HCO3--dependent, dimethylamiloride-insensitive (which at 500 micromol/L completely inhibits all Na+/H+ exchanger isoforms) base influx rates were 4.6 +/- 0.02 and 16.2 +/- 0.04 mmol/L/min in acidified parietal and mucous cells, respectively, and were not significantly different in the absence of Cl-. CONCLUSIONS This study shows that NBC1 and NBC2 are expressed in rabbit stomach, with high levels in mucous cells where Na+/HCO3- cotransport is the major base-importing mechanism in the presence of CO2/HCO3-.

Deletion of the chloride transporter Slc26a7 causes distal renal tubular acidosis and impairs gastric acid secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl−/HCO3− exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.

The switch of intestinal Slc26 exchangers from anion absorptive to HCO3- secretory mode is dependent on CFTR anion channel function

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO(3)(-)) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO(3)(-) secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl(-)/HCO(3)(-) exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO(3)(-) secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO(3)(-) secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na(+)/H(+) exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl(-) absorptive mode and Cl(-) exit via CFTR to operate in the HCO(3)(-) secretory mode.