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Extracellular fluid volume is determined by sodium and its accompanying anions. There are control mechanisms which regulate sodium balance in the body. These include high and low pressure haroreceptors, intrarenal baroreceptors, renal autoregulation, tubuloglomerular feedback, aldosterone, and numerous other physical and hormonal factors. Sodium transport by the nephron involves active and passive processes which occur in several different nephron segments. Mechanisms of cotransport, Na+-H- exchange, antiporters and ion-specific channels are all utilized by the nephron to maintain sodium balance. These regulatory factors and transport mechanisms for sodium in the kidney will he discussed in detail.

The present studies were designed to assess the ways in which antidiuretic hormone (ADH) alters water and solute permeation across isolated, rabbit cortical collecting tubules. In earlier work, it was observed: that ADH produced a tenfold increment inPf(cm per sec), the osmotic water permeability coefficient, and a fourfold increment inPDw(cm per sec), the diffusional water permeability coefficient; that small hydrophilic solutes such as urea, thiourea and acetamide (each having oil/water partition coefficients≦0.0008) had vanishingly low permeation coefficients and unity reflection coefficients, even in the presence of ADH; that lumen to bath osmosis involved a transcellular route; and, that the disparity betweenPfandPDw, either with or without ADH, could be rationalized in terms of cellular diffusion constraints, i.e., that water transport across luminal membranes was diffusional.The present studies were designed to assess the ways in which antidiuretic hormone (ADH) alters water and solute permeation across isolated, rabbit cortical collecting tubules. In earlier work, it was observed: that ADH produced a tenfold increment inPf(cm per sec), the osmotic water permeability coefficient, and a fourfold increment inPDw(cm per sec), the diffusional water permeability coefficient; that small hydrophilic solutes such as urea, thiourea and acetamide (each having oil/water partition coefficients≦0.0008) had vanishingly low permeation coefficients and unity reflection coefficients, even in the presence of ADH; that lumen to bath osmosis involved a transcellular route; and, that the disparity betweenPfandPDw, either with or without ADH, could be rationalized in terms of cellular diffusion constraints, i.e., that water transport across luminal membranes was diffusional.

A unique property of basolateral membrane Cl- channels from the mTAL is that the Cl- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P0). The K1/2 for maximal activation of P0 by Cl- facing intracellular domains of these channels is 10 mm Cl-. The present experiments evaluated the nature of these Cl--interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl- channel faces, could augment P0 with a K1/2 in the range of 10 mm isethionate without affecting conductance (gCl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P0 usually seen when the Cl- concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl- channel activity was increased by first raising the Cl- concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl- channel faces had no effect on P0. We conclude that the intracellular aspects of these Cl- channels contain Cl--interactive loci (termed [Cl]i) which are accessible to impermeant anions in intracellular fluids and which contain arginineand lysine-rich domains which can be inactivated, at low ambient Cl- or isethionate concentrations, by interactions with PGO or TNBS.

Summary We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl- concentrations in modulating the activity of Cl- channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, thecis andtrans solutions face the extracellular and intracellular aspects of these Cl- channels, respectively. Raising thetrans Cl- concentration from 2 to 50mm increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (?G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990.J. Membrane Biol.118:269–278). With 2mmtrans KCl,trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition tocis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50mm)trans Cl- concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl- channel activity by phosphorylation at or near the Cl--sensitive site on the intracellular face of these channels.

Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (Gcmin) of the transcellular conductance, estimated from Ba++ blockade of apical membrane K+ channels, indicated thatGcmin was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K+ was the major conductive species; and ADH increased the magnitude of a Ba++-sensitive K+ conductance under conditions where net Cl- absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl- conductance; this ADH-dependent increase in basal Cl- conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl- absorption. Cl- removal from luminal solutions had no detectable effect onGe, and net Cl- absorption was reduced at luminal K+ concentrations less than 5mm; thus apical Cl- entry may have been a Na+,K+,2Cl- cotransport process having a negligible conductance. The net rate of K+ secretion was approximately 10% of the net rate of Cl- absorption, while the chemical rate of net Cl- absorption was virtually equal to the equivalent short-circuit current. Thus net Cl- absorption was rheogenic; and approximately half of net Na+ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K+ was the principal conductive species in apical plasma membranes, support the view that the majority of K+ efflux from cell to lumen through the Ba++-sensitive apical K+ conductance pathway was recycled into cells by Na+,K+,2Cl- cotransport.

Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,Ve, which attends net Cl- absorption,JClnet, and to assess how ADH might augment bothJClmet andVe in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba++ for 5mm K+ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,Rc/Rbl, and increasing luminal K+ from 5 to 50mm in the presence of luminal 10-4m furosemide resulted in a 53-mV depolarization of apical membrane voltage,Va. Thus K+ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10-4m luminal furosemide reducedVe andJClnet to near zero values and hyperpolarized bothVa andVbl, the voltage across basolateral membranes; however, the depolarization ofVbl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofVa, Thus ADH-dependent increases inVb were referable to greater depolarizations ofVbl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofVa was referable to a decrease in the K+ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofVbl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl- current. Thus an increase in intracellular Cl- activity may have contributed to the depolarization ofVbl during net Cl- absorption, and the intracellular Cl- activity was likely greater with ADH than without hormone. Since ADH increases apical K+ conductance and since the chemical driving force for electroneutral Na+,K+,2Cl- cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba++-sensitive conductance K+ channels and electroneutral Na+,K+,2Cl- cotransport units in apical plasma membranes.

Chloride channels are found throughout the nephron. Many chloride channels expressed in the kidney have now been cloned. The CIC family of voltage-dependent chloride channels accounts for most of the known renal chloride channels. Unfortunately, relatively little is known concerning their functional properties. The role that chloride channels may play in renal disease has only recently been addressed.

Summary We evaluated the effects of vawrying aqueous Cl- concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl- channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl- concentrations and/or PGO on the relation between holding voltageVH-mV) and open-time probability (Po).

Cl- channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl- activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl- in the absence of PKA. The PKA requirement for activation of Cl- channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl- concentrations on the properties of Cl- channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl- channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl- concentration from 2 to 50 mm increased both channel activity (Po) and channel conductance (gCl, pS). Likewise, trans PKA increased mTALH Cl- channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect gCl. Moreover, when mTALH Cl- channels were inactivated by reducing cis Cl- concentrations to 50 mm, cis PKA addition did not affect Po. These results are consistent with the view that these Cl- channels originated from basolateral membranes of the mTALH.

Summary This paper provides the results of studies which characterized conductive36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive36Cl- uptake was studied under two different experimental conditions. In the first,36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive36Cl- uptake. By these two methods, voltage-sensitive36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total36Cl- flux. Separate paired studies demonstrated that the conductive36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 µm. The voltagedependent36Cl- uptake had an activation energy of 6.4 kcal/mole. This36Cl- conductance had an anion selectivity sequence of I->Cl-?NO3-»gluconate.

Summary The present studies examined some of the properties of Cl- channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aPCl/PK ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl- conductance of isolated medullary thick ascending limb segments. The Cl- channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50mm.

Summary ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide-and chloride-sensitive22Na+ uptake and barium-sensitive, voltage-dependent86Rb+-influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1mm ATP, and 50mm KCl, the barium-sensitive86Rb+ influx increased from 361±138 to 528±120pm/mg prot · 30 sec (P<0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 µg/ml) was also present in the vesicle solutions. The stimulation of86Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.

Cl−channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl−concentrations. rbClC-Ka, a ClC Cl−channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl−transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl−uptake and efflux were measured in suspensions of mouse MTAL segments. Cl−uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3′,5-cyclic monophosphate (DBcAMP). Cl−efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 ± 20 to 196 ± 36 nmol ⋅ mg protein−1⋅ 45 s−1(P= 0.003). Cl−efflux was inhibited by the Cl−channel blocker diphenylamine-2-carboxylate (154 ± 26 vs. 70 ± 21 nmol ⋅ mg protein−1⋅ 45 s−1,P= 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl−channels in lipid bilayers, reduced Cl−efflux from intact MTAL segments (154 ± 28 vs. 53 ± 14 nmol ⋅ mg protein−1⋅ 45 s−1,P= 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl−channel that mediates vasopressin-stimulated net Cl−transport in the MTAL segment.

The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s–1) Pfl→b, computed from volume flows from hypotonic lumen to isotonic bath, was 20 ± 4 x 10–4 (SEM); the value of Pfb→l in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 ± 15 x 10–4 cm s–1. We also measured apparent urea permeability coefficients (cm s–1) from 14C-urea fluxes from lumen to bath (PDDureal→b) and from bath to lumen (PDDureab→l). For hypotonic luminal solutions and isotonic bathing solutions, PDDureal→b was 0.045 ± 0.004 x 10–4 and was unaffected by ADH. The ADH-independent values of PDDureal→b and Pureab→l were, respectively, 0.216 ± 0.022 x 10–4 cm s–1 and 0.033 ± 0.002 x 10–4 cm s–1 for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, PDDureal→b did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of Pfb→l in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of Pfl→b in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in luminal plasma membranes.

This paper reports a theoretical analysis of osmotic transients and an experimental evaluation both of rapid time resolution of lumen to bath osmosis and of bidirectional steady-state osmosis in isolated rabbit cortical collecting tubules exposed to antidiuretic hormone (ADH). For the case of a membrane in series with unstirred layers, there may be considerable differences between initial and steady-state osmotic flows (i.e., the osmotic transient phenomenon), because the solute concentrations at the interfaces between membrane and unstirred layers may vary with time. A numerical solution of the equation of continuity provided a means for computing these time-dependent values, and, accordingly, the variation of osmotic flow with time for a given set of parameters including: Pf (cm s–1), the osmotic water permeability coefficient, the bulk phase solute concentrations, the unstirred layer thickness on either side of the membrane, and the fractional areas available for volume flow in the unstirred layers. The analyses provide a quantitative frame of reference for evaluating osmotic transients observed in epithelia in series with asymmetrical unstirred layers and indicate that, for such epithelia, Pf determinations from steady-state osmotic flows may result in gross underestimates of osmotic water permeability. In earlier studies, we suggested that the discrepancy between the ADH-dependent values of Pf and PDDw (cm s–1, diffusional water permeability coefficient) was the consequence of cellular constraints to diffusion. In the present experiments, no transients were detectable 20–30 s after initiating ADH-dependent lumen to bath osmosis; and steady-state ADH-dependent osmotic flows from bath to lumen and lumen to bath were linear and symmetrical. An evaluation of these data in terms of the analytical model indicates: First, cellular constraints to diffusion in cortical collecting tubules could be rationalized in terms of a 25-fold reduction in the area of the cell layer available for water transport, possibly due in part to transcellular shunting of osmotic flow; and second, such cellular constraints resulted in relatively small, approximately 15%, underestimates of Pf.

This paper describes experiments designed to evaluate Na+ and Cl- transport in isolated proximal straight tubules from rabbit kidneys. When the perfusing solution was Krebs-Ringer buffer with 25 mM HCO3- (KRB) and the bath contained KRB plus 6% albumin, net volume reabsorption (Jv, nl min-1 mm-1 was -0.46 ± 0.03 (SEM); Ve, the spontaneous transepithelial potential difference, was -1.13 ± 0.05 mV, lumen negative. Both Jv, and Ve, were reduced to zero at 21°C or with 10-4 M ouabain, but Jv, was not HCO3- dependent. Net Na+ reabsorption, measured as the difference between 22Na+ fluxes, lumen to bath and bath to lumen, accounted quantitatively for volume reabsorption, assuming the latter to be an isotonic process, and was in agreement with the difference between lumen to bath 22Na+ fluxes during volume reabsorption and at zero volume flow. The observed flux ratio for Na+ was 1.46, and that predicted for a passive process was 0.99; thus, Na+ reabsorption was rationalized in terms of an active transport process. The Cl- concentration of tubular fluid rose from 113.6 to 132.3 mM during volume reabsorption. Since Ve, rose to +0.82 mV when tubules were perfused with 138.6 mM Cl- solutions, Ve may become positive when tubular fluid Cl- concentrations rise during volume reabsorption. The permeability coefficients PNa and PCl computed from tracer fluxes were, respectively, 0.23 x 10-4 and 0.73 x 10-4 cm s-1. A PNa/PCl ratio of 0.3 described NaCl dilution potentials at zero volume flow. The magnitudes of the potentials were the same for a given NaCl gradient in either direction and PNa/PCl was constant in the range 32–139 mM NaCl. We infer that the route of passive ion permeation was through symmetrical extracellular interfaces, presumably tight junctions, characterized by neutral polar sites in which electroneutrality is maintained by mobile counterions.

The transport of water across biological membranes is a process of considerable theoretical and practical importance. Classical interpretations of the mechanism of this transport have centered around the hypothesis of aqueous membrane pores. However, more recent evidence indicates that the possible presence of unstirred layers adjacent to membranes must be taken into account. In one sense, the unstirred layers may be considered a vexing technical problem. In these terms, they represent a problem of potential clinical relevance, eg, in the design of more efficient hemodialysis machines. Alternatively, it is not unreasonable to speculate that unstirred layers may result, at least in part, in alterations in the properties of water which result, for example, from hydrophobic interactions between membrane interfaces and vicinal lamellae of water. Considered in this context, unstirred layer phenomenology may be of critical significance, not only to an appreciation of water flows in membranes, but also for the elucidation of the molecular effects of hormones such as antidiuretic hormone (ADH), and, finally, in the rational design of drugs which modify membrane transport processes in a clinically useful way.

Optically black membranes prepared from sheep red cell lipids have a high electrical resistance (1–3 x 108 ohm-cm2). The ionic transference numbers (Ti) for cations (Na+ or K+) are equal to each other but at least four to five times greater than for Cl-. The cyclic depsipeptide valinomycin produces a striking decrease in the membrane resistance when K+, but not when Na+ is in the solutions bathing the membrane. The ratio TNa/TK, estimated from membrane voltages in the presence of ionic concentration gradients, approaches zero. The order of membrane monovalent cation selectivity, in the presence of valinomycin, is H+ > Rb+ > K+ > Cs+ > Na+. Addition of the antibiotic to one side of a membrane which separates identical solutions of NaCl produces a substantial (up to 80 mV) membrane voltage (side opposite valinomycin negative). These data are consistent with the hypothesis that valinomycin can interact with appropriately sized cations (hydrated diameter ??? 6 A) to increase their membrane permeability, perhaps by forming hydrogen bonds between the solvation shell of the cations and carbonyl oxygens in the valinomycin molecule which are directed toward the aperture of the ring.

A cyclic depsipeptide antibiotic, valinomycin, was found to produce increased selective permeability of the plasma membranes of HK and LK sheep red blood cells to potassium but not to sodium ions. The compound had relatively little effect on the active extrusion of sodium from HK sheep red blood cells or on the Na + K-stimulated ATPase activity of membranes derived from these cells. It is proposed that the selective cation permeability produced by this compound depends primarily on steric factors, particularly the relationship between the diameter of the ring and the effective diameter of the ion. The significance of these results for the problem of the mechanism of ionic selectivity in natural membranes is discussed.

This paper reports the electrical properties of thick lipid membranes in the absence and presence of valinomycin. The thick lipid membranes were formed by placing a solution of sheep red cell lipids in decane between two cellophane partitions which formed the interfaces between the membrane and the two aqueous bathing solutions. The DC electrical resistance of these structures was found to be directly proportional to the reciprocal of the concentration of lipids in the decane (CL). The limiting resistance, as (CL-1) approached zero, was 3 x 108 ohm-cm2. Resistance was also found to be linearly related to membrane thickness. The limiting resistance at zero thickness was again 1–3 x 108 ohm-cm2. These data are interpreted to indicate that the DC resistance of thick lipid membranes comprises two surface resistances (RS) at each interface with the aqueous bathing solutions, and a bulk resistance (RB) of the lipid-decane solution, arranged in series. Measurements of the effect of variations of area on resistance were consistent with this interpretation. Valinomycin reduced RS but had no effect on RB. Under certain conditions, thick lipid membranes containing valinomycin behaved like highly selective K+ electrodes.

This paper describes dissipative Cl- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10-7 M amphotericin B. PDCl (cm·s-1), the diffusional permeability coefficient for Cl-, estimated from unidirectional 36Cl- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω-1·cm-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, PDCl was 1.36 x 10-4 cm·s-1 when Gm was 0.02 Ω-1·cm-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of PDCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ∼90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the PDNa/PDCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness.

Lipids were obtained from high potassium (HK) and low potassium (LK) sheep red cells by sequential extraction of the erythrocytes with isopropanol-chloroform, chloroform-methanol-0.1 M KCl, and chloroform. The extract contained cholesterol and phospholipid in a molar ratio of 0.8:1.0, and less than 1% protein contaminant. Stable thin lipid membranes separating two aqueous compartments were formed from an erythrocyte lipid-hydrocarbon solution, and had an electrical resistance of ∼108 ohm-cm2 and a capacitance of 0.38–0.4 µf/cm2. From the capacitance values, membrane thickness was estimated to be 46–132 A, depending on the assumed value for the dielectric constant (2.0–4.5). Membrane voltage was recorded in the presence of ionic (NaCl and/or KCl) concentration gradients in the solutions bathing the membrane. The permeability of the membrane to Na+, K+, and Cl- (expressed as the transference number, Tion) was computed from the steady-state membrane voltage and the activity ratio of the ions in the compartments bathing the membrane. TNa and TK were approximately equal (∼0.8) and considerably greater than TCl (∼0.2). The ionic transference numbers were independent of temperature, the hydrocarbon solvent, the osmolarity of the solutions bathing the membranes, and the cholesterol content of the membranes, over the range 21–38°C. The high degree of membrane cation selectivity was tentatively attributed to the negatively charged phospholipids (phosphatidylethanolamine and phosphatidylserine) present in the lipid extract.

The present experiments were designed to evaluate the effective thickness of the unstirred layers in series with native and porous (i.e., in the presence of amphotericin B) lipid bilayer membranes and, concomitantly, the respective contributions of membranes and unstirred layers to the observed resistances to the diffusion of water and nonelectrolytes between aqueous phases. The method depended on measuring the tracer permeability coefficients for the diffusion of water and nonelectrolytes (PDDi, cm sec-1) when the aqueous phase viscosity (η) was increased with solutes having a unity reflection coefficient, such as sucrose or dextran. The effective thickness of the unstirred layers (αt, cm) and the true, or membrane, permeability coefficients for diffusion of water and nonelectrolytes (Pmmi, cm sec-1) were computed from, respectively, the slope and intercept of the linear regression of 1/PDDi on η. In both the native and porous membranes, αt was approximately 110 x 10-4 cm. The ratio of Pf, the osmotic water permeability coefficient (cm sec-1) to PmmH2O was 1.22 in the native membranes and 3.75 in the porous membranes. For the latter, the effective pore radius, computed from Poiseuille's law, was approximately 5.6 A. A comparison of Pmmi and PDDi, indicated that the porous membranes accounted for 16, 25, and 66% of the total resistance to the diffusion of, respectively, H2O, urea, and glycerol, while the remainder was referable to the unstirred layers.

The present experiments were designed to evaluate coupling of water and nonelectrolyte flows in porous lipid bilayer membranes (i.e., in the presence of amphotericin B) in series with unstirred layers. Alterations in solute flux during osmosis, with respect to the flux in the absence of net water flow, could be related to two factors: first, changes in the diffusional component of solute flux referable to variations in solute concentrations at the membrane interfaces produced by osmotic flow through the unstirred layers; and second, coupling of solute and solvent flows within the membrane phase. Osmotic water flow in the same direction as solute flow increased substantially the net fluxes of glycerol and erythritol through the membranes, while osmotic flow in the opposite direction to glycerol flow reduced the net flux of that solute. The observed effects of osmotic water flow on the fluxes of these solutes were in reasonable agreement with predictions based on a model for coupling of solute and solvent flows within the membrane phase, and considerably in excess of the prediction for a diffusion process alone.

Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (Rm ≅ 108 ohm-cm2) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (Ti) indicate that these membranes are cation-selective (TNa ≅ 0.85; TCl ≅ 0.15). These electrical properties are independent of the cholesterol content of the lipid solutions from which the membranes are formed. Nystatin, and probably amphotericin B, are cyclic polyene antibiotics containing ≈36 ring atoms and a free amino and carboxyl group. When the lipid solutions used to form membranes contained equimolar amounts of cholesterol and phospholipid, these antibiotics reduced Rm to ≈102 ohm-cm2; concomitantly, TCl became ≅0.92. The slope of the line relating log Rm and log antibiotic concentration was ≅4.5. Neither nystatin (2 x 10-5 M) nor amphotericin B (2 x 10-7 M) had any effect on membrane stability. The antibiotics had no effect on Rm or membrane permselectivity when the lipids used to form membranes were cholesterol-depleted. Filipin (10-5 M), an uncharged polyene with 28 ring atoms, produced striking membrane instability, but did not affect Rm or membrane ionic selectivity. These data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions (Cl-, acetate), and, to a lesser degree, cations (Na+, K+, Li+).

Amphotericin B modifies the permeability properties of thin lipid membranes formed from solutions containing sheep red cell phospholipids and cholesterol. At 10-6 M amphotericin B, the DC membrane resistance fell from ≈108 to ≈102 ohm-cm2, and the membranes became Cl--, rather than Na+-selective; the permeability coefficients for hydrophilic nonelectrolytes increased in inverse relationship to solute size, and the rate of water flow during osmosis increased 30-fold. These changes may be rationalized by assuming that the interaction of amphotericin B with membrane-bound sterol resulted in the formation of aqueous pores. N-acetylamphotericin B and the methyl ester of N-acetylamphotericin B, but not the smaller ring compounds, filipin, rimocidin, and PA-166, produced comparable permeability changes in identical membranes, and amphotericin B and its derivatives produced similar changes in the properties of membranes formed from phospholipid-free sterol solutions. However, amphotericin B did not affect ionic selectivity or water and nonelectrolyte permeability in membranes formed from solutions containing phospholipids and no added cholesterol, or when cholesterol was replaced by either cholesterol palmitate, dihydrotachysterol, epicholesterol, or Δ5-cholesten-3-one. Phospholipid-free sterol membranes exposed to amphotericin B or its derivatives were anion-selective, but the degree of Cl- selectivity varied among the compounds, and with the aqueous pH. The data are discussed with regard to, first, the nature of the polyene-sterol interactions which result in pore formation, and second, the functional groups on amphotericin B responsible for membrane anion selectivity.

This paper reports the effects of amphotericin B, a polyene antibiotic, on the water and nonelectrolyte permeability of optically black, thin lipid membranes formed from sheep red blood cell lipids dissolved in decane. The permeability coefficients for the diffusion of water and nonelectrolytes (PDDi) were estimated from unidirectional tracer fluxes when net water flow (Jw) was zero. Alternatively, an osmotic water permeability coefficient (Pf) was computed from Jw when the two aqueous phases contained unequal solute concentrations. In the absence of amphotericin B, when the membrane solutions contained equimolar amounts of cholesterol and phospholipid, Pf was 22.9 ± 4.6 µsec-1 and PDDHDH2O was 10.8 ± 2.4 µsec-1. Furthermore, PDDi was < 0.05 µsec-1 for urea, glycerol, ribose, arabinose, glucose, and sucrose, and σi, the reflection coefficient of each of these solutes was one. When amphotericin B (10-6 M) was present in the aqueous phases and the membrane solutions contained equimolar amounts of cholesterol and phospholipid, PDDHDH2O was 18.1 ± 2.4 µsec-1; Pf was 549 ± 143 µsec-1 when glucose, sucrose, and raffinose were the aqueous solutes. Concomitantly, PDDi varied inversely, and σi directly, with the effective hydrodynamic radii of the solutes tested. These polyene-dependent phenomena required the presence of cholesterol in the membrane solutions. These data were analyzed in terms of restricted diffusion and filtration through uniform right circular cylinders, and were compatible with the hypothesis that the interactions of amphotericin B with membrane-bound cholesterol result in the formation of pores whose equivalent radii are in the range 7 to 10.5 A.

It is becoming increasingly clear that disturbances of membrane structure and function may be of critical relevance to the understanding of a number of phenomena of common clinical concern. The purpose of this symposium is to provide a summary, hopefully palatable, of the current state of the art in the exponentially enlarging area of membrane biology.The papers have been grouped into three sections involving first, the nature of biological and synthetic membranes; second, some aspects of current problems in membrane transport processes; and third, some of the pathologic alterations in membrane transport processes in disease states.I am grateful to Morton D. Bogdonoff, MD, chief editor of the Archives of Internal Medicine for providing this opportunity and to the authors of the manuscripts in this symposium for their interest and cooperation.