Luis Reuss

Mechanisms of cation permeation across apical cell membrane ofNecturus gallbladder: Effects of luminal pH and divalent cations on K+ and Na+ permeability

SummaryConventional 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 inNecturus 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 membranePK. The effects of Ba2+ on membrane potential and relative apical membranePK were larger than those of all other four cations at all concentrations tested (1–10mm). The effect of Sr2+ was significantly larger than those of Mg2+ and Ca2+ at 10mm, but not different at 5mm. The reduction ofPK 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 membranePK 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.

Intracellular ionic activities and transmembrane electrochemical potential differences in gallbladder epithelium

SummaryIntracellular ion activities inNecturus gallbladder epithelium were measured with liquid ion-exchanger microelectrodes. Mean values for K, Cl and Na activities were 87, 35 and 22mm, 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.

Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium.

SummaryConventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCli) inNecturus 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 sidesaCli was about 30mm, i.e., much higher than the activity predicted from equilibrium distribution (aCleq) across either membrane (5–9mm). Removal of Cl from the apical side caused a reversible decrease ofaCli towards the equilibrium value across the basolateral membrane. A new steady-stateaCli was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases ofaCli 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 changeaCli. When K substituted for mucosal Na, the cells depolarized andaCli 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 ofaCli 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 membranePCl.

Effects of amphotericin B on the electrical properties ofNecturus gallbladder: Intracellular microelectrode studies

SummaryIntracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (ΔVms) in the gallbladder ofNecturus. When the tissue was incubated in standard Na-Ringers 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-Ringers 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, whereasPK/PNa doubled. These results indicate that ΔVms is due to an increase of gNa across the luminal membranes of the epithelial cells (Cremaschiet al., 1977,J. Membrane Biol.34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976.J. Membrane Biol.29:1) that ΔVms results from a reduction in shuntPNa/PCl acting in combination with a rheogenic basolateral Na pump.

Triaminopyrimidinium (TAP+) blocks luminal membrane K conductance inNecturus gallbladder epithelium

SummaryThe effect of triaminopyrimidinium (TAP+) on the apical membrane ofNecturus 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.

Chapter 1 Mitogens and Ion Fluxes

Publisher Summary This chapter discusses the mechanisms of the transport of Na + , K + , Cl – , Ca 2+ , and H + by cell membranes and the mechanisms of the maintenance and regulation of cell volume, intracellular pH, and intracellular Ca 2+ activity. The action of mitogens in controlling these processes is reviewed, and the transport across intracellular membranes and the interrelationships between the transport of different substrates is also discussed. Inhibitors are powerful experimental tools in the study of membrane transport processes. However, two kinds of problems are frequently faced when employing these agents. The first problem is related to the lack of specificity of some of these agents and the second problem is related to the lack of consideration of secondary effects. Mitogen-cell interactions are highly complex and subject to both positive and negative regulation. Mitogens alter the rate of various ionic fluxes into cells. For example, Ca 2+ entry increases as a result of the addition of mitogens to cells.

Chapter 9 Control of Mitogenic Activation of Na+-H+ Exchange

Publisher Summary Among the earliest events that follow the binding of mitogens to cell surface receptors is the activation of a variety of transport systems, including those for Na + , K + , Ca 2+ , phosphate, glucose, amino acids, and uridine. The activation of some of these transport events is extremely rapid. The activation of some or all of these transport systems may be permissive for the cells to traverse the cell cycle, but activation of any one of these transport systems is not sufficient by itself to drive the cells through the cell cycle. A convenient method for the measurement of Na + –H + exchange in cells is the use of a dye whose fluorescence is pH sensitive and can be used to determine changes in cytoplasmic pH. Optical methods provide sensitivity and temporal resolution for such measurements. The dye should be nontoxic and have a structure that allows it to be permanently retained in the cytoplasm; that is, the leakage rate should be negligible during the time of the experiment. pH i and Na + –H + exchange activity are controlled, following the addition of mitogens to cells. Protein kinase C acts to modulate the activity of a variety of mitogen receptors, and the tyrosine kinase activity of these receptors is required for their activation of the Na + –H + exchange molecule. The activation of protein kinase C inactivates the epidermal growth factor (EGF) receptor as a tyrosine kinase. EGF activates phosphatidylinositol breakdown in A431 cells, and platelet-derived growth factor (PDGF) has this effect in 3T3 fibroblasts. Thus, each of these mitogens activates an enzyme that renders the mitogen receptor nonfunctional.

Introduction to the Physiology of Body Fluids

Life of complicated pluricellular organisms depends upon the preservation of a very narrow range of volume and composition of body fluids. Water, inorganic substances, and organic molecules are taken up from the external medium and distributed in one or more body fluid compartments. Some of them are eventually utilized for energy production, growth, and repair. Others, such as water and electrolytes, provide the environment in which the physical and chemical processes characteristic of cell, tissue, and organ function take place.

Homeostatic and Excretory Functions of the Kidney

The kidneys have a central role in the homeostasis of water and electrolytes, i.e., in the maintenance of volume and ionic composition of body fluids. This function is accomplished by appropriate changes in the rate of renal excretion of water and electrolytes, controlled by feedback mechanisms which involve participation of the nervous system, the endocrine system, or both. The homeostatic functions of the kidney include the control of the balance of water, sodium, chloride, potassium, calcium, magnesium, hydrogen ions, and phosphate. The adaptability of the kidney to the requirements of homeostasis is demonstrated by the large changes in urine volume and composition which occur in response to alterations in the diet. There is no fixed normal composition of the urine. Normal homeostatic renal function is defined by the capacity of the organ to vary the volume and composition of the urine over a wide range, according to requirements imposed by intake, extrarenal losses, and other factors.

Chapter 14 Ion Conductances and Electrochemical Potential Differences across Membranes of Gallbladder Epithelium

Publisher Summary The epithelial layer is particularly appropriate for intracellular microelectrode studies because of its structural simplicity and the relatively large size of the transporting cells. This chapter describes the equivalent electrical circuit, properties of the luminal and basolateral membrane, intracellular ionic activities, and mechanism of NaCl uptake and extrusion by the cells. Sodium activity in the cells is far below the predicted value for passive distribution, whereas chlorine (Cl) and potassium (K) activities are higher than predicted for passive distribution. Because of the low sodium (Na) conductance of the apical membrane, only a small fraction of Na uptake can be diffusional; however, most is neutral, coupled NaCl entry. The basolateral membrane transports Na actively to the serosal medium. Chlorine transport, although downhill, can be only partly diffusional. The largest moiety of the net Cl flux is electrically silent, either coupled to Na or K extrusion or to the uptake of another anion. Experiments with amphotericin B and transport inhibitors, however, do not support the hypothesis of an electrogenic basolateral Na pump in this tissue.