Cyclooxygenase 1 is required for pH control at the mouse gastric surface.

BACKGROUND: Endogenous cyclooxygenase (COX) activity is required to maintain a relatively alkaline surface pH at the gastric luminal surface. AIMS: The purpose of this study was to determine which COX isoform, COX-1 or COX-2, is responsible for regulating the protective surface pH gradient and to test if COX inhibitors also had non-COX mediated effects in vivo. METHODS: Immunofluorescence and western blot analysis showed constitutive expression of both COX isoforms in the normal mouse stomach. We used in vivo confocal microscopy to measure pH near the mucosal surface of anaesthetised COX-1 (-/-), COX-2 (-/-), or wild-type mice of the same genetic background. RESULTS: When the gastric mucosal surface was exposed and superfused (0.2 ml/min) with a weakly buffered saline solution (pH 3) containing the pH indicator Cl-NERF, the pH directly at the gastric surface and thickness of the pH gradient were similar in wild-type and COX-2 (-/-) mice, but COX-1 (-/-) mice had a significantly thinner pH gradient. Addition of indomethacin had minimal effects on the residual surface pH gradient in COX-1 (-/-) mice, suggesting no role for COX-2 in surface pH regulation. Whole stomach perfusion studies demonstrated diminished net alkali secretion in COX-1 (-/-) mice, and application of SC-560 or rofecoxib to wild-type mice and mutant mice confirmed that only COX-1 inhibition reduced alkali secretion. CONCLUSION: COX-1 is the dominant isoform regulating the normal thickness of the protective surface pH gradient in mouse stomach.

Direct measurement of acid permeation into rat oesophagus.

BACKGROUND AND AIMS: The early responses of the oesophageal mucosa to acid perfusion may predict subsequent pathology. Mucosal responses to luminal acid may result either from acid permeating through the mucosa or from other unknown transduction mechanisms. In order to better understand the dynamics of acid permeation into the oesophageal mucosa, we measured interstitial pH (pH(int)) of the oesophageal basal epithelial layer, pre-epithelial layer thickness, and blood flow in rats in vivo during luminal acid challenge. A novel confocal microscopic technique was used in vitro to measure pH(int) from defined cellular sites in response to luminal and basolateral acidification. METHODS: 5-(and-6)-Carboxyfluorescein (CF) and carboxy-seminapthorhodofluor-1 (SNARF-1) fluorescence was used to measure pH(int) by conventional and confocal microscopy, respectively, in urethane anaesthetised rats. Pre-epithelial layer thickness was measured optically with carbon particles as markers. Blood flow was measured with laser Doppler flowmetry. RESULTS: Luminal acidification failed to alter pH(int) in vivo and in vitro, but pH(int) was lowered by modest serosal acidification. Pre-epithelial layer thickness and blood flow increased significantly during luminal surface acid perfusion. Indomethacin had no effect on any acid related response. CONCLUSION: In this first dynamic measurement of oesophageal acid permeation and pre-epithelial layer thickness, pH(int) was preserved in spite of high luminal acidity by two complementary techniques. Despite the apparent permeability barrier to acid permeation, oesophageal blood flow and thickness responded to luminal acid perfusion.

Intragastric pH regulates conversion from net acid to net alkaline secretion by the rat stomach.

Our previous report showed gastric mucosal surface pH was determined by alkali secretion at intragastric luminal pH 3 but by acid secretion at intragastric pH 5. Here, we question whether regulation of mucosal surface pH is due to the effect of luminal pH on net acid/base secretions of the whole stomach. Anesthetized rats with a gastric cannula were used, the stomach lumen was perfused with weakly buffered saline, and gastric secretion was detected in the gastric effluent with 1) a flow-through pH electrode and 2) a fluorescent pH-sensitive dye (Cl-NERF). During pH 5 luminal perfusion, both pH sensors reported the gastric effluent was acidic (pH 4.79). After perfusion was stopped transiently (stop-flow), net acid accumulation was observed in the effluent when perfusion was restarted (peak change to pH 4.1-4.3). During pH 3 luminal perfusion, both pH sensors reported gastric effluent was close to perfusate pH (3.0-3.1), but net alkali accumulation was detected at both pH sensors after stop-flow (peak pH 3.3). Buffering capacity of gastric effluents was used to calculate net acid/alkaline secretions. Omeprazole blocked acid secretion during pH 5 perfusion and amplified net alkali secretion during pH 3 perfusion. Pentagastrin elicited net acid secretion under both luminal pH conditions, an effect antagonized by somatostatin. We conclude that in the basal condition, the rat stomach was acid secretory at luminal pH 5 but alkaline secretory at luminal pH 3.

Decrease in rat taste receptor cell intracellular pH is the proximate stimulus in sour taste transduction.

Taste receptor cells (TRCs) respond to acid stimulation, initiating perception of sour taste. Paradoxically, the pH of weak acidic stimuli correlates poorly with the perception of their sourness. A fundamental issue surrounding sour taste reception is the identity of the sour stimulus. We tested the hypothesis that acids induce sour taste perception by penetrating plasma membranes as H(+) ions or as undissociated molecules and decreasing the intracellular pH (pH(i)) of TRCs. Our data suggest that taste nerve responses to weak acids (acetic acid and CO(2)) are independent of stimulus pH but strongly correlate with the intracellular acidification of polarized TRCs. Taste nerve responses to CO(2) were voltage sensitive and were blocked with MK-417, a specific blocker of carbonic anhydrase. Strong acids (HCl) decrease pH(i) in a subset of TRCs that contain a pathway for H(+) entry. Both the apical membrane and the paracellular shunt pathway restrict H(+) entry such that a large decrease in apical pH is translated into a relatively small change in TRC pH(i) within the physiological range. We conclude that a decrease in TRC pH(i) is the proximate stimulus in rat sour taste transduction.

pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers.

In the colonic mucosa, short-chain fatty acids change intracellular pH (pH(i)) and extracellular pH (pH(e)). In this report, confocal microscopy and dual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were used for direct evaluation of pH(i) and pH(e) in a simple model epithelium, HT29-C1 cells. Live cell imaging along the apical-to-basal axis of filter-grown cells allowed simultaneous measurement of pH in the aqueous environment near the apical membrane, the lateral membrane, and the basal membrane. Subapical cytoplasm reported the largest changes in pH(i) after isosmotic addition of 130 mM propionate or 30 mM NH(4)Cl. In resting cells and cells with an imposed acid load, lateral membranes had pH(i) values intermediate between the relatively acidic subapical region (pH 6.3-6.9) and the relatively alkaline basal pole of the cells (pH 7.4-7.1). Transcellular pH(i) gradients were diminished or eliminated during an induced alkaline load. Propionate differentially altered pH(e) near the apical membrane, in lateral intracellular spaces between adjacent cells, and near the basal membrane. Luminal or serosal propionate caused alkalinization of the cis compartment (where propionate was added) but acidification of the trans compartment only in response to luminal propionate. Addition of NH(4)Cl produced qualitatively opposite pH(e) excursions. The microscopic values of pH(i) and pH(e) can explain a portion of the selective activation of polarized Na/H exchangers observed in HT29-C1 cells in the presence of transepithelial propionate gradients.

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.

Postnatal maturation of carotid body and type I cell chemoreception in the rat.

The site of postnatal maturation of carotid body chemoreception is unclear. To test the hypothesis that maturation occurs synchronously in type I cells and the whole carotid body, the development of changes in the intracellular Ca2+ concentration responses to hypoxia, CO2, and combined challenges was studied with fluorescence microscopy in type I cells and compared with the development of carotid sinus nerve (CSN) responses recorded in vitro from term fetal to 3-wk animals. Type I cell responses to all challenges increased between 1 and 8 days and then remained constant, with no multiplicative O2-CO2 interaction at any age. The CSN response to hypoxia also matured by 8 days, but CSN responses to CO2 did not change significantly with age. Multiplicative O2-CO2 interaction occurred in the CSN response at 2-3 wk but not in younger groups. We conclude that type I cell maturation underlies maturation of the CSN response to hypoxia. However, because development of responses to CO2 and combined hypoxia-CO2 challenges differed between type I cells and the CSN, responses to these stimuli must mature at other, unidentified sites within the developing carotid body.

Dynamic regulation of gastric surface pH by luminal pH.

In vivo confocal imaging of the mucosal surface of rat stomach was used to measure pH noninvasively under the mucus gel layer while simultaneously imaging mucus gel thickness and tissue architecture. When tissue was superfused at pH 3, the 25 microm adjacent to the epithelial surface was relatively alkaline (pH 4.1 +/- 0.1), and surface alkalinity was enhanced by topical dimethyl prostaglandin E2 (pH 4.8 +/- 0.2). Luminal pH was changed from pH 3 to pH 5 to mimic the fasted-to-fed transition in intragastric pH in rats. Under pH 5 superfusion, surface pH was relatively acidic (pH 4.2 +/- 0.2). This surface acidity was enhanced by pentagastrin (pH 3.5 +/- 0.2) and eliminated by omeprazole, implicating parietal cell H,K-ATPase as the dominant regulator of surface pH under pH 5 superfusion. With either pH 5 or pH 3 superfusion (a) gastric pit lumens had the most divergent pH from luminal superfusates; (b) qualitatively similar results were observed with and without superfusion flow; (c) local mucus gel thickness was a poor predictor of surface pH values; and (d) no channels carrying primary gastric gland fluid through the mucus were observed. The model of gastric defense that includes an alkaline mucus gel and viscous fingering of secreted acid through the mucus may be appropriate at the intragastric pH of the fasted, but not fed, animal.

Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid.

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHi gradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 microM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50 = 1.4 microM, basolateral ID50 = 0.3 microM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHi recovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHi gradients and 2) the predominant site of pHi acidification and pHi recovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHi regulation.

Resetting and postnatal maturation of oxygen chemosensitivity in rat carotid chemoreceptor cells.

1. Carotid chemoreceptor sensitivity is minimal immediately after birth and increases with postnatal age. In the present study we have investigated the peri- and postnatal developmental time course of [Ca2+]i responses to hypoxia in clusters of type I cells isolated from near-term fetal rats and rats that were 1, 3, 7, 11, 14 and 21 days old, using the Ca2+-sensitive fluoroprobe fura-2. 2. In type I cells from all age groups a graded increase in [Ca2+]i occurred in response to lowering the PO2 from 150 mmHg to 70, 35, 14, 7, 2 and 0 mmHg. The graded [Ca2+]i response to hypoxia was hyperbolic at all ages. 3. Type I cells from rats near-term fetal to 1 day old exhibited small [Ca2+]i responses, mainly to the most severe levels of hypoxia. After day 1, an increase in the [Ca2+]i responses to submaximal hypoxia stimulation resulted in a rightward shift in the O2 response curve. Using the Delta[Ca2+]i between 35 and 2 mmHg PO2 as an index of O2 sensitivity, type I cell O2 sensitivity increased approximately 4- to 5-fold between near-term fetal to 1 day old and 11 to 14 days of age. 4. Exposure to elevated extracellular potassium (10, 20 and 40 mM K+) caused a dose-dependent [Ca2+]i rise in type I cells from all age groups. There were no age-related changes in [Ca2+]i responses to any level of K+ between near-term fetal and 21 days. 5. We conclude that the maximal type I cell [Ca2+]i response to anoxia, as well as the sensitivity to submaximal hypoxic stimulation, of rats aged from near-term fetal to 21 days depends on the level of postnatal maturity. The lack of an age-related increase in the [Ca2+]i response to elevated K+ during the timeframe of maximal development of O2 sensitivity suggests that resetting involves maturation of O2 sensing, rather than non-specific developmental changes in the [Ca2+]i rise resulting from depolarization.

GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor.

The incretin hormone glucagon-like peptide-1 (GLP-1)-(7-36) amide is best known for its antidiabetogenic actions mediated via a GLP-1 receptor present on pancreatic endocrine cells. To investigate the molecular mechanisms of GLP-1 action in muscle, we used cultured L6 myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogen synthesis by 140% while stimulating CO2 production and lactate formation by 150%. In the presence of IBMX, GLP-1 diminished cAMP levels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(9-39), inhibited GLP-1-mediated glycogen synthesis in GLP-1 receptor-transfected L6 myotubes. However, in parental L6 myotubes, exendin-4-(9-39) and GLP-1-(1-36) amide, an inactive peptide on pancreatic GLP-1 receptor, displaced 125I-labeled GLP-1 binding and stimulated glycogen synthesis by 186 and 130%, respectively. These results suggest that the insulinomimetic effects of GLP-1 in L6 cells are likely to be mediated by a receptor that is different from the GLP-1 receptor found in the pancreas.

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.

Transepithelial SCFA gradients regulate polarized Na/H exchangers and pH microdomains in colonic epithelia.

Short chain fatty acids (SCFAs) stimulate electroneutral sodium absorption by activation of apical Na/H exchange in colonocytes. It is often assumed that activation of Na/H exchange is via an intracellular acidification caused by SCFA uptake. These lecture notes review shortcomings in this model of SCFA-stimulated sodium absorption, revealed by recent reports in the literature. This is supplemented by information generated in our laboratory using both a tissue culture model of colonocytes (HT29-C1 cells) and a native tissue preparation (mouse distal colonic mucosa). In both preparations, evidence suggests that physiologic SCFA gradients may generate pH heterogeneity in aqueous microdomains near the plasma membrane of colonocytes. Finally, direct observation of such extracellular microdomains with confocal microscopy is used to support a new model, in which pH microdomains play an important role in regulating both SCFA fluxes and sodium absorption.

Transepithelial SCFA fluxes link intracellular and extracellular pH regulation of mouse colonocytes.

We have studied pH regulation in both intracellular and extracellular compartments of mouse colonic crypts, using distal colonic mucosa with intact epithelial architecture. In this work, we question how transepithelial SCFA gradients affect intracellular pH (pHi) and examine interactions between extracellular pH (pHo) and pHi regulation in crypts of distal colonic epithelium from mouse. We studied pH regulation in three adjacent compartments of distal colonic epithelium (crypt lumen, crypt epithelial cell cytosol, and lamina propria) with SNARF-1 (a pH sensitive fluorescent dye), digital imaging microscopy (for pHi), and confocal microscopy (for pHo). Combining results from the three compartments allows us to find how pHi and pHo are regulated and related under the influence of physiological transepithelial SCFA gradients, and develop a better understanding of pH regulation mechanisms in colonic crypts. Results suggest a complex interdependency between SCFA fluxes and pHo values, which can directly affect how strongly SCFAs acidify colonocytes.

Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3-L1 adipocytes.

Glucagon-like peptide-1 (7-36) amide (GLP-1), in addition to its well known effect of enhancing glucose-mediated insulin release, has been shown to have insulinomimetic effects and to enhance insulin-mediated glucose uptake and lipid synthesis in 3T3-L1 adipocytes. To elucidate the mechanisms of GLP-1 action in these cells, we studied the signal transduction and peptide specificity of the GLP-1 response. In 3T3-L1 adipocytes, GLP-1 caused a decrease in intracellular cAMP levels which is the opposite to the response observed in pancreatic beta cells in response to the same peptide. In 3T3-L1 adipocytes, free intracellular calcium was not modified by GLP-1. Peptide specificity was examined to help determine if a different GLP receptor isoform was expressed in 3T3-L1 adipocytes vs. beta cells. Peptides with partial homology to GLP-1 such as GLP-2, GLP-1 (1-36), and glucagon all lowered cAMP levels in 3T3-L1 adipocytes. In addition, an antagonist of pancreatic GLP-1 receptor, exendin-4 (9-39), acted as an agonist to decrease cAMP levels in 3T3-L1 adipocytes as did exendin-4 (1-39), a known agonist for the pancreatic GLP-1 receptor. Binding studies using 125I-GLP-1 also suggest that pancreatic GLP-1 receptor isoform is not responsible for the effect of GLP-1 and related peptides in 3T3-L1 adipocytes. Based on these results, we propose that the major form of the GLP receptor in 3T3-L1 adipocytes is functionally different from the pancreatic GLP-1 receptor.

Gene targeting of a CFTR allele in HT29 human epithelial cells.

HT29 cells endogenously express the cystic fibrosis transmembrane conductance regulator (CFTR) and have been used previously as a model to examine cellular regulation of CFTR expression and chloride secretory function. Homologous recombination has been used to specifically disrupt CFTR transcription in the HT29-18-C1 subclone. Experiments demonstrate successful disruption of a CFTR allele by DNA constructs, which target insertion of the neomycin phosphotransferase gene into CFTR exon 1 via homologous recombination. The mutation of one allele is a partial knockout because this cell line has multiple CFTR alleles. The mutation is confirmed by polymerase chain reaction (PCR) and genomic Southern blot analysis. A 52-68% reduction in CFTR mRNA levels is observed in the mutant cell line by both Northern and PCR analysis. However, Western blots show no decrease in total CFTR protein levels. Consistent with the lack of reduction in CFTR protein, the partial knockout mutant does not demonstrate alterations in cyclic AMP or calcium stimulation of chloride efflux or net osmolyte loss. Results suggest that posttranscriptional regulation of CFTR levels may contribute to maintenance of cellular chloride transport function.