Isolated Frog Gastric Mucosa Research Articles

Working Outside the Cell

Many signaling molecules interact with cell surface receptors to mobilize calcium from intracellular stores, eliciting transient changes in the concentration of intracellular calcium ([Ca 2+ ] i ), which acts as a second messenger. Mobilized cytoplasmic Ca 2+ extruded through plasma membrane Ca 2+ ATPases can increase local extracellular Ca 2+ concentration [Ca 2+ ] o, whereas store-operated Ca 2+ entry can lead to a localized decrease in [Ca 2+ ] o . Indeed, in gastric epithelial cells, the cholinergic agonist carbachol elicits a simultaneous increase in lumenal [Ca 2+ ] o and decrease in serosal [Ca 2+ ] o , because of the polarized distribution of Ca 2+ -handling molecules. Using a proteolytic assay to measure pepsinogen secretion, and ion-sensitive microelectrodes to measure pH and [Ca 2+ ], Caroppo et al. investigated the effects of changing [Ca 2+ ] o on alkaline secretion and pepsinogen secretion in isolated frog gastric mucosa. Simultaneous changes in lumenal and serosal [Ca 2+ ] o comparable to those elicited by carbachol stimulated comparable secretion of pepsinogen and changes in gastric gland lumenal pH. Increasing only lumenal [Ca 2+ ] o had a smaller effect on secretion, whereas decreasing serosal [Ca 2+ ] o alone was ineffective. Pharmacological analysis implicated a previously identified extracellular Ca 2+ receptor (CaR) in the response; moreover, citrate, added to buffer lumenal [Ca 2+ ] o , prevented both the carbachol-dependent increase in lumenal [Ca 2+ ] o and alkaline and pepsinogen secretion. In contrast, stimulating CaR pharmacologically or increasing lumenal [Ca 2+ ] o did not affect [Ca 2+ ] i , as measured either with intracellular microelectrodes or indirectly through activation of a Ca 2+ -sensitive K + current. Thus, the authors propose that, in this system, changes in [Ca 2+ ] o after Ca 2+ mobilization from intracellular stores can act as a third messenger to stimulate secretion. R. Caroppo, A. Gerbino, G. Fistetto, M. Colella, L. Debellis, A. M. Hofer, S. Curci, Extracellular calcium acts as a "third messenger" to regulate enzyme and alkaline secretion. J. Cell Biol . 166 , 111-119 (2004). [Abstract] [Full Text]

A lyophilized aqueous extract of Maytenus ilicifolia leaves inhibits histamine-mediated acid secretion in isolated frog gastric mucosa.

Lyophilized aqueous extract of Maytenus ilicifolia leaves (LAEMIL) is commonly used in Brazilian folk medicine in the treatment of dyspepsia as well as gastric ulcers. We have investigated the effect and the possible mechanism of action of the LAEMIL on acid secretion in isolated frog gastric mucosa incubated in an Ussing chamber. It was observed that LAEMIL (7-28 mg%) as well as cimetidine (125-4,000 microM), a well-known histamine H2 receptor antagonist, decreased basal acid secretion in a concentration-dependent manner. Similarly to cimetidine (190 microM), LAEMIL (21 mg%) also inhibited gastric acid secretion induced by increasing concentrations of histamine (50-800 microM). The EC50 values for histamine alone and histamine in the presence of LAEMIL or cimetidine were 94.6 microM (71.1-125.9 microM), 244.9 microM (209.4-286.4 microM) and 142.2 microM (23.6-855.0 microM), respectively. LAEMIL, histamine and cimetidine were effective on acid secretion only when added to the serosal surface of the mucosa. Furthermore, simultaneous addition of LAEMIL and cimetidine at concentrations, per se, ineffective, caused a 16% reduction in the basal acid secretion [from 8.3 +/- 0.3 to 6.9 +/- 0.2 microEq g(-1) (15 min)(-1), n=4]. Although effects such as inhibition of histamine biosynthesis and/or histamine release can not be ruled out, our data suggest that LAEMIL, like cimetidine, reduces acid secretion in the isolated frog gastric mucosa by antagonising histamine H2 receptors.

Electroneutral Acid Secretion in Frog Gastric Mucosa: An Electron Microprobe Analysis

To elucidate the mode of H secretion in the intact gastric mucosa, the cellular element concentrations and Rb uptake by individual surface and oxyntic cells of the short-circuited isolated frog gastric mucosa <i>(Rana esculenta, Rana temporaria) </i>were determined using electron microprobe analysis. The H secretion rate was measured by the pH stat method. Under normal conditions both surface and oxyntic cells exhibited typical cellular electrolyte patterns with low Na and high K concentrations. The apical parts of surface cells, in which large mucus-containing vesicles are located, were characterized by relatively high S and Ca concentrations. Histamine-stimulated H secretion in the gastric mucosa of R. <i>esculenta </i>and <i>R. temporaria </i>was about 2.0 and 0.5 µmol/cm²·h, respectively. In the presence of 20 m<i>M</i> Rb in the mucosal bathing solution, a substantial portion of cellular K was exchanged for Rb in oxyntic but not in surface cells. This K-Rb exchange was almost completely abolished by inhibition of H secretion with omeprazole indicating a specific H-Rb(K) exchange across the apical membrane of oxyntic cells. At a low H secretion of about 0.5 µmol/cm²·h about 70% of the H secretion rate could be matched by the omeprazole-inhibitable Rb uptake rate. Accordingly H secretion seems to be accomplished by an electroneutral H-Rb(K) exchange. The negligible Rb uptake into oxyntic cells at a mucosal Rb concentration of 5 m<i>M</i> might be explained by K recirculation across the mucosal membrane via a K conductance and the H-K-ATPase.

Conservative and nonconservative inhibitors of gastric acid secretion.

Inhibitors of the initial step (H2-antagonist) and of the final step (thiocyanate, SCN-; and nitrite, NO2-) were used to study the dynamics of acid secretion in isolated frog gastric mucosa. Tissues were mounted in flow-through chambers, and the acid secretion rate (SR) was recorded on a pH-stat microprocessor. Continuous presence of H2-antagonist decreases the SR to a lower steady state, and on removal the SR returns to basal SR, causing a net loss of acid, the nonconservative effect. The amount of lost acid is a unique function of exposure, thus, independent of the patterns (pulses or steps) of inhibition. In contrast, continuous presence of SCN- or NO2- (below 3 mM) results in an undershoot in SR with a return to basal SR, whereas at higher concentrations there is no return. Removal of these inhibitors causes an overshoot in SR with return to basal SR. The rebound acid is equal to acid suppressed by NO2- and low concentration of SCN-, resulting in no net loss of acid, the conservative effect, whereas at high concentrations of SCN- there is an apparent loss of acid. In maximally secreting tissue the overshoot of SR is not observed. However, the acid is not lost, merely delayed. In resting tissue NO2- also merely delays the exit of the acid produced in response to forskolin. The rebound acid is proposed to reside in a sequestered "acid" pool that is stable for at least 120 min. Results with NO2- and SCN- suggest an effect on a saturable exit enzyme, possibly the K+-H+-ATPase.

Histamine: the sole mediator of pentagastrin‐stimulated acid secretion

Isolated frog gastric mucosa was used to study the effect of pentagastrin on histamine release and acid secretion. The release of histamine precedes that of acid secretion. The delay time and time of peak acid secretion occur later than during histamine stimulation. Short exposure (pulse) results in transient increases in both acid secretion and histamine release rates, while longer exposure (step) results in prolonged increases in both variables. Dose-response curves for pentagastrin pulses show that both the total amount of histamine released and the total amount of acid secreted are saturable processes, paralleling each other. Thus, the total amount of acid secreted is a function of histamine (released) exposure (M min-1) in the extracellular space. Repeated exposure will give multiple responses only if the time elapsed between the stimuli is long enough. The magnitude of the responses depends on the length of time intervals between the stimuli and the concentration of the stimuli. Simultaneously administered pulses of histamine and pentagastrin result in merely additive amount of acid being secreted. From these observations I conclude that pentagastrin-induced acid secretion can be understood solely in terms of its effect on histamine release and that depletion of histamine stores is responsible for the observed refractoriness.

Thiocyanate and nitrite inhibit proton translocation in gastric musosa

Isolated frog gastric mucosa was used to study the separation of formation of protons (or their precursors) from proton translocation by using various inhibitors. Both thiocyanate (SCN −) and nitrite (NO 2 −) inhibit the acid secretion in spontanously secreting mucosa. The inhibition is reversed when the inhibitor is removed such that the excess acid secreted above baseline in the ‘off’-period compensates for the amount inhibited in the ‘on’-period. Both agents also inhibit the effect on acid secretion of pulse stimulation with histamine though to a lesser extent. Upon removal of the inhibitor, the total amount of acid secreted in excess of basal is equal to that observed with histamine alone. Likewise, metiamide, an H 2-antagonist, also inhibits acid secretion with or without histamine. However, in contrast to SCN − and NO 2 −, removal of this inhibitor is without effect on the acid-secretion rate. These results indicate that both SCN − and NO 2 − inhibit the proton translocation rather than the formation of protons or their precursors as is the case with metiamide.

Voltage clamping induces resistance and current-voltage plot changes in frog gastric mucosa.

Changing the potential across the isolated frog gastric mucosa by voltage clamping changes the measured resistance of the tissue in two ways. An immediate change in resistance results from changing the measuring position on the nonlinear current-voltage (I-V) plot. Subsequent to this, the resistance changes slowly with a half-time of about 3 min, a change that is not predicted by a previous model for voltage transients and that implies slow changes in membrane resistance following changes in intracellular ion content. The I-V plot over the range examined shows three breakpoints; changing clamp voltage alters the position of two of these breakpoints as well as the slope of the connecting resistances. The central breakpoint agrees with the potential at zero current and varies with it as the clamp potential is changed, as predicted from a diode model for breakpoint generation.

Backdiffusion of H+ in isolated frog gastric mucosa.

Backdiffusion of H+ was studied in isolated bullfrog gastric mucosa using direct titration to evaluate changes in luminal H+ level. In resting mucosae, the resulting permeability coefficient for H+ (uncorrected for electrical effects) was 0.4 X 10(-5) cm X S-1, which is of the same order of magnitude as that estimated from data in the literature for dog and rabbit gastric mucosa in vivo. With maximal stimulation of acid secretion, backdiffusion of H+ was considerably increased. In both resting and stimulated mucosae, an increase in Cl- flux from lumen to serosa was observed to accompany H+ backdiffusion. In experiments in which electrical activity of resting mucosae was monitored, instilled H+ lowered gastric potential difference but had little effect on the normal resistance. Sudden drops in resistance and potential were observed, independent of luminal acid; after such events instilled H+ had no effect on mucosal resistance. It appears that a substantial part of H+ backdiffusion occurs as the ion pair HCl.

Contribution of Ambient HCO3- to Mucosal Protection and Intracellular pH in Isolated Amphibian Gastric Mucosa

The relationship between mucosal protection by ambient HCO3- and its impact on intracellular pH was studied in isolated frog gastric mucosa. Closed sacs of gastric mucosa, containing exogenous HCl and pepsin, readily ulcerated when incubated in Ringer solution devoid of HCO3-, but were effectively protected against ulceration when ambient HCO3- was present. This protection was abolished by blocking anion exchange in cell membrane by 4-acetamido-4'-isothiocyanostillbene-2,2'-disulfonic acid or by inhibiting mucosal carbonic anhydrase by azetazolamide. Intracellular pH, measured by the 14C-labeled 5,5-dimethylxazolidine-2,4-dione method, was significantly higher when open sheets of mucosas were incubated in a medium containing HCO3- than when they were incubated in a medium devoid of HCO3- at the same external pH. Furthermore, a positive pH gradient across the cell membrane (intracellular pH was greater than extracellular pH) was generated only if HCO3- was present in the incubation medium. 4-Acetamido-4'-isothiocyano-stillbene-2,2'-disulfonic acid completely abolished these effects of ambient HCO3- on intracellular pH, suggesting that ambient HCO3- contributes to intracellular pH by acting as an intracellular base. In contrast, acetazolamide had no significant influence on these effects, suggesting that carbonic anhydrase acts in the protective mechanism distally to the entry of HCO3- into the cell. The data indicate that ambient HCO3- significantly contributes to intracellular pH in the gastric mucosa, a feature that may enhance the ability of the mucosa to withstand intraluminal acid.

Characterization of dogfish gastric mucosa under hyperbaric conditions.

Using hyperbaric conditions previously shown to give improved oxygen delivery to the chambered dogfish gastric mucosa, experiments were performed to further characterize this tissue under these conditions. Removal of Cl- depressed but did not abolish acid secretion, raised the transepithelial resistance, and caused the potential to become more negative with reference to the mucosal surface. Voltage clamping to -60 mV inhibited secretion significantly; voltage clamping to +60 mV sometimes gave a stimulation. There was no long time-constant transient response to current passage. Under hyperbaric conditions, addition of the normal 350 mM urea content of elasmobranch plasma stimulated secretion. A further increase in partial pressure of oxygen (PO2) above 1.9 atm had no effect on secretion. The current-voltage plot showed two breakpoints, one about -23 mV and a second (not always detectable) at +50 mV; the negative breakpoint was stable as PO2 changed from 0.9 to 1.9 atm. Many of these observations are different from those reported for normabaric tissue and closely resemble those in the isolated frog gastric mucosa.

Ouabain binding to isolated frog gastric mucosa.

Inhibition of gastric acid secretion by the cardiac glycoside, ouabain, is considerably reduced by elevating external K+ as first shown by Davenport (Proc. Soc. Exp. Biol. Med. 110: 613-615, 1962). To determine the possible role of K+ in this effect, we measured [3H]ouabain binding in isolated bullfrog gastric mucosa. Uptake of the labeled drug showed two components: one that saturated at 0.36 pmol ouabain per milligram wet weight and one that was linear with the external ouabain concentration. The former component is considered to represent specific binding to Na+-K+-ATPase; activity of this enzyme system in mucosal homogenates was 0.2 mumol per milligram protein per hour. Increase of K+ in the nutrient bathing solution from 3 to 30 mM, or replacement of Na+ by K+ in the secretory bathing solution, largely reversed inhibition of acid secretion by ouabain but did not affect maximum specific binding. The results fit a model in which Na+-K+-ATPase is normally required in oxyntic cells to maintain a high K+ level, which in turn supports exchange of K+ for H+ at the apical surface.

Carbon monoxide insensitivity of gastric acid secretion.

By the use of hyperbaric conditions, it is possible to obtain high CO/O2 while maintaining O2 tension at a level that is not in itself inhibitory of acid secretion in the isolated frog gastric mucosa. Free solution CO/O2 of up to 10:1 only inhibit acid secretion about 20%, less than expected. Considerations of O2 diffusion into a respiring tissue bounded by unstirred layers predict a CO/O2 at the terminal oxidase ranging from 12 to 64 under various conditions and positions in the tissue, with predicted inhibitions between 55 and 86%. The inhibition observed is light reversible, affects potential difference and resistance in a consistent manner but is never very large. In the absence of O2, the CO-cytochrome alpha 3 complex can be demonstrated spectrophotometrically. Among other possible explanations for these observations, it is suggested that this tissue may contain an inhibitor-insensitive oxidase for a portion of the cytochrome system directly involved in acid secretion.

Ulceration of Isolated Amphibian Gastric Mucosa

Protective mechanisms in gastric mucosa were investigated with the use of closed sacs and open sheets of isolated frog gastric mucosa. Closed sacs ulcerated at a high rate (80%) in a phosphate-buffered serosal medium devoid of HCO3- and gassed with 100% O2. In a nutrient solution buffered with CO2-HCO3-, the incidence of ulceration was significantly lower (10%). The protection afforded by HCO3- in the serosal solution was the same regardless of the presence or absence of CO2. Alkalinization of the serosal medium per se did not protect the mucosa, whereas buffering the luminal contents of the sacs to pH 7 completely abolished ulceration. Acetazolamide, and, to a lesser degree, SITS, increased the susceptibility of the sacs to ulceration. Open sheets of frog gastric mucosa exposed to an acidic mucosal solution (pH 2) gassed with N2 on the mucosal surface to simulate the hypoxic conditions prevailing inside the sacs showed significantly lesser decreases in PD and Isc when the serosal solution contained HCO3- than when phosphate buffer was used. Gross ulceration did not occur, however, unless the concentration of pepsin in the acidic mucosal solution was elevated to the level encountered within closed sacs. Ulceration could be induced in open sheets gassed on both sides with 100% O2 in the absence of nutrient HCO3- only upon the addition of luminal pepsin with a mucosal pH of 2.0. The data indicate that both luminal H+ and pepsin are needed for ulceration to occur in the present model. The presence of nutrient HCO3- significantly protects the mucosa, possibly by providing an intracellular buffer to neutralize the in fluxing luminal H+ and/or by maintaining a higher rate of alkali (HCO3-) secretion by the surface cells of the mucosa.

Mechanism of the Inhibitory Action of Electric Current on H+ Secretion by Frog Gastric Mucosa

The mechanism of the inhibitory action of electric current on gastric H+ secretion was studied using isolated frog gastric mucosa. Electric current, 500 μA/cm2, passed from mucosa to serosa (mucoso positive with respect to serosa), reversibly inhibited net H+ secretion, leading to complete cessation of luminal H+ appearance usually within 1 hr. The decrease in luminal H+ appearance was associated with an almost equal decrease in serosal HCO3− appearance. This is consistent with the suggestion that the inhibition of net H+ secretion was due to decrease in active H+ transport rather than to passive electrochemical back flux of the secreted H+ from the luminal bathing solution. Ultrastructural analysis of the tissues supported this view by demonstrating conversion of oxyntic cells into the resting (nonsecreting) morphologic configuration during the passage of current. Blocking of the conductive pathways for K+ in the nutrient (serosal) cell membrane by adding Ba++ to the serosal bathing solution significantly opposed the inhibitory action of electric current on H+ secretion. When electric current was passed through a mucosal solution containing a high concentration of K+, slight stimulation rather than inhibition of active H+ transport occurred (as judged from serosal HCO3− appearance). In this circumstance, the oxyntic cells retained their active secretory configuration despite the opposing current. The data indicate that electric current, passed from mucosa to serosa, inhibits active H+ transport by oxyntic cells. The data also suggest that the inhibitory action of an opposing current may be accomplished by electrolytic removal of K+ from an intracellular compartment essential for the function of the H+ secretory pump.

Kinetics of Acetylsalicylate and D-lactate Transport across Isolated Frog Gastric Mucosa

Luminal to submucosal migration of 14C-acetylsalicylate and D-lactate and some electrical properties were studied in isolated frog gastric mucosae. With both compounds, the unionized acids permeated much more rapidly than the ionized conjugate bases. The permeability coefficient for unionized acetylsalicylic acid increased from 0.27 to 0.43 mumoles, h-1, cm-2, mM-1 when its luminal concentration was increased above 3 mM. Simultaneously there was an increase in mucosal ion conductance. Acetylsalicylic acid has been shown previously to increase gastric mucosal permeation of ions and uncharged larger molecules. The present results indicate that above a threshold concentration the unionized form also enhances absorption of this drug per se. Unionized D-lactic acid had no effect of mucosal ion conductance and the permeability coefficient (0.07 mumoles, h-1, cm-2, mM-1) was independent of its luminal concentration.

Osmotic flow of water in isolated frog gastric mucosa.

A gravimetric procedure was used to measure net volume flow across bullfrog gastric mucosa mounted between chambers. A portion of the net volume flow towards the lumen was coupled to acid production. With an isotonic solution instilled on the luminal surface, the secreted acidity (ratio of increase in acid output to increase in volume flow) was hypertonic, in agreement with previous reports in mammalian stomach. Dilution of the secretory solution to 10% of normal nearly abolished the net volume flow coupled to acid production so that the mean secreted acidity rose to 1.87 M. Other experiments in which gastric juice was collected from this preparation showed that secretion into an initially empty lumen was only slightly hypertonic, as in mammalian stomach. The results indicate that instillation of secretory solution dilutes the endogenous osmotic gradient due to secreted HC1. This gradient is probably just outside the apical surface of the oxyntic cells of stomach.

Chloride Transport and Acid Secretion in Stomach

Chloride transport in isolated frog gastric mucosa is known to include major components due to active transport and exchange diffusion, and a minor component due to passive diffusion, but the sites responsible for moving Cl- within the mucosa have not been identified. The present study shows that the method used by Zerahn and others to measure transport pool yields an apparent Cl- pool which is close to the Cl- content of mucosal cells measured by chemical or radioactive means. Uptake of Cl- by the mucosal cells across the luminal surface in 2 min is rapid, constituting over one-half of the steady state cell content. The results suggest that much of this uptake occurs by exchange diffusion of Cl- at the secretory surface.

Influence of amytal and menadione on high-energy phosphates and acid secretion in frog gastric mucosa

Measurement of ATP and creatine phosphate in isolated frog gastric mucosa showed that amytal depressed the level of both high-energy compounds and inhibited acid secretion. Addition of menadione restored the ATP level to control values and partially restored the creatine phosphate levels but did not support acid secretion. Menadione was found to support that portion of active chloride transport which is not associated with acid output. It is concluded that an amytal-sensitive reaction, presumably mitochondrial coupling site I, is required for acid secretion to occur and that ATP is not a sufficient source of energy for this secretory process.

Hydrolysis of exogenous ATP by isolated frog gastric mucosa

ATP, added to the solution bathing the nutrient (serosal) surface of isolated frog gastric mucosa, was found to break down rapidly to ADP, inorganic phosphate and other products. This activity is due to an ectoenzyme, i.e., to an enzyme system easily accessible to the bathing solution. This conclusion follows from experiments which showed that penetration of ATP into the mucosal cells occurred at a much slower rate: leakage of inorganic phosphate and adenine nucleotides from mucosal cells was also minor. The surface ATPase may reflect the operation of a mechanism at the nutrient surface involved in acid secretion.

Response of active transport of ions and spontaneous water flux to osmotic gradients in gastric mucosa.

The effects of symmetric changes of the mucosal and serosal bathing solution on cell water content, net ion flux, and net water movement were studied in the isolated frog gastric mucosa. Similar to transmucosal concentration gradients that induce water movement and changes in cell water content, symmetric osmolality changes of the bathing solutions also produce changes in these functional parameters. Thus, increments from 165 to 286 mosmol/kg water in the osmolality of both solutions reduce cell water content from 2.37 plus or minus 0.12 to 1.30 plus or minus 0.20 ml/g wt, the net ion flux (acid secretion plus short-circuit current) from 4.83 plus or minus 0.36 to 3.44 plus or minus 0.26 mueq/cm2 per h, and the net water flux from 10.6 plus or minus 1.1 to 2.4 plus or minus 1.2 mul/cm2 per h. These osmotically induced flux changes of water and ions must be considered when osmotic gradients are being used to generate and to evaluate water movement across the gastric mucosa.