Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Summary Microelectrodes were used to investigate the effect of 0.5mm mucosal lanthanum (La³⁺) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potentialV _o ^sc (mean value=–65.4±3.2 mV, inside negative) hyperpolarized to –108.7±2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potentialV _o or the inner membrane potentialV _t . The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La³⁺ to the outer medium increased the short-circuit current to 190% of the control value.V _o ^sc concomitantly changed to –28±3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La³⁺ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La³⁺ on short-circuit current has been observed. It is ascribed to the penetration of La³⁺ into the sodium channels.     

Ouabain on active transepithelial sodium transport in frog skin: studies with microelectrodes

Studies were done with isolated frog skin to determine the effects of 10(-4) M ouabain on the electrophysiological parameters of outer and inner barriers of the Na-transporting cells. Microelectrodes were used to impale the skins from the outer surface to determine the intracellular voltages (Vsco) under conditions of short-circuiting and under conditions where a voltage clamp was used to vary the transepithelial voltage, VT. From this, the electrical resistances of outer (Rfo) and inner (RI) barriers were estimated. In addition, the driving force for active transepithelial Na transport (ENa = E'1) was estimated from the values of VT when the Vo = 0 mV (Helman and Fisher. 1977. J. Gen. Physiol. 69: 571-604). Studies were done with skins bathed with the usual 2.4 meq/liter [K]i in the inner solution as well as with reduced [K]i of 0.5 and 0 meq/liter. Characteristically, the responses to ouabain could be described by an initial rapid phase (5-10 min) during which time the Ri was increased markedly and the E'1 was decreased from control values. Thereafter, during the slow phases of the response, the resistances of both outer and inner barriers increased continuously and markedly with time leading ultimately to essentially complete inhibition of the short-circuit current. Similar studies were done with skins exposed to 10(-4) M amiloride in the outer solution. Although estimates of Ri could not be obtained under these conditions, the effects on the Vsco and E'1 were similar to those observed for the Na-transporting skins. However, the magnitudes of the effects were less and relatively slower than observed for the Na-transporting skins. The results of these studies were analyzed within the context of a proposed electrical model that takes into account the observation that the magnitude of the voltage at the inner barrier appears to exceed the equilibrium potential for K especially when transepithelial Na transport is inhibited at the apical barrier of the cells.     

Effects of antidiuretic hormone upon electrical potential and resistance of apical and basolateral membranes of frog skin

Summary The effect of ADH upon the intracellular potential and the resistance of inner and outer borders of the transport pathway was investigated on isolated skins ofRana temporaria. Within 40 min after ADH (100-300 mU/ml), the intracellular potential under short-circuit conditions decreased to about 40% of the control value (–79±4 mV), concomitant with an increase in the short-circuit current to about 160% of the control value. Amiloride, applied when steady values under ADH had been reached, caused an immediate rise of the intracellular potential to values typical for control conditions. This confirms (i) the intracellular location of the microelectrode and the absence of impalement artifacts, and (ii) the ineffectiveness of ADH upon the electromotive forces of the inner border. ADH had no effect upon the intracellular potential after blockage of the Na entry by Amiloride. The equilibrium potential of the outer border was estimated to be about +20 mV under the influence of ADH. As this value is considerably less positive than might be expected for the chemical potential of Na, a significant contribution of ions other than Na to the outer border conductance and equilibrium potential is implicated. The resistance of the outer border was more significantly decreased than that of the active transcellular pathway after ADH due to an increase in the inner border resistance, which exceeded that of the outer border after ADH. The effect of ADH upon the outer membrane characteristics would be underestimated by a factor of two, if the alterations of the electrical potential difference were not taken into consideration.     

Model of the outer membrane potential generation by the inner membrane of mitochondria.

Voltage-dependent anion channels in the outer mitochondrial membrane are strongly regulated by electrical potential. In this work, one of the possible mechanisms of the outer membrane potential generation is proposed. We suggest that the inner membrane potential may be divided on two resistances in series, the resistance of the contact sites between the inner and outer membranes and the resistance of the voltage-dependent anion channels localized beyond the contacts in the outer membrane. The main principle of the proposed mechanism is illustrated by simplified electric and kinetic models. Computational behavior of the kinetic model shows a restriction of the steady-state metabolite flux through the mitochondrial membranes at relatively high concentration of the external ADP. The flux restriction was caused by a decrease of the voltage across the contact sites and by an increase in the outer membrane potential (up to +60 mV) leading to the closure of the voltage-dependent anion channels localized beyond the contact sites. This mechanism suggests that the outer membrane potential may arrest ATP release through the outer membrane beyond the contact sites, thus tightly coordinating mitochondrial metabolism and aerobic glycolysis in tumor and normal proliferating cells.     

Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin

Isolated skins from the frog Rana pipiens were mounted on Ussing-type chambers and bathed with Amphibian Ringer's solution on both sides. Electrical potential difference, resistance, and short-circuit current (SCC) were measured by using calomel electrodes, Ag-AgCl electrodes, Ringer's-agar bridges, and Tektronix digital multimeters. Under the conditions employed, SCC is a measure of net Na+ transport. The frog skin peptides bombesin, caerulein, and physalaemin were administered to the serosal side at concentrations of 0.5, 5.0, and 50 ng/ml. Control electrophysiological parameters were: potential difference, 23 +/- 2 mV; resistance, 738 +/- 59 ohms cm2; and SCC 32 +/- 3 microA/cm2. Although bombesin and caerulein had no significant, reversible effect on potential difference, resistance, or SCC, physalaemin significantly, and reversibly, depressed SCC by 22%. Caerulein did significantly depress SCC, but the response was not reversible.     

Heptaminol hydrochloride as an epithelium transporter

Electrophysiological and transport effects induced by heptaminol hydrochloride were studied in frog epithelium. This tissue, which can easily be maintained in vitro, is a valuable model for studying sodium active transport with hormone-dependent characteristics that reproduce mammalian nephron behavior (notably in areas with tight gap junctions). The two following techniques were used: the Ussing short-circuit current method and the swept-frequency impedance measurement method. Our findings indicate the following. (i) Heptaminol hydrochloride significantly increases the short-circuit current and transepithelial polarization. (ii) This effect develops progressively as the molecule is introduced on the serous side (3Na+/2K+ active countertransport sites). Time to maximum development is approximately 20 min and the electrophysiological effect lasts from 60 to 90 min. (iii) The mean equivalent cationic current rise is larger in sulfate-Ringer (+23 +/- 4.6 microA, p less than 0.01) than in chloride-Ringer (+14 +/- 4.9 microA, p less than 0.05). The increase in short-circuit current is approximately 0.9 muequiv. cm-2 h-1 in sulfate-Ringer. (iv) The increase in mean polarization is greater in chloride (+21 +/- 6.2 mV, p less than 0.02) than in sulfate (+6 +/- 1.5 mV, p less than 0.01) following a diphasic effect on potential. (v) Changes in apical impedance Z are small (-454 +/- 323 omega, nonsignificant) compared with transepithelial resistance in sulfate (-1065 +/- 359 omega, p less than 0.05). (vi) Changes in membrane capacitance reflect changes in the membrane surface. However, no significant capacitance changes are produced in sulfate and chloride solution by heptaminol hydrochloride (-0.04 +/- 0.11 microF and 0.05 +/- 0.11 microF, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Does intracellular sodium regulate sodium transport across the mucosal surface of frog skin?

A method has been devised to functionally remove the serosal membrane of frog skin. Skins treated in this way have no spontaneous potential. However, if sodium gradients are placed across the tissues diffusion potentials and hence short-circuit currents of either sign, depending on the direction of the gradient, could be recorded. These short-circuit currents were completely imhibited by amiloride only from the mucosal face. However, the concentration of amiloride causing 50% inhibition of the short-circuit curent (Km) in treated skins was 2.3 . 10(-3)M, when a sodium gradient was applied from serosa to mucosa, whereas both in untreated skins without a sodium gradient and in treated skins with a mucosal to serosal sodium gradient, the Km of amiloride was 2 . 10(-7)-4 . 10(-7)M. The mechanism by which amiloride is able to inhibit the short-circuit currents of either sign is discussed.     

Cobalt-dependent stimulation of sodium transport in the amphibian skin and nephron

One to ten millimolar CoCl2, when applied to the outer surface of the apical membrane of the frog skin (Rana temporaria), reversibly increased the potential differences and short-circuit current. These observations suggest that Co2+ may control the gating system of the sodium channel. In the kidney of the newt (Triturus vulgaris), proximal reabsorption is increased under the influence of 0.5 mM CoCl2 injected into the lumen. When CoCl2 (0.5 mM) was injected into the lumen of the distal tubule of the newt kidney, Na+ and Cl- reabsorption was stimulated simultaneously Ca2+ transport was inhibited. The data obtained suggested that Co2+ may affect the state of the sodium and calcium channels of nonexcitable membranes of amphibia, and thus may be involved in the regulation of the function of the renal tubules.     

A study of the perturbation effects of the local anesthetic procaine on human erythrocyte and model membranes and of modifications of the sodium transport in toad skin

The interaction of the local anesthetic procaine with human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), isolated toad skins, and molecular models is described. The latter consisted of phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that procaine induced the formation of stomatocytes. Experiments performed on IUM at 37 degrees C by fluorescence spectroscopy showed that procaine interacted with the phospholipid bilayer polar groups but not with the hydrophobic acyl chains. X-ray diffraction indicated that procaine perturbed DMPC structure to a higher extent when compared with DMPE, its polar head region being more affected. Electrophysiological measurements disclosed a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of procaine to isolated toad skin, reflecting inhibition of active ion transport.     

Na+/H+ exchangers in the human eccrine sweat duct

Using an anti-NHE1 antibody, we demonstrate the presence of a Na+/H+ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1basal), at the junction between inner and outer cells layers (NHE1inter), and along the lateral membranes (NHE1later) of all cells of the duct. At the luminal membrane, no staining was demonstrated either for NHE1 or NHE3. To investigate Na+/H+ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO3-free conditions. In the presence of basolateral 5-ethyl-N-isopropyl amiloride (EIPA), an acidification of 0.29 +/- 0.03 pH units was observed, whereas no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 +/- 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinization, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in intracellular pH regulation.     

Postcatelectrotonic potentials and trace changes in the nerve fiber excitability

When cathode subthreshold impulse was turned off, excitable membranes of isolated nerve fibres and nervous trunk show postelectrotonic depolarisation (PED), that is a slow recovery of membrane potential to the resting level. PED of the single nodes of Ranvier and nervous trunk is registered not only in normal conditions, but also after complete block of sodium channels. The size and duration of nervous trunk PED under subthreshold depolarising current increase along with duration of applied depolarisation: when cathode current 1 ms in duration was used, they were 0.093 +/- +/- 0.004 mV and 7.123 +/- 0.576 ms, respectively; when current was 5 ms in duration, they were 0.189 +/- 0.005 mV and 23.212 +/- 1.186 ms, whereas a 10-ms depolarisation yields values of 0.220 +/- 0.011 mV and 68.721 +/- 3.389 ms. Application of the train of catelectrotonic impulses leads to PED built-up. As PED is found not only in normal conditions but also after complete block of sodium channels, it is reasonable to suggest that the most probable reason for PED is an outward potassium current.     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Effects of the local anesthetic benzocaine on the human erythrocyte membrane and molecular models

The interaction of the local anesthetic benzocaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphospatidylcholine (DMPC), and phospholipid multilayers of DMPC and dimyristoylphospatidyletanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that benzocaine induced the formation of echinocytes. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy showed that benzocaine interacted with the phospholipid bilayer polar groups and hydrophobic acyl chains. X-ray diffraction analysis of DMPC confirmed these results and showed that benzocaine had no effects on DMPE. The effect on sodium transport was also studied using the isolated toad skin. Electrophysiological measurements indicated a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of benzocaine, reflecting inhibition of active ion transport.     

Role of Ca2+ and prostaglandin in regulation of active Na+ transport in frog skin

1. The role of prostaglandins and intracellular Ca2+ in regulation of active transepithelial sodium transport in frog skin were studied by examinations of effects of the calcium ionophore A23187 on short-circuit current (SCC) and intracellular voltage. 2. A23187 and arachidonic acid induced a marked increase in both SCC and prostaglandin E2 synthesis. 3. In indomethacin treated skins A23187 did not stimulate but on the contrary inhibited the basal SCC. 4. The A23187-induced increase in SCC was associated with a decrease in the fractional resistance of the apical membrane and a depolarization of the cells. 5. In skins pretreated with indomethacin, the A23187 induced inhibition of SCC coincided with a slight hyperpolarization of the cellular potential and an increase in fractional resistance of the apical membrane.     

Effects of Pb<Superscript>2+</Superscript> ions on Na<Superscript>+</Superscript> transport in the isolated skin of the toad <Emphasis Type="Italic">Pleurodema thaul</Emphasis>

The effects induced by lead ions on the short-circuit current (SCC) and on the potential difference (V) of the toad Pleurodema thaul skin were investigated. Pb²⁺ applied to the outer (mucosal) surface increased SCC and V and when applied to the inner (serosal) surface decreased both parameters. The stimulatory effect, but not the inhibitory action, was reversible after washout of the metal ion. The amiloride test showed that the increase was due principally to stimulation of the driving potential for Na⁺ (V-E_Na+) and that inhibition was accompanied by a reduction in the V-E_Na+ and also by a significant decrease in skin resistance indicating possible disruption of membrane and/or cell integrity. The effect of noradrenaline was increased by outer and decreased by inner administration of Pb²⁺. The results suggest that mucosal Pb²⁺ activates toad skin ion transport by stimulating the V-E_Na+ and that serosal Pb²⁺, with easier access to membrane and cellular constituents, inactivates this mechanism, revealing greater toxicity when applied to the inner surface of the skin. Abbreviations: SCC – short-circuit current; V – potential difference; V-E_Na+– driving potential for Na⁺; ENaC – epithelial sodium channel; R_Na+– active sodium resistance; RS – passive or shunt resistance; G_Na– active sodium conductance; GS – passive or shunt conductance; Gmax – total conductance; EC₅₀– half-maximal excitatory concentration; IC₅₀– half maximal inhibitory concentration; NA – noradrenaline.     

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex.

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria by means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified iron-sulfur protein, +75 (+/- 5) mV, and in prepared mitochondrial outer membrane, +62 (+/- 6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.     

β-adrenergic receptors and enzymes in rat myocardial membranes: implications of fractionation procedures andβ-adrenoceptor antagonists

1. We performed an enzymatic characterization of two different fractionation procedures of ventricles from rat hearts. The enzymatic assays covered succinic dehydrogenase as a marker for inner mitochondrial membranes, monoamine oxidase as a marker for outer mitochondrial membranes, NADPH-cytochrome c reductase and RNA as endoplasmatic reticular markers, acid phosphatase as a lysosomal marker, and lactic dehydrogenase as a marker for the "soluble" compartment; DNA was estimated for nuclear contamination. 2. The plasma membrane markers 5'-nucleotidase, Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and adenylate cyclase were determined. 3. The roughly prepared membrane fractions showed increased yields of the membrane markers; the number of beta receptors, determined with (-)-[3H] dihydroalprenolol and DL-propranolol, amounted to 68 +/- 6 fmol/mg protein (KD = 3390 +/- 450 pmol, Hill coefficient = 1.5). 4. The membrane fraction prepared with a linear sucrose gradient showed an increased inner mitochondrial membrane marker; presumably the outer mitochondrial membrane was stripped off. The beta-receptor number was 39 +/- 3 fmol/mg protein (KD = 6250 +/- 300 pmol; Hill coefficient = 1.2).