Thomas L. Holloman

Effects of ouabain on frog gastric mucosa in vitro

The effect of the addition of ouabain to the nutrient solution was determined on resistance, potential difference (p.d.) and H+ secretion rate. In NaCl media, 10(-3) M ouabain decreased significantly the p.d. from 25.6 mV to 16.1 mV in 30 min and to 11.0 mV in 60 min. No significant changes occurred in resistance and H+ secretion rate. In Na2SO4 (Cl(-)-free) media, ouabain produced a biphasic effect on p.d. The p.d. changed from -28.0 mV (nutrient-negative) to a nadir of -37.4 mV in 7 min and then increased to -16.4 mV in 60 min. At the nadir there was no significant change in resistance or H+ secretion rate but at 60 min, unlike Cl- media, resistance increased by 36% and H+ secretion rate decreased by 43%. To decide whether the ouabain-caused decrease in H+ rate in Na2SO4 media was due to an effect on the H+ pump or on resistance of the return pathways, the voltage was clamped at 0 and 40 mV. Clamping the voltage showed that in the case of a marked decrease in the H+ secretion rate, the H+ transport mechanism itself was inhibited (and not the parallel pathway). The decrease in p.d. due to ouabain in Cl- and SO42- media indicates that the (Na+ + K+)-ATPase mechanism may be electrogenic.

Potency of Barbiturates in Inhibition of Frog Gastric Secretion

Previously Saidman et al. (1) found a correlation between anesthetic potency and the oil/gas partition coefficient. Their measure of potency was the MAC, i.e., the minimum anesthetic concentration required to prevent a muscular response to a skin incision in 50% of the subjects to whom the anesthetic was administered. Schwartz and MacKrell (2) showed that the percentage of anesthetic needed to produce a given decrease in the H+ secretory rate of frog gastric mucosa in vitro was proportional to the MAC in man and on this basis predicted the MAC values for chloroform and Compound 347 (Ethrane). These results suggested lipid solubility as a factor influencing the potency of anesthetics in inhibition of acid secretion. In the work above (2) on frog gastric mucosa the anesthetics had molecules with no more than four carbon atoms. Mullins (3) points out that for compounds with more than four carbon atoms the anesthetic potency declines and that by the time one reaches a compound of C10 or C12 an anesthetic series is inert and higher analogues, whether they be alcohol, ether, or paraffin, have no anesthetic activity. Hence it is inferred that there is a site size limit for anesthetic molecules in biological membranes. In contrast, other investigators (4) found that the hypnotic activity of narcotics such as barbiturates was closely related to their relative lipophilic character as defined by the logarithm of the octanol-water partition coefficient, log P. Consequently, it was of interest to compare the behavior of barbiturates with anesthetics under similar conditions, namely, their effects on frog gastric mucosa. The question then is: to what extent is lipid solubility of barbiturates as defined by log P indicated as a factor in inhibition of acid secretion.

FURTHER EVIDENCE FOR A SIMPLE CL CONDUCTANCE PATHWAY IN NUTRIENT MEMBRANE OF FROG STOMACH

A decrease in nutrient Cl- increases the negativity of the nutrient relative to the secretory side. It seemed possible that Cl- transport could result from a neutral Cl- mechanism in the nutrient membrane coupled to a simple Cl- conductance pathway in the secretory membrane. Experiments in HCO3(-)-free, K(+)-free and Na(+)-free solutions in both bathing media gave for a 10-fold change in nutrient Cl- a PD change of 9.5 mV. Similar experiments with 0.5 mM DIDS in the nutrient solution gave for a 10-fold change in nutrient Cl- a PD change of 7.9 mV. These experiments eliminated a neutral Cl(-)-HCO3- exchanger, a NaCl and a KCl symport. Thus the change in PD could best be explained by a simple Cl- conductance in the nutrient membrane.

Models for Gastric Proton Pump in Light of the High Oxyntic Cell Conductance

The secreting in vitro frog fundus has a low transmucosal resistance (Rt), and the low Rt is due to a low resistance of the lumen-tubular (oxyntic) cell pathway (RLC = RL + R3 + lQ; see FIG. 1). The parallel paths, that is, the surface cell (R,,, = R, + RZ; see FIG. l), and the transintercellular (paracellular) Rnc and R+IF paths have high resistances. These conclusions are based on many lines of evidence that we believe are well accepted by workers in this field. l~~ Contemporary models2 for gastric HCl secretion are diagrammed in FIGURE 2. The question arises as to which of these models are compatible with the low resistance of the tubular cells. In model 2A it is postulated that there are two parallel neutral mechanisms in the secretory (luminal) membrane of the tubular cells; KCl moves from cell to lumen via a neutral symport and K+ is then actively exchanged with protons by a second neutral mechanism (one K+ for one proton). With this model a low value for R L ~ would not be anticipated; it would be necessary to make the ad hoc postulate of a low resistance path for some other ion not necessarily essential for secretion. This model has been suggested in the past but has no advocates today, so we will not further comment on it. In model 2B there are conductive pathways for K+ and Clfor transport from cell to lumen and a parallel neutral active K+-H+ exchange mechanism across the membrane. In model 2C there are two high conductance paths, a C1path and an electrogenic proton path. It is postulated that regardless of the complexity of the proton mechanism, it is electrogenic, that is, there is a transfer of H+ from the membrane to the lumen without the involvement of another ion at this site. A completed circuit for the secretion of HCl is necessary, and with C1media, movement of C1from cell to lumen completes the circuit. Model 2B (NP = neutral proton pump) and model 2C (EP = electrogenic proton pump) are both compatible with a low RLC . Our main thrust in this paper is to attempt to distinguish between the NP and EP models on the basis of other experimental evidence.

Further evidence of Na+ conductance in frog stomach in Cl- -free media.

Summary The replacement of choline ions by Na+ ions in the secretory solution of the frog gastric mucosa of Rana pipiens in Cl--free media increases the resistance, the PD, and the H+ secretory rate. The replacement of Na+ by choline ions produces generally the reverse changes of the three electrophysiological parameters. The experimental data involving the PD and H+ secretory rate can be explained in terms of a Na+ emf in a Na+ conductance pathway opposing the H+ emf in a H+ conductance pathway on the secretory (apical) membrane.

Electrophysiological effects of Ca ionophore A23187 on frog stomach.

Summary The Ca ionophore A23187 at a concentration of 10−6 M in a Cl− nutrient solution containing 1 mM Ca2+ or at a concentration of 10−5 M in a Cl− nutrient solution containing 0.04, 1 or 3 mM Ca2+ decreased the resistance and increased the H+ secretory rate of the frog gastric mucosa of Rana pipiens. Similar effects were obtained with 10−5 M ionophore in secretory solutions. The ionophore exhibited inhibitory effects in Cl−-free media. These results together with those involving Ca2+-free Cl− solutions and Ba2+ studies suggested that the ionophore increased Cl− conductance, particularly of the secretory membrane.

Cationic conductances of nutrient and secretory membranes of frog stomach in Cl--free solutions.

Summary The K+ conductance in Cl- -free media of the frog gastric mucosa of Rana pipiens was found to be relatively high and essentially the same for both the nutrient and secretory membranes. The replacement of Na+ with either Mg2+ or Ca2+ in either the nutrient or secretory solution and in the presence of 4 mM K+ in both solutions increased the resistance and decreased the PD. An analysis of the results for the nutrient membrane suggests that Mg2+ and Ca2+ block the K+ pathway.

EFFECTS OF OUABAIN ON IN VITRO FROG STOMACH

Publisher Summary This chapter discusses the effects of ouabain on in vitro frog stomach. Both H + and Cl are actively secreted by the mammalian and frog stomach. It has been proposed that the secretion of Cl takes place through a NaCl cotransport system located in the nutrient (basolateral) membrane. The energy for the transport comes from a Na–K ATPase pump located also in the nutrient membrane. To investigate the extent to which this pump controls secretion of ions, experiments were designed using ouabain, a well-known inhibitor of the Na–K ATPase pump. An in vitro method was used on gastric mucosa of Rana pipiens mounted between a pair of cylindrical chambers. All experiments were started with physiological solutions. The transmembrane resistance, the transmembrane potential difference (PD), and the H + secretory rate were recorded. Two pairs of electrodes were used, one for sending current and the other for measuring the PD. Histamine was added to the nutrient solution. Ouabain was added to the nutrient solution. It was found that the addition of ouabain resulted in a substantial decrease in PD. The PD remained positive after 60 min. No major change was observed in H + secretory rate and resistance.

Effects of β-Adrenergic Amines on Frog Gastric Mucosa

Summary Beta adrenergic amines like alpha adrenergic amines decreased the H+ secretory rate and increased the transmucosal resistance of the gastric mucosa of Rana pipiens. A concentration of 0.1 mM propranolol in the nutrient solution followed by a combination of 0.1 mM propranolol and either 0.25 mM isoproterenol or 1.0 mM epinephrine reduced the effects of the beta agonists, isoproterenol and epinephrine. However, a concentration of 0.25 mM isoproterenol or 1.0 mM epinephrine followed by the same combination did not reduce the effects of isoproterenol or epinephrine alone. These results suggested the existence of beta receptor sites in addition to non-specific sites.

Immediate Short-Time Effects of Ethacrynic Acid on Frog Gastric Mucosa in Chloride-Free Solutions

Summary The presence of ethacrynic acid in the nutrient sulphate solution produces, generally in less than 3 min, a decrease in resistance accompanied by an increase in transmucosal PD. These results strongly suggest that the dominant effect of ethacrynic acid is not on the Na+ pathway of the nutrient membrane, but without further knowledge of the resistance of other ionic pathways, it cannot be inferred whether the effect predominates on the K+ pathway as in Cl- media.