An update on acid secretion.

Gastric acid secretion is a complex process that requires hormonal, neuronal, or calcium-sensing receptor activation for insertion of pumps into the apical surface of the parietal cell. Activation of any or all these pathways causes the parietal cell to secrete concentrated acid with a pH at or close to 1. This acidic fluid combines with enzymes that are secreted from neighbouring chief cells and passes out of the gland up through a mucous gel layer covering the surface of the stomach producing a final intragastric pH of less than 4 during the active phase of acid secretion. Defects in either the mucosal barrier or in the regulatory mechanisms that modulate the secretory pathways will result in erosion of the barrier and ulcerations of the stomach or esophagus. The entire process of acid secretion relies on activation of the catalytic cycle of the gastric H+,K+-ATPase, resulting in the secretion of acid into the parietal cell canaliculus, with K+ being the important and rate-limiting ion in this activation process. In addition to K+ as a rate limiter for acid production, Cl- secretion via an apical channel must also occur. In this review we present a discussion of the mechanics of acid secretion and a discussion of recently identified transporter proteins and receptors. Included is a discussion of some of the recent candidates for the apical K' recycling channel, as well as two recently identified apical proteins (NHE-3, PAT-1), and the newly characterized calcium-sensing receptor (CaSR). We hope that this review will give additional insight into the complex process of acid secretion.

Fluid absorption in isolated perfused colonic crypts.

A spatial segregation of ion transport processes between crypt and surface epithelial cells is well-accepted and integrated into physiological and pathophysiological paradigms of small and large intestinal function: Absorptive processes are believed to be located in surface (and villous) cells, whereas secretory processes are believed to be present in crypt cells. Validation of this model requires direct determination of fluid movement in intestinal crypts. This study describes the adaptation of techniques from renal tubule microperfusion to hand-dissect and perfuse single, isolated crypts from rat distal colon to measure directly fluid movement. Morphologic analyses of the isolated crypt preparation revealed no extraepithelial cellular elements derived from the lamina propria, including myofibroblasts. In the basal state, crypts exhibited net fluid absorption (mean net fluid movement = 0.34 +/- 0.01 nl.mm-1.min-1), which was Na+ and partially HCO3- dependent. Addition of 1 mM dibutyryl-cyclic AMP, 60 nM vasoactive intestinal peptide, or 0.1 mM acetylcholine to the bath (serosal) solution reversibly induced net fluid secretion (net fluid movement approximately -0.35 +/- 0.01 nl.mm-1.min-1). These observations permit speculation that absorption is a constitutive transport function in crypt cells and that secretion by crypt cells is regulated by one or more neurohumoral agonists that are released in situ from lamina propria cells. The functional, intact polarized crypt described here that both absorbs and secretes will permit future studies that dissect the mechanisms that govern fluid and electrolyte movement in the colonic crypt.

Distal tubule acidification.

The distal tubule defines the final section of the renal tubule, and can be subdivided into four segments: distal tubule, connecting segment (which was previously considered part of the distal tubule), cortical collecting duct and medullary collecting tubule. This section of the nephron is the area that has been considered to have the maximal concentrating ability and maximal Acidification. This results from the fact that most reabsorption takes place in the proximal tubule and other nephron segments so that this final portion of the nephron is the final concentrating and acidifying region of the nephron. In this review I will briefly go over the distribution of the various cell types in the outer and inner medullary region as well as discuss some of the modifications that occur in the functional distribution of acid related proteins, during metabolic disturbances or during hormonal stimulation.

Role of calcium and other trace elements in the gastrointestinal physiology.

Calcium is an essential ion in both marine and terrestrial organisms, where it plays a crucial role in processes ranging from the formation and maintenance of the skeleton to the regulation of neuronal function. The Ca(2+) balance is maintained by three organ systems, including the gastrointestinal tract, bone and kidney. Since first being cloned in 1993 the Ca(2+)-sensing receptor has been expressed along the entire gastrointestinal tract, until now the exact function is only partly elucidated. As of this date it still remains to be determined if the Ca(2+)-sensing receptor is involved in calcium handling by the gastrointestinal tract. However, there are few studies showing physiological effects of the Ca(2+)-sensing receptor on gastric acid secretion and fluid transport in the colon. In addition, polyamines and amino acids have been shown to activate the Ca(2+)-sensing receptor and also act as allosteric modifiers to signal nutrient availability to intestinal epithelial cells. Activation of the colonic Ca(2+)-sensing receptor can abrogate cyclic nucleotide-mediated fluid secretion suggesting a role of the receptor in modifying secretory diarrheas like cholera. For many cell types changes in extracellular Ca(2+) concentration can switch the cellular behavior from proliferation to terminal differentiation or quiescence. As cancer remains predominantly a disease of disordered balance between proliferation, termination and apoptosis, disruption in the function of the Ca(2+)-sensing receptor may contribute to the progression of neoplastic disease. Loss of the growth suppressing effects of elevated extracellular Ca(2+) have been demonstrated in colon carcinoma, and have been correlated with changes in the level of CaSR expression.

Cl-dependent Na-H exchange. A novel colonic crypt transport mechanism.

This communication summaries a series of observations of the transport function of the crypt of the rat distal colon. Development of methods to study both 22Na uptake by apical membrane vesicles prepared from crypt cells and intracellular pHi (pHi), fluid movement (Jv), and bicarbonate secretion during microperfusion of the crypt has led to the identification of (1) a novel Cl-dependent Na-H exchange (Cl-NHE) that most likely represents the coupling of a Cl channel to a Na-H exchange isoform that has not as yet been identified and (2) bicarbonate secretion that appears to be most consistent with HCO3 uptake across the basolateral membrane by a mechanism that is closely linked to Cl transport and its movement across the apical membrane via an anion channel. Na-dependent fluid absorption is the constitutive transport process in the crypt, while fluid secretion is regulated by one or more neurohumoral agonists. Cl-NHE is responsible for both the recovery/regulation of pHi in crypt cells to an acid load and fluid absorption.

Impaired hepatocyte glucose transport protein (GLUT2) internalization in chronic pancreatitis.

Chronic pancreatitis (CP) is associated with impaired glucose tolerance and with reduced hepatic sensitivity to insulin. We have previously shown that in normal and sham-operated rats, insulin suppresses hepatic glucose production, and this suppression is associated with a decrease in the hepatocyte plasma membrane-bound quantity of the facilitative glucose transport protein GLUT2. The insulin-mediated reduction in membrane-bound GLUT2 is impaired in CP, and may play a role in the glucose intolerance associated with CP. To determine whether GLUT2 is actively internalized and whether this mechanism is disordered in CP, livers from fed and fasting rats in whom CP had been induced 2-3 months earlier by pancreatic duct oleic acid infusion, and in sham-operated (sham) rats, were fractionated to yield endosome (E)- and plasma membrane (PM)-enriched fractions. Forty-five minutes after duodenal intubation alone (fasting) or intubation plus duodenal feeding, livers were removed, homogenized and ultracentrifuged, and microsomal pellets were separated by sucrose density gradient ultracentrifugation. GLUT2 content of fractions was determined by Western blotting and scanning densitometry. The E:PM ratio of GLUT2 increased from 0.68 +/- 0.11 (mean +/- SEM) in fasting sham livers (n = 8) to 1.04 +/- 0.09 in fed sham livers (n = 8; p < 0.05). However, there was no change in the E:PM ratio of GLUT2 in CP livers after duodenal feeding (0.90 +/- 0.12 vs. 0.86 +/- 0.10; n = 8,8; p = NS). To test our findings using confocal laser scanning microscopy, liver specimens from fed and fasting CP and sham rats were minced, fixed in 4% paraformaldehyde, sectioned, and stained with rabbit antirat GLUT2 antibody followed by rhodamine-labeled secondary antibody. GLUT2 was quantified by mean pixel intensity in an 8 x 16-pixel area of PM and a 16 x 16-pixel area of cytosol (CYT) in each of 30 random cells/field (400x) in each of three rats per group. As in the fractionation study, duodenal feeding increased the CYT:PM ratio of GLUT2 from 0.75 +/- 0.01 in fasting sham liver to 0.86 +/- 0.01 in fed sham liver (p < 0.0001), while the CYT:PM ratio in CP remained unchanged. We conclude that feeding induces a shift in GLUT2 from the plasma membrane to the endosomal pool. The feeding-induced internalization of GLUT2 is absent in livers from rats with CP and may play a role in the glucose intolerance associated with CP.

Characteristics of the K+-competitive H+,K+-ATPase inhibitor AZD0865 in isolated rat gastric glands.

The gastric H+,K+-ATPase of the parietal cell is responsible for acid secretion in the stomach and is the main target in the pharmacological treatment of acid-related diseases. Omeprazole and other benzimidazole drugs, although having delayed efficacy if taken orally, have high success rates in the treatment of peptic ulcer disease. Potassium competitive acid blockers (P-CAB) compete with K+ for binding to the H+,K+-ATPase and thereby they inhibit acid secretion. In this study, the in vitro properties of AZD0865, a reversible H+,K+-ATPase inhibitor of gastric acid secretion, are described. We used a digital-imaging system and the pH sensitive dye BCECF to observe proton efflux from hand-dissected rat gastric glands. Glands were stimulated with histamine (100 microM) and exposed to a bicarbonate- and Na+-free perfusate to induce an acid load. H+,K+-ATPase inhibition was determined by calculating pHi recovery (dpH/dT) in the presence of omeprazole (10-200 microM) or AZD0865 (0.01-100 microM). The efficacies of both drugs were compared. Our data show that acid secretion is inhibited by both the proton pump inhibitor omeprazole and the P-CAB AZD0865. Complete inhibition of acid secretion by AZD0865 had a rapid onset of activation, was reversible, and occurred at a 100-fold lower dose than omeprazole (1 microM AZD0865 vs. 100 microM omeprazole). This study demonstrates that AZD0865 is a potent, fast-acting inhibitor of gastric acid secretion, effective at lower concentrations than drugs of the benzimidazole class. Therefore, these data strongly suggest that AZD0865 has great potential as a fast-acting, low-dose inhibitor of acid secretion.

Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-).

The role of H(+)-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused "in vitro" while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na(+)- and Cl(-)-free medium, cell pH fell by a mean of 0.37+/-0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 +/-0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl(-) channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na(+) solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018+/- 0.0021 min(-1), not significantly different from zero (P>0.42). For superfusion with Na(+)0 Ringer, this variation was 0.081+/-0.015 min(-1), P<0.001 against 0. These slopes were markedly reduced by the Cl(-) channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na(+)-free medium is probably due to a vacuolar H(+)-ATPase, whose activity is stimulated in the presence of Cl(-), and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl(-) and on the integrity of the cytoskeleton.

Differential activities of H+ extrusion systems in MDCK cells due to extracellular osmolality and pH.

The aim of the present study was to obtain detailed information on MDCK cell proton secretion characteristics under various growth conditions. Confluent monolayers cultured on glass coverslips were adapted over 48 h to media with different osmolality and pH (200 mosmol/kgH2O, pH 7.4; 300 mosmol/kgH2O, pH 7.4; and 600 mosmol/kgH2O, pH 6.8) corresponding to the luminal fluid composition of the collecting duct segments found in the in renal cortex, the outer stripe of outer medulla and inner medulla. Proton fluxes were determined from the recovery of intracellular pH following an acid load induced by an NH4Cl pulse times the corresponding intrinsic buffering power (beta(i)). The intracellular buffering power was found to change only with culture medium osmolality but not with culture medium pH. In addition to an amiloride and Hoe-694-sensitive Na+/H+ exchange, Madin-Darby canine kidney (MDCK) cells possess a Sch-28080-sensitive, K+-dependent H+ extrusion mechanism that is increased upon adaptation of monolayers to hyperosmotic-acidic culture conditions. A significant contribution of the bafilomycin A1-sensitive vacuolar H+-ATPase could be found only in cells adapted to hyposmotic culture conditions. Exposure of MDCK cells to 10(-5) or 10(-7) M aldosterone for either 1 or 18 h did not alter the H+ extrusion characteristics significantly. The results obtained show that different extracellular osmolality and pH induce different MDCK phenotypes with respect to their H+-secreting systems.

Na(+)-dependent fluid absorption in intact perfused rat colonic crypts.

BACKGROUND & AIMS: The traditional paradigm of fluid movement in the mammalian colon is that fluid absorption and secretion are present in surface and crypt cells, respectively. We have recently demonstrated Na(+)-dependent fluid absorption in isolated crypts that are devoid of neurohumoral stimulation. We now explore the mechanism of Na(+)-dependent fluid absorption in isolated rat colonic crypts. METHODS: Net fluid absorption was determined using microperfusion techniques and methoxy[(3)H]inulin with ion substitutions and transport inhibitors. RESULTS: Net fluid absorption was reduced but not abolished by substitution of either N-methyl-D-glucamine- Cl(-) or tetramethylammonium for Na(+) and by lumen addition of 5-ethylisopropyl amiloride, an amiloride analogue that selectively inhibits Na(+)-H(+) exchange. Net fluid absorption was also dependent on lumen Cl(-) because removal of lumen Cl(-) significantly (P < 0.001) reduced net fluid absorption. DIDS at 100 micromol/L, a concentration at which DIDS is an anion exchange inhibitor, minimally reduced net fluid absorption (P < 0.05). In contrast, either 500 micromol/L DIDS, a concentration at which DIDS is known to act as a Cl(-) channel blocker, or 10 micromol/L NPPB, a Cl(-) channel blocker, both substantially inhibited net fluid absorption (P < 0.001). Finally, both the removal of bath Cl(-) and addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl(-) secretion, resulted in a significant increase in net fluid absorption. CONCLUSIONS: (1) Net Na(+)-dependent net fluid absorption in the isolated colonic crypt represents both a larger Na(+)-dependent absorptive process and a smaller secretory process; and (2) the absorptive process consists of a Na(+)-dependent, HCO(3)(-)-independent process and a Na(+)-independent, Cl(-)-dependent, HCO(3)(-)-dependent process. Fluid movement in situ represents these transport processes plus fluid secretion induced by neurohumoral stimulation.

HCO(3)(-) secretion in the rat colonic crypt is closely linked to Cl(-) secretion.

BACKGROUND & AIMS: The mechanism of colonic HCO(3)(-) secretion has not been established largely because of a lack of experimental methods for its detailed study. The present studies were designed to establish whether the isolated, perfused crypt of the rat distal colon is an excellent model to study HCO(3)(-) movement and the mechanism of colonic HCO(3)(-) secretion. METHODS: HCO(3)(-) secretion was determined in isolated, microperfused crypts by measuring [HCO(3)(-)] by microcalorimetry on nanoliter samples. RESULTS: Net HCO(3)(-) absorption was observed during lumen and bath perfusion with an HCO(3)(-)-Ringer solution. Vasoactive intestinal polypeptide (60 nmol/L), acetylcholine (100 nmol/L), or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mmol/L) induced active HCO(3)(-) secretion that required bath but not lumen HCO(3)(-)/CO(2). DBcAMP-stimulated HCO(3)(-) secretion was not affected by acetazolamide, an inhibitor of carbonic anhydrase. Removal of lumen Cl(-) did not alter DBcAMP-stimulated HCO(3)(-) secretion but reduced fluid secretion. DBcAMP-stimulated HCO(3)(-) secretion was closely linked to active Cl(-) secretion because HCO(3)(-) secretion was substantially reduced by removal of bath Cl(-), by addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl(-) secretion, and by addition of lumen NPPB, a Cl(-) channel inhibitor. CONCLUSIONS: These studies establish that colonic crypt HCO(3)(-) secretion (1) is not a result of an apical membrane Cl(-)-HCO(3)(-) exchange, (2) is tightly associated with Cl(-) secretion, and (3) primarily occurs via an apical membrane Cl(-) channel.

Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis.

The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily due to limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. "Pits" measuring 500-2000 nm and containing 3-20 depressions measuring 100-180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in "depression" size correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be the fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time.

An apical permeability barrier to NH3/NH4+ in isolated, perfused colonic crypts.

Fermentation of nonabsorbed nutrients in the colon generates high concentrations of NH3/NH4+ in the colonic lumen. NH3 is a small, lipophilic neutral weak base that readily permeates almost all cell membranes, whereas its conjugate weak acid NH4+ generally crosses membranes much more slowly. It is not known how colonocytes maintain intracellular pH in the unusual acid-base environment of the colon, where permeant acid-base products of fermentation exist in high concentration. To address this issue, we hand dissected and perfused single, isolated crypts from rabbit proximal colon, adapting techniques from renal-tubule microperfusion. Crypt perfusion permits control of solutions at the apical (luminal) and basolateral (serosal) surfaces of crypt cells. We assessed apical- vs. basolateral-membrane transport of NH3/NH4+ by using fluorescent dyes and digital imaging to monitor intracellular pH of microvacuolated crypt cells as well as luminal pH. We found that, although the basolateral membranes have normal NH3/NH4+ permeability properties, there is no evidence for transport of either NH3 or NH4+ across the apical borders of these crypt cells. Disaggregating luminal mucus did not increase the transport of NH3/NH4+ across the apical border. We conclude that, compared to the basolateral membrane, the apical border of crypt colonocytes has a very low permeability-area product for NH3/NH4+. This barrier may represent an important adaptation for the survival of crypt cells in the environment of the colon.

Unusual permeability properties of gastric gland cells.

Physiologists have long pondered the riddle of why the stomach is itself not digested by the very juice it secretes. One explanation is that a mucus-bicarbonate barrier, coating the stomach lumen as well as superficial portions of gastric glands, prevents autodigestion. However, this leaves unanswered the question of what protects cells deeper in the glands, which seem to lack a mucus barrier. These are the parietal and chief cells, which secrete acid and pepsin. Using perfused single gastric glands from rabbit, we recently found that intracellular pH is uniquely resistant to extreme degrees of luminal acidification, suggesting that the apical (luminal) barrier might also exclude ammonia and carbon dioxide, to which cell membranes are generally highly permeable. We now show that this is indeed the case. There are three reports of membranes with very low permeabilities to NH3 (refs 5-7), and none of membranes impermeable to CO2.

Novel transport properties of colonic crypt cells: fluid absorption and Cl-dependent Na-H exchange.

Colonic ion transport is heterogeneous including the long-accepted spatial separation of absorptive and secretory processes between surface and crypt cells. We recently described the isolation of individual crypts from the rat distal colon that were studied using microperfusion technology. Na-dependent fluid absorption was consistently demonstrated in these crypts during perfusion with a Ringer-like solution; dibutyryl cyclic AMP, VIP and acetylcholine, when added to the bath solution, all induced net fluid secretion. As several morphologic techniques, including immunocytochemistry, failed to provide evidence for the presence of myofibroblasts in the isolated crypt preparation, we propose that a Na-dependent absorptive process is a constitutive transport mechanism in crypt cells, while secretory processes are regulated by the release of one or more neurohumoral agonists from lamina propria cells including myofibroblasts. The mechanism of Na-dependent fluid movement was also studied by determining [H] gradient stimulation of 22Na uptake in isolated apical membrane vesicles (AMV) from crypt cells. In contrast to Na-H exchange in surface cell AMV, Na-H exchange in crypt cells is Cl-dependent. Intracellular pH determined in crypt cells using video-imaging fluorescence microscopy established that the response to an acid load requires both lumen Na and Cl. As a result, these studies have identified a novel Cl-dependent Na-H exchange in crypt AMV that may mediate apical membrane Na uptake and regulate pHi.

Localization of amiloride-sensitive sodium channels in A6 cells by atomic force microscopy.

Atomic force microscopy (AFM) was used for high-resolution imaging of the apical distribution of epithelial Na+ channels in A6 renal epithelial cells. A6 cells grown on coverslips were labeled with antibodies generated against an amiloride-sensitive epithelial Na+ channel complex purified from bovine renal medulla that had been conjugated to 8-nm colloidal gold particles before preparation for AFM. AFM revealed that there was a marked increase in the height of the microvilli in cells labeled with the anti-epithelial Na+ channel antibodies compared with unlabeled cells or cells labeled with rabbit nonimmune immunoglobulin G conjugated to colloidal gold particles. We interpret this apparent increase in microvillar height to be due to anti-epithelial Na+ channel antibody binding to the apical microvilli. These data demonstrate that epithelial Na+ channels are restricted to the apical microvilli in Na+-transporting renal epithelial cells. Furthermore, they demonstrate the applicability of using AFM for high-resolution imaging of the cell surface distribution of epithelial ion channels.

Unique permeability barrier of the apical surface of parietal and chief cells in isolated perfused gastric glands.

Although many factors can influence intracellular pH (pHi), some of the most important are those that involve the movement of acids and bases across the cell membrane. We will discuss recent results concerning barriers to the movement of H+, NH3 and CO2 across the apical cell membranes of gastric gland cells. Cell membranes are generally highly permeable to small, lipophilic molecules such as NH3 and CO2. In fact, only two examples are known of membranes relatively impermeable to NH3 and none membranes permeable to CO2. We recently developed a technique for perfusing the lumen of a single hand-dissected gastric gland on the stage of a microscope, while monitoring pHi with a fluorescent dye. We observed the expected pHi changes when we exposed the basolateral (i.e. blood-side) membrane to a pH 6.4 solution (a large, rapid pHi decrease), to a pH 7.4 solution containing approximately 0.3 mmoll-1 NH3 (a large and rapid pHi increase) or to a pH7.4 solution equilibrated with 1% CO2 (a rapid pHi decrease of -0.08). However, pHi was not significantly affected by perfusing the lumen with a pH 1.4 solution, with a pH 7.4 solution containing as much as 2.7 mmoll-1 NH3 or with a pH 6.1 solution equilibrated with 100% CO2. These data indicate that a barrier at or near the apical membrane has a uniquely low permeability to H+, NH3 and CO2.

Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells.

Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl-, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl-, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl- channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl--dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3- reabsorption along the proximal tubule when tubules are exposed to AII.

Direct effects of dopamine on colonic mucosal pH: implications for tonometry.

Tonometric measurements of colonic and gastric mucosa pH are used as indirect determinants of splanchnic perfusion in shocked patients or those undergoing aortic cross-clamp. Mucosal acidification in response to splanchnic vasodilators such as dopamine has been assumed to signify ischemia. However, cellular acidification may occur independent of oxygenation and the direct effects of dopamine on mucosal acid-base are unknown. We examined the effects of dopamine on cellular pH (independent of oxygenation) of intestinal mucosa in vitro. Crypts isolated from the distal colon of Sprague-Dawley rats were loaded with a pH-sensitive fluorescent probe, perfused with a Hepes-buffered Ringers solution, and imaged with confocal laser scanning microscopy. In separate experiments, crypts were loaded with a calcium-sensitive probe (Fura-2) and concentrations of free cytosolic calcium were measured with fluorescence imaging. Dopamine perfusion produced a reversible cytosolic acidification of crypts which was not significantly affected by (i) the nominal absence of bicarbonate, (ii) alpha- and beta-adrenergic receptor blockade, or (iii) protein kinase C inhibition. Dopamine did not significantly affect intracellular calcium concentrations. However, dopamine-induced acidification was inhibited by (a) blocking sodium-hydrogen exchange with amiloride, (b) prior exposure to adenosine 3', 5'-cyclic monophosphate (cAMP), or (c) protein kinase A blockade (all P < 0.01). Dopamine directly acidifies mucosal crypt cells in a mechanism that involves a cAMP-mediated inhibition of sodium-hydrogen exchange. This finding accounts for the acidification of intestinal mucosa during low-dose dopamine infusion despite a demonstrable improvement in splanchnic perfusion. Direct mucosal effects of pharmacological agents must be considered in the evaluation of perfusion parameters based on tonometric data.

Calcium, ATP and nuclear pore channel gating.

Nuclear envelope (NE) cisternal Ca2+ and cytosolic ATP are required for nuclear-pore-complex-(NPC-) mediated transport of DNAs, RNAs, transcription factors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. The large ion conductance (gamma) activity of the NPC channel (NPCC) is blocked by the NPC monoclonal antibody mAb414, known to block MMT, and is also silenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. To test the role of Ca2+ and ATP in NPCC activity, we carried out the present patch-clamp study with the pipette attached to the outer NE membrane of nuclei isolated from cultured Dunning G prostate cancer cells. Our investigations demonstrate that in these isolated nuclei neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. However, when simultaneously applied to the bath and pipette, they transiently silence NPCC activity through stimulation of MMT by raising the Ca2+ concentration in the NE cisterna ([Ca2+]NE). Our fluorescence microscopy observations with nuclear-targeted macromolecular fluorochromes (B-phycoerythrin and plasmid for the enhanced green fluorescence protein EGFP, pEGFP-C1) and with FITC-labeled RNA support the view that channel silence accompanies MMT. Repeated Ca2+ loading of the NE with Ca2+ and ATP, after unloading with 1-5 microM inositol 1,4,5-trisphosphate (IP3), thapsigargin (TSG) or 5 mM BAPTA or EGTA, failed to affect channel gating. This result indicates that other factors are involved in this phenomenon and that they are exhausted during the first cycle of NE Ca2+ loading/unloading--in agreement with current theories of NPC-mediated MMT. The results explain how Ca2+ and IP3 waves may convert the NE into an effective Ca2+ barrier and, consequently, affect the regulation of gene activity and expression through their feedback on MMT and NPCC gating. Thus, [Ca2+]NE regulation by intracellular messengers is an effective mechanism for synchronizing gene activity and expression to the cellular rhythm.