Initial events in the interaction of epidermal growth factor with cells.
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Animals↗
Biomedical subjects
L Reuss
Publications and source records attributed to L Reuss.
Serum and epidermal growth factor transiently depolarize quiescent BSC-1 epithelial cells.
Continuous intracellular recording of membrane potential with microelectrodes in BSC-1 epithelial cells has been used to study the early ionic effects of the interaction of mitogens with cell surface receptors. Initial results show that (i) there is no significant difference in membrane potential between growing and quiescent cells, (ii) addition of epidermal growth factor or serum to quiescent BSC-1 cells induces a brief and transient depolarization, (iii) the mitogenic response of BSC-1 cells to epidermal growth factor and the transient depolarization show similar concentration dependences, and (iv) serum addition to quiescent BSC-1 cells induces a sustained increase in Na+ influx that is electroneutral and amiloride sensitive. Intracellular pH changes may be a primary event triggering the response of quiescent cells to mitogenic polypeptides.
Amiloride↗
Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies.
The mechanism of luminal solution acidification was studied in Necturus gallbladder by measurement of mucosal solution and intracellular pH with glass electrodes. When the gallbladder was bathed by a Na-Ringer's solution it acidified the luminal side by a Na+-dependent, amiloride-inhibitable process. In the presence of ouabain, acidification was reduced but could be stimulated to a rate greater than that under control conditions by the imposition of an inwardly directed Na+ gradient. These results suggest that luminal acidification results from Na+-H+ exchange at the apical membrane and not by diffusion of metabolic CO2. Li+ can substitute for Na+ but K+, Rb+, Cs+, and tetramethylammonium (TMA+) cannot. The maximal rate of exchange was about five times greater for Na+ than for Li+. Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes; with the tissue bathed in Na-Ringer's solution (pH 7.75), pHi was 7.51 +/- 0.04. After inhibition of Na+-H+ exchange by mucosal perfusion with amiloride (1 mM) or by complete Na+ replacement with TMA+, phi fell reversibly by 0.15 and 0.22 pH units, respectively. These results support the conclusion that Na+-H+ exchange at the apical membrane is the mechanism of luminal acidification and is involved in the maintenance of steady state pHi.
Acids↗
Mechanisms of cation permeation across apical cell membrane of Necturus gallbladder: effects of luminal pH and divalent cations on K+ and Na+ permeability.
Conventional microelectrode techniques were combined with unilateral mucosal ionic substitutions to determine the effects of luminal pH and luminal alkali-earth cation concentrations on apical membrane cation permeability in Necturus gallbladder epithelium. Acidification of the mucosal solution caused reversible depolarization of both cell membranes and increase of transepithelial resistance. Low pH media also caused: (a) reduction of the apical membrane depolarization induced by high K, and (b) increase of the apical membrane hyperpolarization produced by Na replacement with Li or N-Methyl-D-glucamine. These results, in conjunction with estimates of cell membrane conductances, indicate that acidification of the luminal solution produces a reduction of apical membrane K permeability (PK). Addition of alkali earth cations (Mg2+, Ca2+, Sr2+, or Ba2+) produced cell membrane depolarization, increase of relative resistance of the luminal membrane and reduction of the apical membrane potential change produced by a high-K mucosal medium. These results, as those produced by low pH, can be explained by a reduction of apical membrane PK. The effects of Ba2+ on membrane potential and relative apical membrane PK were larger than those of all other four cations at all concentrations tested (1-10 mM). The effect of Sr2+ was significantly larger than those of Mg2+ and Ca2+ at 10 mM, but not different at 5 mM. The reduction of PK produced by mucosal acidification appears to be mediated by: (a) nonspecific titration of membrane fixed negative charges, and (b) an effect of luminal proton activity on the apical K channel. Divalent cations reduce apical membrane PK probably by screening negative surface charges. The larger magnitude of the effects of Ba2+ and Sr2+ can be explained by binding to membrane sites, in the surface or in the K channel, in addition to their screening effect. We suggest that the action of luminal pH on K secretion in some segments of the renal tubule could be mediated in part by this pH-dependent K permeability of the luminal membrane.
Animals↗
Mechanism of the effect of cyanide on cell membrane potentials in Necturus gall-bladder epithelium.
1. Addition of sodium cyanide to the mucosal or the serosal medium bathing the isolated gall-bladder of Necturus maculosus causes hyperpolarization of both apical and basolateral membrane of the epithelial cells. The effect of cyanide is practically immediate, reversible (if exposure is brief), and long-lasting (greater than 30 min). 2. The hyperpolarization is accompanied by: (a) reduction of the equivalent resistance of the cell membranes, as shown by cable analysis and input resistance measurements, and (b) increase of the potassium selectivity of both cell membranes, as evidenced by the effects of external substitutions of potassium for sodium on cell membrane potentials. We conclude that the cyanide-induced hyperpolarization is caused mainly or exclusively by an increase of the potassium permeability of the cell membranes. 3. Addition of the calcium ionophore A23187 (5 microM) to the mucosal medium in the presence of 1 mM-calcium caused similar effects to those produced by cyanide. After either cyanide or A23187, addition of the other agent did not cause further membrane potential changes. 4. Quinine (100 microM, mucosal medium) reduced the potassium permeability of the apical membrane both under control conditions and during exposure to cyanide. 5. We suggest that the cyanide-induced increase of the potassium permeability of the cell membrane is mediated by an elevation of intracellular calcium ion activity, attributable to release from mitochondrial sources.
Absorption↗
Potassium transport mechanisms by amphibian gallbladder.
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Amines↗
Mechanisms of the mucosa-negative transepithelial potential produced by amphotericin B in gallbladder epithelium.
Exposure of the apical (luminal) surface of gallbladder epithelium to the polyene antibiotic amphotericin B produces a mucosa-negative change of the transepithelial potential. The mechanism of this effect has been studied by both electrophysiologic techniques and tracer flux measurements. The main effect of amphotericin B appears to be an increase in the permeability of the apical membrane to small monovalent cations. During exposure to Na-Cl-Ringer's, this results in cell depolarization and in K loss and Na uptake by the cells. The loss of apical membrane K selectivity appears to account for most of the change in the transepithelial potential. Accumulation of K in the mucosal un-stirred layers can contribute to this change as well. The evidence for stimulation of an electrogenic basolateral Na pump (by the net Na entry caused by amphotericin B) is not conclusive due to a) indirect effects of the polyene antibiotic on the resistance of basolateral membrane, b) lack of reduction of the amphotericin B-dependent change of transepithelial potential when the pump is inhibited, and c) lack of effect of NaCl addition on basolateral membrane potential in tissues preincubated in high K-media and exposed to amphotericin B on the luminal side.
Amphotericin B↗
Intracellular K+ activity and its relation to basolateral membrane ion transport in Necturus gallbladder epithelium.
A study of the mechanisms of the effects of amphotericin B and ouabain on cell membrane and transepithelial potentials and intracellular K activity (alpha Ki) of Necturus gallbladder epithelium was undertaken with conventional and K-selective intracellular microelectrode techniques. Amphotericin B produced a mucosa-negative change of transepithelial potential (Vms) and depolarization of both apical and basolateral membranes. Rapid fall of alpha Ki was also observed, with the consequent reduction of the K equilibrium potential (EK) across both the apical and the basolateral membrane. It was also shown that, unless the mucosal bathing medium is rapidly exchanged, K accumulates in the unstirred fluid layers near the luminal membrane generating a paracellular K diffusion potential, which contributes to the Vms change. Exposure to ouabain resulted in a slow decrease of alpha Ki and slow depolarization of both cell membranes. Cell membrane potentials and alpha Ki could be partially restored by a brief (3-4 min) mucosal substitution of K for Na. Under all experimental conditions (control, amphotericin B, and ouabain), EK at the basolateral membrane was larger than the basolateral membrane equivalent emf (Eb). Therefore, the K chemical potential difference appears to account for Eb and the magnitude of the cell membrane potentials, without the need to postulate an electrogenic Na pump. Comparison of the rate of Na transport across the tissue with the electrodiffusional K flux across the basolateral membrane indicates that maintenance of a steady-state alpha Ki cannot be explained by a simple Na,K pump-K leak model. It is suggested that either a NaCl pump operates in parallel with the Na,K pump, or that a KCl downhill neutral extrusion mechanism exists in addition to the electrodiffusional K pathway.
Animals↗
Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium.
Conventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCli) in Necturus gallbladder epithelium. As shown previously (Reuss, L., Weinman, S.A., 1979; J. Membrane Biol. 49:345), when the tissue was exposed to NaCl-Ringer on both sides aCli was about 30 mM, i.e., much higher than the activity predicted from equilibrium distribution (aCleq) across either membrane (5--9 mM). Removal of Cl from the apical side caused a reversible decrease of aCli towards the equilibrium value across the basolateral membrane. A new steady-state aCli was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases of aCli when Li, choline, tetramethylammonium or N-methyl-D-glucamine (NMDG) were employed to replace Na. During bilateral Na substitutions with choline the cells depolarized significantly. However, no change of cell potential was observed when NMDG was employed as Na substitute. Na replacements with choline or NMDG on the serosal side only did not change aCli. When K substituted for mucosal Na, the cells depolarized and aCli rose significantly. Combinations of K for Na and Cl for SO4 substitutions showed that net Cl entry during cell depolarization can take place across either membrane. The increase of aCli in depolarized cells exposed to K2SO4-Ringer on the mucosal side indicates that the basolateral membrane Cl permeability (PCl) increased. These results support the hypothesis that NaCl entry at the apical membrane occurs by an electroneutral mechanism, driven by the Na electrochemical gradient. In addition, we suggest that Cl entry during cell depolarization is downhill and involves an increase of basolateral membrane PCl.
Animals↗
Management of pregnancy after cesarean section.
Management of 226 patients with previous low transverse cesarean section in a special obstetric clinic for high-risk pregnancies significantly and safely reduced the incidence of purely elective cesarean section to less than 10%. A substantial incidence (25%) of other high-risk factors dictating antepartum indicated cesarean section were found in this population. There was 38.5% incidence of vaginal delivery in 145 patients who underwent a trial of labor. Patients delivered vaginally followed a normal labor curve up to full dilatation. Oxytocin was required in three cases of prolonged second stage of labor and resulted in instrumental vaginal delivery in all instances. There were no uterine ruptures on vaginal exploration after delivery or at the time of repeat cesarean section during labor. There was no maternal death or perinatal loss. No justification for the present clinical practice of a 99% inicidence of elective repeat cesarean section could be found. Substantial savings in hospital cost, as well as a decreased number of diagnostic tests for the otherwise normal gravid woman, can be safely achieved.
Birth Weight↗
Intracellular ionic activities and transmembrane electrochemical potential differences in gallbladder epithelium.
Intracellular ion activities in Necturus gallbladder epithelium were measured with liquid ion-exchanger microelectrodes. Mean values for K, Cl and Na activities were 87, 35 and 22 mM, respectively. The intracellular activities of both K and Cl are above their respective equilibrium values, whereas the Na activity is far below. This indicates that K and Cl are transported uphill toward the cell interior, whereas Na is extruded against its electrochemical gradient. The epithelium transports NaCl from mucosa to serosa. From the data presented and the known Na and Cl conductances of the cell membranes, we conclude that neutral transport driven by the Na electrochemical potential difference can account for NaCl entry at the apical membrane. At the basolateral membrane, Na is actively transported. Because of the low Cl conductance of the membrane, only a small fraction of Cl transport can be explained by diffusion. These data suggest that Cl transport across the basolateral membrane is a coupled process which involves a neutral NaCl pump, downhill KCl transport, or a Cl-anion exchange system.
Animals↗
Triaminopyrimidinium (TAP+) blocks luminal membrane K conductance in Necturus gallbladder epithelium.
The effect of triaminopyrimidinium (TAP+) on the apical membrane of necturus gallbladder epithelial cells was investigated with intracellular microelectrode techniques. TAP+, added to the mucosal bathing solution only, produced the following effects (all rapid and reversible): (i) cell depolarization, (ii) increase of apical membrane resistance, and (iii) decrease of the apical membrane potential change produced by K for Na substitution on the mucosal side. These results can be explained by a decrease of apical membrane K conductance. The paracellular effects of TAP+ were similar to the ones previously described by Moreno (J.H. Moreno, 1974; Nature (London) 251:150; J.H. Moreno, 1975. J. Gen. Physiol. 66:97). These results indicate that the change of transepithelial potential produced by TAP+ cannot be ascribed solely to its effect on the paracellular pathway.
Animals↗
Electrical properties of the cellular transepithelial pathway in Necturus gallbladder: III. Ionic permeability of the basolateral cell membrane.
The ionic permeability of the basolateral membrane of Necturus gallbladder epithelium was studied with intracellular microelectrode techniques. After removal of most of the subepithelial tissue (to reduce unstirred layer thickness), impalements were performed from the serosal side, and ionic substitutions were made in the serosal solution while a microelectrode was kept in a cell. Thus, it was possible to obtain continuous (and reversible) records of transepithelial and cell membrane potentials and to measure intermittently the transepithelial resistance and the ratio of cell membrane resistances. From these data and the mean value of the equivalent resistance of the cell membranes in parallel (obtained from cable analysis in a different group of tissues), absolute cell membrane and shunt resistances and equivalent electromotive forces (emfs) were calculated. From the changes of basolateral membrane emf (Eb) produced by the substitutions, the conductance (G) and permeability (P) of the membrane for K, Cl and Na were estimated. Potassium-for-sodium substitutions produced large reductions of both cell membrane potentials, of Eb, and of the resistance of the basolateral membrane (Rb), indicating high GK and PK. Chloride substitution with isethionate or sulfate resulted in smaller changes of cell membrane potentials and Eb and in no significant change of Rb, indicating small but measurable values of GCl and PCl. Sodium substitutions with N-methyl-D-glucamine (NMDG) resulted in cell potential changes entirely attributable to the biionic potential produced in the shunt pathway (PNa greater than PNMDG), and in no significant changes of Rb or Eb, indicating that GNa and PNa are undetectable. The question of the mechanism of Cl transport across the basolateral membrane was addressed by comparing the mean rate of transepithelial Cl transport : formula, see text: and the predicted passive Cl flux across the basolateral membrane (from the membrane Cl conductance, potential, and Cl equilibrium potential). The conclusion is that only a very small fraction of the Cl flux across the basolateral membrane can be electrodiffusional. Since the paracellular Cl conductance is also too low to account for : formula, see text:, these results suggest the presence of a neutral mechanism of Cl extrusion from the cells. This could be a NaCl pump, a downhill KCl transport mechanism, or a Cl-HCO3 exchange mechanism.
Animals↗
Effects of ouabain on fluid transport and electrical properties of Necturus gallbladder. Evidence in favor of a neutral basolateral sodium transport mechanism.
Net fluid transport (Jv) and electrical properties of the cell membranes and paracellular pathway of Necturus gallbladder epithelium were studied before and after the addition of ouabain (10(-4) M) to the serosal bathing medium. The glycoside inhibited Jv by 70% in 15 min and by 100% in 30 min. In contrast, the potentials across both cell membranes did not decrease significantly until 20 min of exposure to ouabain. At 30 min, the basolateral membrane potential (Vcs) fell only by ca 7 mV. If basolateral Na transport were electrogenic, with a coupling ratio (Na:K) of 3:2, the reductions of Vcs at 15 and 30 min should be 12--15 and 17--21 mV, respectively. Thus, we conclude that the mechanism of Na transport from the cells to the serosal bathing solution is not electrogenic under normal transport conditions. The slow depolarization observed in ouabain is caused by a fall of intracellular K concentration, and by a decrease in basolateral cell membrane K permeability. Prolonged exposure to ouabain results also in an increase in paracellular K selectivity, with no change of P Na/P Cl.
Animals↗
Amphotericin B and K+ transport across excised toad urinary bladder.
The transmural electric PD of bladders bathed by Na2SO4 Ringer was not affected by amphotericin (5 x 10(-6) M, mucosal) but the PD followed the direction for K+ diffusion in the presence of a transmural K+ gradient. Increases in bathing solution K+ increased conductance. Ouabain pretreatment did not affect drug-induced changes in PD or conductance. Unidirectional fluxes of radiolabeled Na+ and K+ but not SO42- across the short-circuited bladder were increased by amphotericin. Ninety percent of the rise in the serosal-to-mucosal flow of Na+ disappeared when mucosal Na+ was replaced by choline. Amphotericin induced a 20-fold increase in mucosal-to-serosal K+ flux but K+ serosal-to-mucosal flow increased 200-fold. This flux asymmetry persisted for 110 min, was abolished by pre- or posttreatment with ouabain, and was immeasurable when bathing solution K+ was increased from 2.4 to 59 meq/liter. With 2.4 meq K+/liter the ratio of active Na+ reabsorption to K+ secretion was 8 to 1, but K+ secretion was not closely linked to Na+ transport. The results suggest that amphotericin induces a paracellular K+-selective path, Na+ isotope exchange, and K+ secretion.
Amphotericin B↗
Mechanisms of sodium and chloride transport by gallbladder epithelium.
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Animals↗
Electrophysiology of epithelial transport: introduction.
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Animals↗
Effects of amphotericin b on the electrical properties of Necturus gallbladder: intracellular microelectrode studies.
Intracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (deltaVms) in the gallbladder of Necturus. When the tissue was incubated in standard Na-Ringer's solution, the antibiotic reduced the apical membrane potential by about 40 mV, and the basolateral membrane potential by about 35 mV, whereas the transepithelial potential increased by about 5 mV. The electrical resistance of the apical membrane fell by 83%, and that of the basolateral membrane by 40%; the paracellular resistance remained unchanged. Circuit analysis indicated that the equivalent electromotive forces of the apical and basolateral membranes fell by 35 and 11 mV, respectively. Changes in potentials and resistances produced by ionic substitutions in the mucosal bathing medium showed that amphotericin B produces a nonselective increase in apical membrane small monovalent cation conductance (K, Na, Li). In the presence of Na-Ringer's on the mucosal side, this resulted in a reduction of the K permselectivity of the membrane, and thus in a fall of its equivalent emf. During short term exposure to amphotericin B, PNa/PCl across the paracellular pathway did not change significantly, whereas PK/PNa doubled. These results indicate that deltaVms is due to an increase of gNa across the luminal membranes of the epithelial cells (Cremaschi et al., 1977. J. Membrane Biol. 34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976. J. Membrane Biol 29:1) that deltaVms results from a reduction in shunt PNa/PCl acting in combination with a rheogenic basolateral Na pump.
Amphotericin B↗