Intracellular ionic compartmentation, electrical membrane properties, and cell membrane permeability before and during first cleavage in the Ambystoma egg.

The intracellular ionic distribution in uncleaved and cleaving Ambystoma eggs was investigated by analysing the influx of ³H₂O, by determining the total content of Na⁺, K⁺ and Cl^? in extracts of eggs at different stages by both flame spectrophotometry and ion-selective microelectrodes, and by the continuous measurement of the Na⁺, K⁺ and Cl^? activities (a_Naⁱ, a_Kⁱ and a_Clⁱ) using intracellular ion-selective microelectrodes. The electrical membrane potential (E_m) and membrane resistance (R_m) were measured continuously in uncleaved and normally cleaving eggs as well as in eggs cleaving after removal of the vitelline membrane. The latter eggs expose their newly formed cleavage membrane to the external medium. Ionic permeability of the cell membrane before and during cleavage was analysed by a statistical comparison of the experimentally determined relationship between E_m and the ionic gradients across the cell membrane with those predicted theoretically from a constant field equation in dependence on the relative permeability, through insertion of the measured intracellular ion activities.³H₂O influx revealed the existence of a single intracellular water compartment (3.06 μl/egg) and a low water permeability (5.35 × 10^?5 cm sec^?1). Na⁺, K⁺ and Cl^? concentrations were constant at 54.1, 72.1 and 73.1 mM respectively, while a_Naⁱ, a_Kⁱ and a_Clⁱ were constant at 5.8, 51.8 and 59.7 mM respectively. It was concluded that all Cl^? ions are in solution, while 12.5% of all K⁺ and 86% of all Na⁺ is bound. The uncleaved egg showed a positive E_m of ca 40 mV and a specific membrane resistance of 39 kOhm cm². E_m could be described by a constant field equation with a permeability ratio $\text{P}{}_{\mathrm{<mtext>K</mtext>}}\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0.073}$. Shortly after the onset of first cleavage, E_m rapidly decreased concomitant with a rise in R_m (68.5 kOhm cm²). This was interpreted as a drop in Na⁺ permeability. During the cleavage process E_m progressively hyperpolarized and R_m decreased due to the insertion of a small fraction (3.3%) of the newly formed intercellular membrane into the cleavage furrow. This new membrane had a low specific resistance (0.69 kOhm cm²). Both in normally cleaving eggs and in eggs cleaving in the absence of the vitelline membrane E_m behaved according to the constant field equation, $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ being 0.69 and 0.39, respectively. The differences with other amphibian eggs were discussed.     

Fluctuation analysis of Na+ channels modified by batrachotoxin in myelinated nerve

(1) Single myelinated nerve fibres of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na⁺ currents and Na⁺-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na⁺-current fluctuations was fitted by the sum of a $\text{1}\text{f}$ component and a Lorentzian function. The time constant $\text{τ}{}_{\mathrm{<mtext>c</mtext>}}\text{= 1/(2π?}{}_{\mathrm{<mtext>c</mtext>}}\text{)}$ obtained from the corner frequency ?_c of the Lorentzian function approximately agreed with the activation time constant τ_m of the macroscopic currents. (3) The conductance γ of a single Na⁺ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na⁺ current. At the resting potential V = O we obtained γ = 1.6 pS, higher γ-values of 3.2 and 3.45 pS were found at V = ?8 and ?16 mV, respectively. (4) The conductance of a modified Na⁺ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na⁺ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an ‘all-or-none’ manner on Na⁺ channels and creates a distinct population of modified channels.     

Energetics of coupled Na+ and Cl− entry into epithelial cells of bullfrog small intestine

Na⁺, K⁺ and Cl^? concentrations ($\text{c}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$) and activities ($\text{a}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$), and mucosal membrane potentials ($\text{E}{}_{\mathrm{<mtext>m</mtext>}}$) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na⁺ and Cl^? and 5.4 mM K⁺. Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry and Cl^? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO₃. ¹⁴C-labelled inulin was used to determine extracellular volume. $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ was measured with conventional open tip microelectrodes, $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with solid-state Cl^?-selective silver microelectrodes and $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with Na⁺- and K⁺-selective liquid ion-exchanger microelectrodes. The average $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ recorded was ?34 mV. $\text{c}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{c}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{c}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 51, 105 and 52 mM. The corresponding values for $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na⁺ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K⁺ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl^?. $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na⁺ and Cl^? is implicated in intracellular Cl^? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na⁺ and Cl^? electrochemical potential differences (Δμ&#x0304;Na and Δμ&#x0304;Cl). Δμ&#x0304;Na (?7000 J · mol^?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ&#x0304;Cl (1000–2000 J · mol^?1).     

Further studies on the effect of aldosterone on electrical resistance of toad bladder

The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na⁺ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na⁺ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na⁺ uptake, produced changes in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ that were linearly correlated with its effects on tissue conductance. On the basis of the $\text{conductance}\text{-I}{}_{\mathrm{<mtext>sc</mtext>}}$ relationship with amiloride, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na⁺ currents and Na⁺-current fluctuations were measured in myelinated frog nerve fibres at 15°C during 7.7 ms depolarizations to $\text{V = 40, 60}\text{and}\text{80}\text{mV}$. (2) The conductance γ of a single Na⁺ channel and the number $\text{N}{}_0$ of channels per node were calculated from ensemble average values of the mean Na⁺ current and the variance of Na⁺-current fluctuations. (3) For a hyperpolarizing holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ the mean values of the channel conductance and number were $\text{γ = 9.8}\text{pS}$ and $\text{N}{}_0\text{= 74 000}$. (4) After changing the holding potential to the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) the conductance γ increased by a factor of 1.37 whereas the number $\text{N}{}_0$ decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ increased γ by a factor of 1.55 and reduced $\text{N}{}_0$ by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na⁺ channels in the nodal membrane.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions I. Influence of sodium ions in bath medium on the uptake and run out of fluorescein and uranin

With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na⁺ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na⁺ concentration permitted to define values of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}$ for uranin and fluorescein transport in the medium with different Na⁺ content. It was shown that a decrease of Na⁺ concentration in the medium increases $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and lowers the $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio with uncharged $\text{V}$. By varying the Na⁺ concentration in the medium with a constant concentration of the marker anion the and values for fluorescein and uranin transport were determined. A value for fluorescein in twice as much that for uranin. The $\text{1/K}{}_{\mathrm{<mtext>m</mtext>}}$ value for uranin transport is a linear function of Na⁺ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na⁺ from the medium participate in active transfer of one fluorescein anion whereas only one Na⁺ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na⁺ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na⁺-dependent, and Na⁺ from the medium is likely to participate directly in formation of a transport complex. When Na⁺ is absent in the medium a carrier fulfils a facilitated diffusion only.     

Retention of fully differentiated electrophysiological properties of chick embryonic heart cells in culture.

Many cultured cells in spherical reaggregates (100–1000 μm diameter), prepared using cells isolated from embryonic chick ventricles (16 days in ovo), retain great sensitivity to tetrodotoxin (TTX; 0.1 μg/ml), rapidly rising action potentials (up to 200 V/sec), high resting potentials (up to ?90 mV), and they lack automaticity. The Na⁺ channels generating the action potential upstroke inactivate totally at about ?50 mV. The chronaxie (hence, excitability), the ratio of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$, and the intracellular K⁺ concentration are about the same as in adult cells. Following blockade of the Na⁺ channels with TTX, norepinephrine produces slowly rising overshooting responses, indicating the presence of functional beta-adrenergic receptors. Thus, trypsin-dispersed myocardial cells can be made to retain highly differentiated membrane properties in vitro.     

The effect of n-alkanols on the stationary current voltage behavior and action potential of myelinated nerve

Stationary current voltage characteristics and the action potential of single myelinated nerve fibres were measured to examine the effect of $\text{n-}\text{alkanols}$ (methanol to octanol) on the electrophysiological function of the axon membrane. K⁺-depolarized membranes show alkanol-dependent shifts of $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, the membrane transition voltage, whereas in veratridine-depolarized membranes such $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}\text{-}\text{shifts}$ are not observed. In the latter case, $\text{n-}\text{alkanols}$ reduce both the stationary Na⁺ current and the conductivity step between the high- and low-ohmic conductivity state of the membrane. Action potential amplitude, however, is less affected by the alkanols as is the stationary Na⁺ current. The results are compared with the alkanol-dependent changes of the thermotropic phase transition in phospholipid bilayers.     

Effect of calcium ionophore A23187 on electrogenic acid-base transport in turtle bladder Inhibition of acidification and stimulation of alkalinization

Turtle bladders bathed on both surfaces with identical HCO^?₃/CO₂-rich, Cl^?-free Na⁺ media and treated with ouabain and amiloride exhibit a transepithelial potential serosa electronegative to mucosa and a short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) which is a measure of the net luminal acidification rate. Addition of calcium ionophore A23187 (10 μM) to the mucosal side of the epithelium rapidly reverses the direction of the potential difference and $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ and decreases tissue resistance. The resulting positive $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ resembles that previously observed in response to isobutylmethylxanthine (IBMX) and cAMP analogs. Reversal of the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is enhanced in bladders from severely alkalotic turtles. In contrast, in severely acidotic turtles, ionophore A23187 decreases, but does not reverse, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$. The data suggest that, like IBMX and cAMP analogs, the Ca ionophore stimulates an electrogenic alkalinization mechanism, but, unlike the former agents inhibits the concurrent acidification process as well.     

Effect of N-ethylmaleimide on leucine transport in the chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na⁺-independent leucine transport system is resolved into two components by their different affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 44 μM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with $\text{N-}\text{ethylmaleimide}$ (1 mM) specifically stimulates the high-affinity component of the Na⁺-independent system by greatly increasing its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value, whereas the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of the low-affinity component is markedly lowered. The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na⁺-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na⁺-independent leucine uptake itself. We conclude that the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Amiloride stimulation of sodium transport in the presence of calcium and a divalent cation chelator

Amiloride in nM to μM concentrations stimulates the short circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) of the toad urinary bladder by as much as 120% when applied in conjunction with apical Ca²⁺ and a divalent cation chelator. A significant decrease in transepithelial resistance ($\text{R}{}_{\mathrm{<mtext>t</mtext>}}$) is observed simultaneously. This response is spontaneously reversible and its amplitude is dependent upon apical sodium concentrations. The stimulated $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ persisted when acetazolamide (1 mM) was introduced, HPO₄^2? substituted for HCO₃^? or SO₄^2? replaced Cl^?. Consequently, the increase in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is not due to the change of Cl^?, H⁺ or HCO₃^? flux. This behavior in a ‘tight’ epithelium may be related to the mechanism controlling apical sodium permeability.     

Phlorizin as a probe of the small-intestinal Na+,d-glucose cotransporter. A model

(1)‘Uptake’ of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of d-glucose, by the simultaneous presence of Na_out⁺ and $\text{Δψ?0}$. However, phlorizin contrary to d-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na⁺,d-glucose cotransporter. (2) The stoicheiometry of Na⁺/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Na_out⁺-dependence of phlorizin binding. (3) The preferred order of binding at $\text{Δψ?0}$ is Na⁺ first, phlorizin second (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to Δψ. These observations while agreeing with the effect of $\text{Δψ?0}$ on the $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na⁺,Δψ-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na⁺,d-glucose cotransport. (6) The $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$, $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ constants of phlorizin interaction with the Na⁺,d-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.