Lupeol: A Triterpenoid Isolated from the Stem Bark of Hymenocardia Acida (tul.) Exhibits a van der Waal Antagonism on the Alpha Subunit of Gastric H+K+Atpase - A Promising Antiulcer Principle.

BACKGROUND: Hymenocardia acida (HA) is one of the numerous medicinal plants in Nigeria with ethnomedicinal history of usage in the treatment of ulcer. The study aimed at isolating antiulcer principle(s) from the stem bark of HA as well as the mechanism of action determination. METHODS: Antiulcer screenings of the crude extract, aqueous fraction, and bulked VLC fractions were performed using in vivo and in vitro models. Docking was carried out by using PyRx. RESULTS: Crude extract (HA; 1 mg/mL) and the aqueous fraction of H. acida (HAA; 1 mg/mL) showed an acid neutralizing capacity (MEq) of 0.3948 and 0.4035, respectively which is significantly different from 0.431 MEq showed by negative control (distilled water) at p<0.05. BVLC 3 (1 mg/mL) showed a significant value of 0.4049 MEq. However, HA showed a dose-dependent decrease in activity across doses examined, with 100 mg/kg showing an ulcer index of 10.00±2.89 (61.50%) and cimetidine (positive control; 100 mg/kg), also showed the highest ulcer index of 3.67±0.88 (85.9%), which is significantly different from ulcer index of 26.00±6.35 (0.00%) p<0.05 observed in the negative control (5% dimethylsulphoxide). The highest ulcer index of 8.00±1.32 (65.10%) was noted in BVLC 3. Bioactive BVLC 3, resulted in an isolated compound (BF3B2A). The compound was suggested to be lupeol, with a docking score of -7.7. It showed a van der Waal interaction with some key amino acid residues in the vonoprazan binding site. CONCLUSION: The experimental studies justify the ethnomedicinal claim of usage among locals.

Effects of high fat diet on kidney lipid content and the Na, K-ATPase activity

ABSTRACT It is widely known that high fat diet (HFD) can contribute to the advent of health problems. Recent studies have indicated that obesity imposes a hemodynamic overload to the kidneys. In order to further investigate such injuries, two groups of six Swiss mice each were fed with a controlled AIN93G diet or a high fat (AIN93G modified) diet for eight weeks. Blood samples were collected to determine the hormonal, lipid profile, glucose, urea, and creatinine levels. Histopathological and immunohistochemical analysis were carried out to analysis the kidney damage. Fractions of renal membranes were prepared to assess the Na,K-ATPase activity, lipid peroxidation, total cholesterol, and phospholipid content. The results indicated that the blood lipid profile, urea and creatinine was not altered by the HFD. On the other hand, it was observed in HFD diet mice elevated glucose blood levels along with an augment on insulin and a decrease on corticosterone release. HFD provoked a reduction in the diameter of the convoluted tubules and cell volume in Bowman's capsule and an increased number of positive cells with Na,K-ATPase, but reduced the Na,K-ATPase activity and the cholesterol content in the kidney cell membrane but favored the lipid peroxidation.

Western blot confirmation of the H+/K+-ATPase proton pump in the human larynx and submandibular gland.

OBJECTIVE: The authors have previously demonstrated the H(+)/K(+)-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis. STUDY DESIGN: Prospective controlled tissue analysis study. SETTING: Academic medical institution. METHODS: Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or ß subunits of the H(+)/K(+)-ATPase. Western blot analysis was performed on all specimens. RESULTS: Consistent IHC staining was observed in the submandibular gland specimens for both α and ß subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated ß subunit protein, as well as the 52-kDa ß subunit precursor for all specimens. CONCLUSION: The H(+)/K(+)-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.

The renal H,K-ATPases.

PURPOSE OF REVIEW: We integrate recent evidence that demonstrates the importance of the gastric (HKalpha1) and nongastric (HKalpha2)-containing hydrogen potassium adenosine triphosphatases (H,K-ATPases) on physiological function and their role in potassium (K), sodium (Na), and acid-base balance. RECENT FINDINGS: Previous studies focused on the primary role of H,K-ATPases as a mechanism of K conservation during states of K deprivation. Both isoforms function in H secretion and K absorption in vivo during K deprivation, but recent findings show that these pumps also function in acid secretion in animals fed normal K-replete diets. The complicated pharmacological inhibition of both pumps is reviewed. Interestingly, HKalpha2-null mice have a reduced expression and activity of the renal epithelial Na channel alpha subunit in the colon. When the human nongastric isoform was studied in a heterologous expression system with its cognate beta subunit (NaKbeta1), the pump exhibited substantial Na affinity at the 'K'-binding site. Evidence cited herein raises the possibility that either directly or indirectly the renal HKalpha2-containing H,K-ATPase may affect Na balance. SUMMARY: Both H,K-ATPase isoforms are active in normal animals and not just under conditions of K depletion. The possibility that either one or both isoforms contribute to Na absorption, particularly in humans, raises important clinical implications for these pumps in the kidney.

Effects of streptozotocin-induced long-term diabetes on parietal cell function and morphology in rats.

Gastric pathology is a common complication in diabetes mellitus. The aim of the study was to evaluate the functions and morphological changes of the parietal cells of the rat stomach after streptozotocin-induced diabetes. Diabetes mellitus was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (60 mg/kg body weight). The rats were weighed weekly and sacrificed after 6 months. The glandular portion of the stomach was removed and processed for H(+)-K(+)-ATPase immunohistochemistry and light and electron microscopy studies. Acid secretion was measured in vivo. After 6 months of diabetes, the mean weight of the rats was significantly lower (P < 0.001) compared to control. The mean weight of the stomach to body weight percentage increased significantly (P < 0.001) compared to control. The blood glucose level in diabetic rats was significantly higher (P < 0.001) than in normal control. Diabetic rats showed significant (P < 0.001) decrease in basal and stimulated acid secretion when compared to control. Electron micrographs of the parietal cells of glandular stomach of diabetic rats revealed significant (P < 0.0002) reduction in the number of mitochondria and a small though not significant increase in the number of canaliculi in the parietal cells compared with normal. Immunohistochemistry showed reduced H(+)-K(+)-ATPase (P < 0.00001) compared to control. Long-term diabetes induces morphological as well as functional changes in gastric parietal cells. The decrease in the number of mitochondria accompanied by reduced in H(+)-K(+)-ATPase in parietal cells may explain the reduced acid secretion observed in diabetics.

H,K-ATPase protein localization and Kir4.1 function reveal concordance of three axes during early determination of left-right asymmetry.

Consistent laterality is a fascinating problem, and study of the Xenopus embryo has led to molecular characterization of extremely early steps in left-right patterning: bioelectrical signals produced by ion pumps functioning upstream of asymmetric gene expression. Here, we reveal a number of novel aspects of the H+/K+-ATPase module in chick and frog embryos. Maternal H+/K+-ATPase subunits are asymmetrically localized along the left-right, dorso-ventral, and animal-vegetal axes during the first cleavage stages, in a process dependent on cytoskeletal organization. Using a reporter domain fused to molecular motors, we show that the cytoskeleton of the early frog embryo can provide asymmetric, directional information for subcellular transport along all three axes. Moreover, we show that the Kir4.1 potassium channel, while symmetrically expressed in a dynamic fashion during early cleavages, is required for normal LR asymmetry of frog embryos. Thus, Kir4.1 is an ideal candidate for the K+ ion exit path needed to allow the electroneutral H+/K+-ATPase to generate voltage gradients. In the chick embryo, we show that H+/K+-ATPase and Kir4.1 are expressed in the primitive streak, and that the known requirement for H+/K+-ATPase function in chick asymmetry does not function through effects on the circumferential expression pattern of Connexin43. These data provide details crucial for the mechanistic modeling of the physiological events linking subcellular processes to large-scale patterning and suggest a model where the early cytoskeleton sets up asymmetric ion flux along the left-right axis as a system of planar polarity functioning orthogonal to the apical-basal polarity of the early blastomeres.

Helicobacter pylori eradication restored sonic hedgehog expression in the stomach.

BACKGROUND/AIMS: Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Shh expression is reportedly to be reduced in Helicobacterpylori-associated gastritis. The aim of this study was to assess the restoration of Shh expression after H. pylori eradication. METHODOLOGY: Twenty H. pylori-positive patients who underwent upper gastrointestinal endoscopy before and after the eradication were studied. Biopsy specimens were taken from the greater curvature of the middle third of the stomach body. The specimens were evaluated for the severity of acute and chronic inflammation and for that of mucosal atrophy, based on the updated Sydney system. Immunohistochemistry for Shh and H(+)-, K(+)-ATPase was also performed; the percentages of positively stained epithelial cells for the two molecules were expressed as the Shh index and H(+)-, K(+)-ATPase index, respectively. RESULTS: There was a significant decrease in the acute and chronic inflammation scores and also in the mucosal atrophy score following the eradication. The Shh and H(+)-, K(+)-ATPase index were significantly increased following the eradication. CONCLUSIONS: Suppressed Shh expression in the gastric mucosa by H. pylori infection was significantly restored following eradication of the infection.

Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling.

Gastric parietal cells possess an amplified apical membrane recycling system dedicated to regulated apical recycling of H-K-ATPase. While amplified in parietal cells, apical recycling is critical to polarized secretory processes in most epithelial cells. To clarify putative regulators of apical recycling, we prepared immunoisolated parietal cell H-K-ATPase-containing recycling membranes from human stomachs and analyzed protein contents by tryptic digestion and mass spectrometry. We identified and validated by Western blots many of the proteins previously identified on immunoisolated rabbit tubulovesicles, including Rab11, Rab25, syntaxin 3, secretory carrier membrane proteins (SCAMPs), and vesicle-associated membrane protein (VAMP)2. In addition, we detected several previously unrecognized proteins, including Rab10, VAMP8, syntaxin 7, and syntaxin 12/13. We also identified the K(+) channel component KCNQ1. Immunostaining of human gastric mucosal sections confirmed the presence of each of these proteins in parietal cells and their colocalization with H-K-ATPase on tubulovesicles. To investigate the role of the identified soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in apical recycling, we transfected them as DsRed2 fusions into an enhanced green fluorescent protein (EGFP)-Rab11a-expressing Madin-Darby canine kidney (MDCK) cell line. Syntaxin 12/13 and VAMP8 caused a collapse of the EGFP-Rab11a compartment, whereas a less dramatic effect was observed in cells transfected with syntaxin 3, syntaxin 7, or VAMP2. The five DsRed2-SNARE chimeras were also transfected into a MDCK cell line overexpressing Rab11-FIP2(129-512). All five of the chimeras were drawn into the collapsed apical recycling system. This study, which represents the first proteomic analysis of an immunoisolated vesicle population from native human tissue, demonstrates the diversity of putative regulators of the apical recycling system.

Dynamic functional and ultrastructural changes of gastric parietal cells induced by water immersion-restraint stress in rats.

AIM: To investigate the dynamic functional and ultrastructural changes of gastric parietal cells induced by water immersion-restraint stress (WRS) in rats. METHODS: WRS model of Sprague-Dawley (SD) rats was established. Fifty-six male SD rats were randomly divided into control group, stress group and post-stress group. The stress group was divided into 1, 2 and 4 h stress subgroups. The post-stress group was divided into 24, 48 and 72 h subgroups. The pH value of gastric juice, ulcer index (UI) of gastric mucosa and H(+), K(+)-ATPase activity of gastric parietal cells were measured. Ultrastructural change of parietal cells was observed under transmission electron microscope (TEM). RESULTS: The pH value of gastric juice decreased time-dependently in stress group and increased in post-stress group. The H(+), K(+)-ATPase activity of gastric parietal cells and the UI of gastric mucosa increased time-dependently in stress group and decreased in post-stress group. Compared to control group, the pH value decreased remarkably (P = 0.0001), the UI and H(+), K(+)-ATPase activity increased significantly (P = 0.0001, P = 0.0174) in 4 h stress subgroup. UI was positively related with stress time (r = 0.9876, P < 0.01) but negatively with pH value (r = -0.8724, P < 0.05). The parietal cells became active in stress group, especially in 4 h stress subgroup, in which plenty of intracellular canalicular and mitochondria were observed under TEM. In post-stress group, the parietal cells recovered to resting state. CONCLUSION: The acid secretion of parietal cells is consistent with their ultrastructural changes during the development and healing of stress ulcer induced by WRS and the degree of gastric mucosal lesions, suggesting gastric acid play an important role in the development of stress ulcer and is closely related with the recovery of gastric mucosal lesions induced by WRS.

Promiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells.

"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.

Attenuating effect of H+K+ATPase inhibitors on airway cough hypersensitivity induced by allergic airway inflammation in guinea-pigs.

BACKGROUND: Gastrooesophageal reflux (GER) is a frequent cause of chronic cough. Several investigators have indicated that inhibitors of H(+)K(+)ATPase (proton pump inhibitors; PPIs) could relieve coughing via inhibition of acid reflux. However, we considered that PPIs might directly inhibit increased cough reflex sensitivity. OBJECTIVE: The present study was designed to examine whether PPIs directly inhibit antigen-induced increase in cough reflex sensitivity and to elucidate the mechanism. METHODS: Actively sensitized guinea-pigs were challenged with aerosol antigen (ovalbumin, OVA) and cough reflex sensitivity to inhaled capsaicin was measured 24 h later. The PPIs (omeprazole and rabeprazole) or the histamine H(2) blocker cimetidine were administered intraperitoneally 1 h before OVA challenge and before measuring cough reflex sensitivity, then bronchoalveolar lavage fluid (BALF) was immediately collected. The pH of the fluid obtained by bronchial washing was determined after examining the effect of rabeprazole on the cough response to capsaicin. RESULTS: The number of coughs elicited by capsaicin was significantly increased 24 h after challenge with OVA compared with saline, indicating antigen-induced increase in cough reflex sensitivity. Both PPIs dose dependently and significantly inhibited antigen-induced cough hypersensitivity. Omeprazole did not influence the antigen-induced increase in the total number of cells or ratio (%) of eosinophils in BALF. Cimetidine did not affect the antigen-induced cough hypersensitivity or cellular components of BALF. The pH of the bronchial washing fluid was significantly decreased in antigen-challenged animals. Rabeprazole did not affect the antigen-induced decrease in the pH of bronchial washing fluid. CONCLUSION: These findings show that PPIs, but not histamine H(2) blockers, can directly decrease antigen-induced cough reflex hypersensitivity, while the mechanism remains unclear.

Retinol enhances differentiation of the gastric parietal cell lineage in developing rabbits.

In the gastric glands, parietal cells are the targets for anti-ulcer drugs because they contain the proton pump or HK-ATPase responsible for acid secretion. Little is known about factors influencing developmental expression and activity of HK-ATPase. In this study, the parietal cell lineage was investigated in rabbits at post-natal days 0 (P0) to P60 by using morphological and biochemical methods. Immunohistochemical and ultrastructural studies show that the HK-ATPase-expressing cells that appear at P0 and P3 are pre-parietal cells. However, terminally differentiated, mature parietal cells make their appearance at P7. These data correlate with the activity of HK-ATPase, measured as K(+)-dependent hydrolysis of p-nitrophenyl phosphate. Three-day-retinol treatment of P3-P30 rabbits induced an increase in the (i) production of parietal cells, (ii) intensity of the HK-ATPase immunostaining per cell, (iii) activity of HK-ATPase and (iv) amount of HK-ATPase protein measured by Western blotting. In conclusion, retinol upregulates the development of HK-ATPase in rabbits, perhaps due to precocious acceleration of the differentiation program of parietal cell lineage.

Non-gastric H+/K+ ATPase is present in the microvillous membrane of the human placental syncytiotrophoblast.

In humans, the non-gastric H(+)/K(+)ATPase (ATP1AL1) has previously been shown to be expressed in the epithelia of skin, kidney and colon. In this study we tested the hypothesis that the non-gastric H(+)/K(+)ATPase is localized to the syncytiotrophoblast, the transporting epithelium of the human placenta. Microvillous (MVM) and basal plasma membranes (BM) of the syncytiotrophoblast were isolated from term placenta and membrane proteins were separated using SDS-PAGE. The ATP1AL1 protein was identified as a 114 kD band in both MVM and BM by Western blot, however, the protein was more abundant in the MVM. Using immunocytochemistry H(+)/K(+)ATPase protein was localized in MVM but not BM. We constructed primers specific for ATP1AL1 and performed RT-PCR on RNA isolated from human placenta and human kidney. A product of the expected size could be detected in both tissues after 30 cycles of amplification. The sequence identity of this 517 nucleotide product was confirmed by sequencing and found to be identical to the human non-gastric H(+)/K(+)ATPase. The activity of this proton pump appears to be low in normal healthy placental at term, however, it is speculated that MVM non-gastric H(+)/K(+)ATPase may be important in pathological states. In conclusion, non-gastric H(+)/K(+)ATPase is present in the microvillous plasma membrane of the transporting epithelia of the human placenta.

Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance.

Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.

Nongastric H-K-ATPase in rodent prostate: lobe-specific expression and apical localization.

The molecular basis of active ion transport in secretory glands such as the prostate is not well characterized. Rat nongastric H-K-ATPase is expressed at high levels in distal colon surface cell apical membranes and thus is referred to as "colonic." Here we show that the ATPase is expressed in rodent prostate complex in a lobe-specific manner. RT-PCR and Western blot analyses indicate that rat nongastric H-K-ATPase alpha-subunit (alpha(ng)) mRNA and protein are present in coagulating gland (anterior prostate) and lateral and dorsal prostate and absent from ventral lobe, whereas Na-K-ATPase alpha-subunit is present in all lobes. RT-PCR analysis shows that Na-K-ATPase alpha(4) and alpha(3) and gastric H-K-ATPase alpha-subunit are not present in significant amounts in all prostate lobes. Relatively low levels of Na-K-ATPase alpha(2) were found in lateral, dorsal, and anterior lobes. alpha(ng) protein expression is anteriodorsolateral: highest in coagulating gland, somewhat lower in dorsal lobe, and even lower in lateral lobe. Na-K-ATPase protein abundance has the reverse order: expression in ventral lobe is higher than in coagulating gland. alpha(ng) protein abundance is higher in coagulating gland than distal colon membranes. Immunohistochemistry shows that in rat and mouse coagulating gland epithelium alpha(ng) protein has an apical polarization and Na-K-ATPase alpha(1) is localized in basolateral membranes. The presence of nongastric H-K-ATPase in rodent prostate apical membranes may indicate its involvement in potassium concentration regulation in secretions of these glands.

CaMKII is activated and translocated to the secretory apical membrane during cholinergically conveyed gastric acid secretion.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is thought to be activated during the cholinergic stimulation of gastric acid secretion. The carbachol-induced acid production of cultured rabbit parietal cells was dose-dependently inhibited by the CaMKII inhibitor KN-62 as measured by accumulation of the weak base [(14)C]aminopyrine ([(14)C]-AP). Inhibition by KN-62 was most efficient at concentrations of carbachol >10(-6) M. After carbachol stimulation, we observed an activation of CaMKII activity, and its translocation to the apical membrane of gastric mucosal cells. We found a doubling of the abundance of CaMKII to the stimulus-associated apical membrane (SA vesicles) compared to the apical membrane from the resting state after carbachol induction. This was shown by both an anti-CaMKII serum and the 1.8-fold increase of the CaMKII phosphotransferase activity in vitro. The SA vesicles exhibited a strong increase of autoactivated CaMKII probed with an anti-autoactivated CaMKII antibody. Additionally, we observed a colocalization of both CaMKII and the H(+)-K(+)-ATPase of SA vesicles similar to the colocalization of both enzymes to the tubulovesicles suggesting them as at least one pool for the SA vesicular CaMKII. Our data indicate that the activation of CaMKII and the carbachol-dependent redistribution of CaMKII to the SA vesicles are distinct processes that occur in parallel to regulate the activity and localization of CaMKII. These findings contribute to the model implicating an involvement for CaMKII in the intracellular dynamics of the acid secretion.

Regulation of intracellular pH in rat renal inner medullary thin limbs of Henle's loop.

Regulation of intracellular pH (pHi) was studied in isolated rat renal inner medullary thin limbs of Henle's loop in bicarbonate/phosphate-buffered medium with high pCO2, high osmolality ( congruent with670 mosmol/kg H2O; 270 mM urea; 180 mM NaCl), organic osmolytes, and a pH of 6.8 to approximate the physiological in vivo environment. The pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi. Resting pHi was always acid and significantly more acid in descending thin limb (DTL) cells than in ascending thin limb (ATL) cells from pure or mixed-type thin limbs. Resting pHi was slightly but significantly higher in both DTLs and ATLs in high osmolality ( approximately 670 mosmol/kg H2O) than in low osmolality ( approximately 290 mosmol/kg H2O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pHi following additional acidification with an NH4Cl pulse was reduced by Na+ removal from the medium and by the addition of 60 microM HOE642 (an inhibitor of the Na+/H+ exchanger, NHE1), 55 microM S1611 (inhibitor of Na+/H+ exchanger, NHE3), 1 microM bafilomycin A1 (an inhibitor of vacuolar H+ -ATPase), or 20 microM Schering 28080 (an inhibitor of H+ -K+ -ATPase) to the medium. Resting pHi was also reduced by 60 microM HOE642, 55 microM S1611, and 20 microM Schering 28080. In both DTLs and ATLs, RT-PCR revealed message for NHE1, NHE3, and vacuolar H+ -ATPase; immunocytochemistry demonstrated the expression of the protein for NHE1 (basolateral membrane), NHE3 (luminal membrane), and H+ -K+ -ATPase (luminal membrane). These data suggest that pHi in rat inner medullary thin limbs is regulated by urea and by basolateral and luminal H+ extrusion via NHE1, NHE3, vacuolar H+ -ATPase, and H+ -K+ -ATPase.

Apical ouabain-sensitive and ouabain-insensitive ATPases in rat colonic epithelium.

Active resorption of potassium ions in the colon is mediated by ouabain-sensitive and ouabain-insensitive H,K-ATPases localized in the apical membrane of colonic enterocytes (colonocytes). The present study was performed to investigate distribution patterns of apical ATPases using catalytic histochemistry and immunohistochemistry. Activity of the ATPases was localized both at the apical and basolateral regions of these cells. The basolateral activity was almost completely inhibited by ouabain, but apical ATPase activity was only partially inhibited by ouabain. Ultracytochemically, activity was localized at the cytoplasmic side of the apical and basolateral membrane and thus we assume that the activity represents isoforms of apical H,K-ATPases and basolateral Na,K-ATPase. With the use of a polyclonal antibody raised against H,K-ATPase alpha-subunit, we demonstrated immunostaining only in the apical region of colonic enterocytes whereas positive staining was not observed at the basolateral membrane. On the other hand, when antibodies against alpha1-subunit Na,K-ATPase were applied, immunostaining was localized only in the basolateral membrane domain. Therefore, we conclude that ATPases as demonstrated histochemically in the present study were identified immunohistochemically as colonic alpha-subunit H,K-ATPase (in the apical cell membrane of colonocytes along the entire length of crypts) and as alpha1-subunit Na,K-ATPase (in the basolateral membrane).

Detection and localization of H+-K+-ATPase isoforms in human kidney.

An H+-K+-ATPase contributes to hydrogen secretion and potassium reabsorption by the rat and rabbit collecting ducts. Transport of these ions appears to be accomplished by one or both of two isoforms of the H+-K+-ATPase, HKalpha(1) and HKalpha(2,) because both isoforms are found in the collecting ducts and transport of hydrogen and potassium is attenuated by exposure to inhibitors of these transport proteins. To evaluate whether an H+-K+-ATPase is present in the human kidney, immunohistochemical studies were performed using normal human renal tissue probed with antibodies directed against epitopes of three of the known isoforms of the H+-K+-ATPase , HKalpha(1), HKalpha(2), and HKalpha(4), and the V-type H+-ATPase. Cortical and medullary tissue probed with antibodies against HKalpha(1) showed cytoplasmic staining of intercalated cells that was less intense than that observed in the parietal cells of normal rat stomach stained with the same antibody. Also, weak immunoreactivity was detected in principal cells of the human collecting ducts. Cortical and medullary tissue probed with antibodies directed against HKalpha(4) revealed weak, diffuse staining of intercalated cells of the collecting ducts and occasional light staining of principal cells. Cortical and medullary tissue probed with antibodies directed against the H+-ATPase revealed staining of intercalated cells of the collecting ducts and some cells of the proximal convoluted tubules. By contrast, no discernible staining was noted with the use of the antibody against HKalpha(2). These data indicate that HKalpha(1) and HKalpha(4) are present in the collecting ducts of the human kidney. In this location, these isoforms might contribute to hydrogen and potassium transport by the kidney.

Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.

BACKGROUND & AIMS: Gastric epithelial renewal is an asymmetric process. A stem cell located halfway up the tubular unit gives rise to both a basal gland region and a luminal pit compartment, but the mechanisms responsible for the maintenance of this asymmetry are obscure. We investigated whether Sonic hedgehog (Shh), an established polarizing signal protein during development, is expressed and functional in the adult human and murine stomach. METHODS: Expression of Shh and putative transcriptional targets was investigated using immunoblot and immunohistochemistry. Mice were treated with the Shh inhibitor cyclopamine and examined for expression levels of Shh targets and proliferation of gastric epithelial cells. RESULTS: Shh was expressed in the stomach. In cyclopamine-treated mice, we observed decreased expression of HNF3beta, Islet (Isl)-1 and BMP4, 3 putative Shh target genes. Inhibition of Shh markedly enhanced gastric epithelial proliferation and affected the cell cycle of gastric epithelial gland cells, whereas pit cells remained unaffected. CONCLUSIONS: Shh controls the expression of at least 3 factors important for epithelial differentiation and is a negative regulator of gastric gland cell proliferation. Shh is a candidate polarizing signal in the maintenance of gastric pit-gland asymmetry.