Positive Potential Difference Research Articles

Electrogenic Transport of Protons Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish

Mg:ATP-dependent H(+) pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Delta psi). DeltapH was measured as the decrease of absorbance of Acridine orange and Delta psi as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Delta pH and Delta psi generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Delta pH generation is not inhibited by NO(3) (-). These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H(+)-ATPases. Both intravesicular acidification and Delta psi generation are influenced by anions: Delta pH increases and Delta psi decreases following the sequence SO(4) (2-), Cl(-), Br(-), NO(3) (-). ATP-dependent H(+) pumping strictly requires Mg(2+). It is very specific for ATP (apparent K(m) 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Delta pH generation is inhibited by CuSO(4) and diethylstilbestrol as well as vanadate. Delta pH generation is specificially stimulated by K(+) (+ 80%) and to a lesser extent by Na(+) and choline (+28% and +14%, respectively). The characteristics of H(+) pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.

Effect of amphotericin B and gestational age on sodium transport across the rat visceral yolk sac placenta in vitro.

The transport properties of the rat visceral yolk sac placenta from Days 14.5 to 18.5 of gestation were studied in vitro. All tissues had a positive potential difference, fetal side relative to maternal side, and showed net Na transport towards the fetus. Basal short-circuit current and net Na flux increased rapidly with gestational age over the period studied. Amphotericin B applied to the maternal surface of the yolk sac stimulated current and net Na flux, indicating that the apical membrane Na permeability limited transport and revealing a reserve capacity for transport. Contrary to their basal values, current and Na flux following treatment with amphotericin were independent of gestational age.

Monocular and binocular evoked average potential field topography: upper and lower hemiretinal stimuli.

Scalp potentials evoked by monocular and binocular 2/s checkerboard reversals with checks of 56 or 14 min were recorded from four midline electrodes between inion and 7.5 cm above inion vs a common anterior reference in 31 normals. The electrode location and latency of the evoked maximal occipitally positive potential difference between any 2 of the 5 electrodes, between 80 and 140 ms latency was determined in each run. Mean maximum locations over subjects for upper hemiretinal, binocular stimuli were more anterior than for monocular stimuli; for lower hemiretinal stimuli, inverted location differences were found. Binocular responses also had shorter latencies than monocular responses. Since area 18 is more anterior to 17 for upper, more posterior to 17 for lower hemiretinal projection to cortex, the results suggest that neurons which respond only if both eyes are simultaneously stimulated are more frequent in higher than lower order visual areas.

Cellular Mechanism of the furosemide sensitive transport system in the kidney.

Experiments were performed in the distal tubule of the doubly-perfused kidney of Amphiuma to determine active and passive forces, involved in the transport processes of potassium, sodium and chloride. Ion-sensitive microelectrodes and conventional microelectrodes were applied to estimate intracellular ion activities, cell membrane potentials and net flux of potassium and chloride under control conditions and during inhibition of active transport. Sodium chloride cotransport, located in the luminal cell membrane is postulated, based on the following observations: Total omission of sodium from the tubular lumen inhibits furosemide sensitive chloride reabsorption, decreases the lumen positive transepithelial potential difference and leads to a dramatic decrease of intracellular chloride. The experiments further suggest that potassium ions are involved in the sodium chloride transport system because potassium reabsorption is inhibited by furosemide and because intracellular sodium falls significantly when potassium ions are removed from the tubular fluid. Furthermore, there is experimental evidence that the luminal potassium uptake mechanism is suppressed after potassium adaptation. Under these conditions potassium transport is found to be insensitive to furosemide. The data suggest a furosemide sensitive cotransport system for sodium, chloride and potassium, operative in the luminal cell membrane. The energy for this carrier-mediated transport process is provided by the large "downhill" gradient of sodium across the luminal cell membrane which is maintained by the sodium pump located in the peritubular cell membrane.

Ion flux changes induced by voltage clamping or by amphotericin B in the isolated urinary bladder of the trout.

1. When bathed with Ringer solutions on both sides in Ussing chambers, isolated urinary bladders of trout carry out an electrically silent transport of Na+ and Cl- ions from mucosa to serosa. In the present experiments, an electrical gradient was set up across the epithelium mounted under these conditions either by using a voltage-clamp technique or by submitting the mucosal side to the polyene antibiotic Amphotericin B. 2. With stable potential differences between -40 and +40 mV applied to the tissue, both unidirectional fluxes (Jms and Jsm) for Na+ and for Cl- varied as a function of the electrical gradient. This indicates that a sizeable fraction of these ionic exchanges is due to passive diffusion across a low-resistance pathway. 3. Amphotericin B (40 micrograms/ml.) applied to the mucosal side induced an immediate and large (up to 50 mV, serosa positive) potential difference and a sustained diminution in tissue resistance. These effects were strictly Na+-dependent and were reduced in the presence of ouabain. Therefore they are due to increased passive entry of Na+ along its electrochemical gradient across the cellular apical membrane. 4. The effect of Amphotericin B was enhanced when Cl- was replaced by an impermeant anion such as gluconate. Therefore the shunting effect of Cl- on the induced potential observed in the control conditions appears to be due to an increase in Cl- permeability across an anion pathway. Flux data indicate that this pathway is transcellular.

The influence of pH on phosphate transport into rat renal brush border membrane vesicles.

Sodium-dependent transport of inorganic phosphate into brush border membrane vesicles is strongly influenced by altering pH of the incubation medium (pHo). At constant total phosphate concentration an increase in pHo leads to an increase in the uptake of inorganic phosphate. Uptake of inorganic phosphate, however, is not affected by the intravesicular pH (pHi) or by transmembrane pH differences (pHo--pHi). If initial phosphate uptake is studied as a function of total phosphate concentration in the medium the half saturation concentration increases when pHo is raised from 6.3--6.9 but remains unaltered between pHo 6.9 and 7.8 Vmax increases about 3-fold between pHo 6.3 and 6.9 and by a factor of about 1.6 between pHo 6.9 and 7.4. The pHo- dependence of phosphate uptake is diminished by increasing sodium concentrations. Altering transmembrane electrical potential difference by potassium + valinomycin-induced diffusion potentials or by anion replacement fails to demonstrate electrogenicity of sodium-phosphate cotransport. Experiments using a potential-sensitive fluorescent dye, however, indicate a vesicle inside positive electrical potential difference when inorganic phosphate is added. The phosphate-induced alterations in the electrical potential difference are sodium-dependent and more pronounced at low pHo values. Together with earlier observations these results suggest that translocation of inorganic phosphate across the proximal tubular brush border membrane is mediated by cotransport of 2 sodium ions with one either monovalent or divalent phosphate molecule according to its availability in the tubular fluid. The pH sensitivity of this transport system is rather due to alterations in the transport system itself than to pH- dependent alterations in the ratio of monovalent to divalent phosphate.

Origins of positive potential difference of frog gastric mucosa in Cl--free solutions.

Experiments were performed on gastric mucosae of Rana pipiens with an in vitro method. Bathing both sides of the mucosae in Cl--free solutions resulted in an inverted potential difference (PD) (i.e., nutrient negative). Cimetidine in a concentration of 10(-4) or 10(-3) M in the nutrient solution gave a positive PD when the H+ secretory rate was zero or near zero and confirmed that the PD versus H+ rate was linear. the origins of the positive PD were examined by ion replacement, e.g., choline for Na+ in both solutions. When both solutions had 25 mM HCO(-)3 (i.e., no HCO(-)3 gradient), the difference in PD between choline sulfate and Na2SO4 solutions indicated a possible small active transport of Na+ from the secretory to nutrient side. Similar experiments indicated no active K+ transport. A residual PD occurred without Na+. With H+ rate zero, 100% O2, and no HCO(-)3, a base secretion of about 0.4 mueq.h-1.cm-2 was obtained in a direction to contribute to the positive PD. Active SO2(-)4 transport may occur.

Light-Activated H+Transport into the Vacuole ofValonia ventricosa

Using glass capillary microelectrodes for the measurement of potential differences (PD) and antimony microelectrodes for the measurement of pH, we investigated the light-induced changes of PD between the central vacuole and the external medium, of pH in the vacuole (pHv), as well as of pH in the external medium (pH0) of the green marine alga Valonia ventricosa. PD in the dark was about +30 to +40 mV (vacuole positive), pHv 6-3, and the resistance of the protoplast (cell wall-plasmalemma-tonoplast) 17-8 kOhm cm2. Illumination caused an increase of the positive PD (after a few oscillations) up to +80 to +100 mV, acidification of the vacuolar sap, alkalinization of the external medium, and a decrease in the resistance of the protoplast to 7-6 kOhm cm2. The kinetics of the changes of PD, pHv, and pH0 were similar to each other. It is concluded that a light-stimulated active H+ flow occurs from the external medium into the central vacuole of Valonia ventricosa as a result of the onset of photosynthetic activity.

Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle's loop.

Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.

Characterization of the light-driven sodium pump of Halobacterium halobium. Consequences of sodium efflux as the primary light-driven event.

A class of cell envelope vesicles (M vesicles) prepared from Halobacterium halobium Rl contain a light-driven sodium pump that serves to eject sodium ions and to create an electrical potential difference in direct proportion to the intensity of illumination. The M vesicles take up protons as an immediate consequence of this electrical potential difference. Predictably, the sodium efflux rate and extent are not inhibited by proton ionophores, whereas the proton uptake is enhanced. In the presence of valinomycin and K+, the electrical potential difference and the proton uptake are abolished, but the light-driven sodium extrusion is only slightly decreased. M vesicles are essentially free (>95%) from contamination by a light-driven proton pump. The participation of a reversed bacteriorhodopsin pump is unlikely since absolutely no internally positive electrical potential difference is detectable. The ejection of sodium by M vesicles is independent of protonmotive force and the rate of the Na+ efflux does not show a gating effect in response to membrane potential. In sharp contrast, another class of cell envelope vesicles (L vesicles) accomplish sodium extrusion via an electrogenic H’/Na’ antiporter that is driven by protonmotive force and activated by membrane potential (Lanyi, J. K., and MacDonald, R. E. (1976) Biochemistry, 15, 4608-4614). We conclude that at least two mechanisms exist in H. halobium to create electrical and chemical potential energy by the transduction of light energy: the bacteriorhodopsin proton pump and a light-driven sodium pump. The consequences of this second pump to the circulation of ions and to Na’/ amino acid symport are considerable and cannot be neglected.

The effect of amiloride on the transepithelial potential difference of the distal tubule of the rat kidney.

Microelectrodes with relatively large tips (3-5 mu O.D.) were used to measure the transepithelial potential difference (PD) of the distal tubule of the rat kidney in the control state and following i.v. administration of amiloride. This drug produced an increase in the magnitude of the positive PD in early distal segments (from +8.0 to +10.5 mV) and a change in polarity of the PD in late distal segments (from -18.0 to +2.5 mV). These data suggest that the potassium-conserving properties of the drug are due to its ability to induce an unfavorable electrochemical gradient opposing passive potassium secretion along the distal tubule.

Studies of the electrical potential difference in rat proximal tubule.

The electrical potential difference (PD) in the rat proximal convoluted tubule was investigated in vivo as a function of distance from the glomerulus. The PD was found to be invariably negative (up to -4.5 mV) in the earliest segments (less than 0.5 mm from the glomerulus) and rose to positive values (+2 to +4) in the later segments (1 mm beyond the glomerulus). This change in PD correlated with the bubule fluid-to-plasma (TF/P) chloride ratios, which rose from unity in the early segments to approximately 1.3 in the late. Corresponding changes in PD and chloride ratios could be elicited by single-nephron stop-flow techniques in the early segments. Luminal perfusion techniques demonstrated a direct relationship between PD and tubule fluid chloride concentration. Acetazolamide was found to significantly reduce both late proximal PD (less than +2 mV) and TF/P chloride ratios (less than 1.06). Split-drop studies demonstrated that the negative PD in the early proximal tubule was dependent on the presence of glucose and alanine and the absence of a chloride gradient, whereas in the late proximal tubule under the same conditions the PD was not significantly different from zero. In this segment of the nephron the positive PD in free flow appeared to result from the chloride diffusion potential generated by preferential HCO3 reabsorption. These results provide further demonstration of intrinsic differences in the transport properties along the length of the proximal convoluted tubule.

Lack of relationship of potential difference to fluid absorption in the proximal renal tubule

Fluid absorption by isolated perfused rabbit proximal convoluted tubules is accompanied by an electrical potential difference (PD), negative in the lumen, when the tubule is bathed by rabbit serum and perfused with an ultrafitrate of that serum. In contrast the PD is positive when the perfusate composition approximates that of fluid in the late proximal tubule in vivo, which lacks glucose, amino acids and bicarbonate. The principal purpose of the present studies was to investigate the characteristics of fluid absorption under these conditions. Proximal convoluted tubules were dissected from rabbit kidneys and perfused in vitro. When the PD was positive, the mean net fluid absorption was 81 nl mm minus 1 min minus 1. The positive PD is caused by a chloride concentration difference across the tubule epithelium (higher in the lumen than in the bath). Elimination of the chloride concentration difference by replacing the bicarbonate in the bath as well as in the perfusate with chloride caused the PD to fall to zero without a significant change in the rate of fluid absorption. Therefore, neither the positive PD nor the chloride concentration difference is significantly related to the fluid absorption. Ouabain inhibited fluid absorption under all of the above conditions, making it likely that the fluid absorption is due to active sodium transport. Although the results are consistent with the generally accepted view that active sodium transport is a major driving force for fluid absorption, the mechanism of anion (chloride) transport is uncertain owing to the lack of correlation between fluid absorption and the transepithelial PD.

The magnetohydrodynamic thrust bearing with variable film thickness

Thrust bearings with converging and diverging film thicknesses under external azimuthal magnetic fields are analysed. With constant flow rate and positive terminal potential difference load capacity diminishes to a minimum then increases with increase of Hartmann number. With negative terminal potential difference load capacity increases with increase of Hartmann number. Under short circuit conditions the load capacity increases with increase of Hartmann number. Load capacity is unaffected by magnetic field under an open circuit condition. For constant flow rate and Hartmann number load capacity increases with increase of current; current increases as the terminal potential difference diminishes.

Factors governing the transepithelial potential difference across the proximal tubule of the rat kidney.

Previous measurements of the transepithelial potential difference (PD) of the proximal tubule have yielded widely conflicting values (range -20 to +3 mV). In a recent study, Kokko has demonstrated that the PD of the in vitro perfused isolated proximal tubule of the rabbit varies in a predictable way from -6 to +3 mV, depending on the concentration of chloride, bicarbonate, glucose, and amino acids in the perfusing solution. The present micropuncture study examines the effect of tubular fluid composition on the PD profile along the proximal tubule of the in vivo rat kidney. Low resistance measuring electrodes with large tips (3-5 microns OD) filled with 3 M KCl, were used to provide stable PD recordings. Experiments were performed to validate the use of these electrodes. Transepithelial PD measurements were made in immediate postglomerular segments identified by injection of dye into Bowman's space of accessible surface glomeruli and in randomly selected more distal segments of the proximal tubule. In the control state, the first loop was found to have a small but consistently negative PD which could be obliterated by an infusion of phloridzin. In contrast, the PD in later segments was consistently positive. Infusion of acetazolamide abolished the positive PD in the later segments. Acetazolamide and glucose infusion resulted in a negative PD which was abolished by the additional infusion of phloridzin. These data provide evidence that glucose reabsorption is electrogenic and can account for the small negative PD normally present in the early proximal tubule. The positive PD in later segments appears to be a passive chloride diffusion potential. This positive potential is discussed as an important electrochemical driving force for significant passive reabsorption of sodium in the proximal tubule.

Proximal tubule potential difference. Dependence on glucose on glucose, HCO 3 , and amino acids.

The effect of various intraluminal substrates on the magnitude of the transepithelial potential difference (PD) across the proximal convoluted tubule (PCT) of the mammalian kidney was investigated in two ways. First, the transepithelial PD was measured before and after the removal of glucose, bicarbonate, and alanine from the lumen. Second, the effects of specific transport inhibitors-ouabain, phloridzin, and acetazolamide-was ascertained when placed either on luminal or blood side. Isolated segments of rabbit PCT were perfused in vitro. Tubules perfused with isosmolar ultrafiltrate (UF) at rates > 10 nl/min had a mean PD of - 5.8+/-0.2 mV (lumen negative). Normal UF was simulated by an artificial perfusion solution. Using the latter, observed PD was - 5.4+/-0.2 mV. A significant reversible decrease in PD was noted when the following constituents were removed singly: glucose (from - 5.7+/-0.4 to - 3.5+/-0.4 mV); alanine (from - 5.8+/-0.4 to - 4.7+/-0.3 mV); and bicarbonate (from - 5.3+/-0.3 to - 3.3+/-0.5 mV). The combined removal of alanine and glucose (replaced with mannitol) reduced the transepithelial PD to - 0.5+/-0.1 mV with removal of glucose and alanine (replaced with mannitol) and decrease of NaHCO(3) to 5.6 meq/liter (replaced with NaCl), as normally occurs in early part of in vivo PCT, resulted in reversible change of PD from - 5.1+/-0.2 to + 3.2+/-0.2 mV. Ouabain (10(-5) M) reversibly decreased the negative control PD from blood side, but had no effect from luminal side. Phloridzin (10(-5) M) reversibly decreased PD from - 6.4+/-0.3 to - 3.7+/-0.4 mV when placed on luminal side but had minimal effect from blood side, - 6.3+/-0.4 to - 5.8+/-0.4 mV. Acetazolamide (Diamox) was without effect from either side. Reversal of bulk flow of water by addition of 31 mosmol/liter raffinose to perfusion ultrafiltrate did not significantly decrease the PD. It is concluded that specific pumps for transport of glucose, amino acids, and bicarbonate exist on the luminal surface. All three constituents are necessary for expression of maximum PD. Removal of these substrates by transport changes PD from - 5.1 mV to + 3.2 mV (lumen positive). This 3.2 mV positive PD is secondary to a chloride diffusion potential and is not effected by ouabain from the blood side.

Effects of nonpolar environments on the redox potentials of heme complexes.

A method has been described for measurement of the oxidation-reduction potentials of redox couples in nonaqueous solutions. This method has been applied to a study of the effect of a nonpolar environment on the redox potential of a heme complex. A positive potential difference of some 300 mV is observed for the measured redox potentials by comparison with reported values for the corresponding heme complex in aqueous solution. The proposal is made that the redox potentials of many high-potential cytochromes may be accounted for by a local heme environment of low-dielectric constant, characteristic of nonpolar amino-acid side chains, and that this factor may play a dominant role in the determination of the oxidation-reduction properties of these proteins.

Protoplasmic Potentials in Halicystis. VI. Role of Ammonia in Potential Reversal by Perfusion.

Earlier papers1 have described the reversal of the normally positive potential difference across the protoplasm of impaled cells of Halicystis osterhoutii (Bermuda) both by exposure to ammonia (or other weak bases) and by perfusion of the vacuole with more alkaline sap or sea water. The author was then inclined to explain the former effect by the latter, since penetration of ammonia raised the vacuolar pH. One difficulty of this interpretation was that the Californian species, H. ovalis, did not show such reversal on perfusion with sea water at pH 8.0 or higher, although reversing well on exposure to ammonia. This difference between the species now seems reconciled by referring the effects in both cases to the penetration of ammonia into the protoplasm. This is shown in H. ovalis by introducing into the perfusing fluid a trace of NH4Cl; at pH 5.0, the normal acidity of the sap, no effect is observed, but if sea water now is perfused at pH 8.0, reversal of potential often results. Then as perfusion is cont...

THE FORM AND MAGNITUDE OF THE ELECTRICAL RESPONSE OF THE EYE TO STIMULATION BY LIGHT AT VARIOUS INTENSITIES

1. The photo‐electric reaction of an isolated eye is specially adapted to the study of the effect of stimuli of very different intensities. In our experiments light stimuli have been used whose energy varies from 3·05 × 10−12 to 3·95 × 10−2 g. cal. per sec.Short illumination with the strongest light has not been found to damage the eye, while the weakest light was capable of producing a fairly considerable photo‐electric reaction.2. The form under which the photo‐electric reaction manifests itself under different conditions gives ground for the supposition that there occur in the eye three separate processes, and each of these may be dependent upon a separate substance. We speak of three substances for the sake of convenience.3. The first substance reacts more rapidly than the other two. On lighting it develops a negative, on darkening a positive potential difference. Its action comes strongly into prominence in a light eye and appears almost unmixed on sudden darkening of short duration (a flash of darkness).The second substance reacts less rapidly than the first, and in an opposite sense. On lighting it develops a positive, on darkening a negative potential difference. Its action appears almost unmixed in a dark eye which is illuminated for a short time with weak light.The third substance reacts in the same sense as the second, but much more slowly. Its action fails in a completely light eye, and also in a dark eye which is illuminated very weakly for a short time.4. The latent period of the photo‐electric reaction is in a high degree dependent upon the intensity of the stimulus. With strong stimuli it is of the order of 0·01 sec., while with very weak stimuli it may be lengthened to more than 2 sec. These values are in agreement with the latent periods of light perception in the human eye.5. For each of the three substances the rule holds good that with moderate and strong light the energy of the stimulus increases much more quickly than the energy of the reaction.6. Although the eye is far more sensitive than the most sensitive artificial bolometer, the energy of the reaction remains below that of the stimulus even in the most favourable circumstances. The photo‐electric reaction of the eye is in this respect comparable with the action current of a muscle or nerve.