Retrospective data analysis of animal poisoning events in Liguria.

Intentional poisoning represents a serious risk to domestic and wild animals, and it can be an environmental and human health issue as well . This paper is a retrospective study, which covers a decade, based on animal poisoning cases and poisoned baits that were submitted for diagnostic examinations to the Veterinary Medical Research Institute for Piedmont, Liguria and the Aosta Valley (IZS-PLVA) in Liguria region. All data were collected through a passive surveillance system introduced in Italy by a decree of the Ministry of Health in January 2009. 43.2% of the animal poisoning cases were confirmed by toxicological analysis, whereas toxic agents were detected in 31.1% of the baits. The most affected animal species were dogs and cats, followed by synanthropic birds,. Only 4% of the total poisoning events analysed involved wild animals and cases of livestock poisoning were minimal. An increased number of cases in January, March, April and August was noticed, but no seasonal trend was detected. The most affected areas were the ones with the highest level of urbanization and population density. The major cause of the poisonings and the most common substances detected in the examined baits were anticoagulants whereas cholinesterase inhibitors, organochlorine pesticides and carbamates were detected in a minor number of cases. This study raises concerns about deliberate animal poisoning in ligurian region and highlights the necessity to fight this phenomenon as it endangers animals, humans and environment.

Anion transport processes in the mammalian superficial proximal straight tubule.

The experiments reported in this paper were designed to evaluate some of the characteristics of anion transport processes during fluid absorption from superficial proximal straight tubules isolated from rabbit kidney. We measured net chemical C1- flux during fluid absorption from tubules perfused and bathed with Krebs-Ringer buffers containing 113.6 mM C1-, 10 mM acetate, and 25 mM HCO-/3 at pH 7.4; assessed the effects of carbonic anhydrase inhibitors on net fluid absorption in the presence and absence of CO2; and evaluated the influx and efflux coefficients for [14C]-acetate transport at 37degreesC, at 21degreesC, and in the presence of carbonic anhydrase inhibitors. The experimental data shown that, for this nephron segment, net C1- flux accompanies approximately 27.5% of net Na+ absorption; and net C1- absorption may be accounted for by a passive transport process, primarily diffusional in nature. Fluid absorption in this nephron segment is reduced 40-60% by carbonic anhydrase inhibitors, but only when the tubules are exposed to 95% O2-5% CO2 rather than 100% O2. Thus, it seems probably that approximately half of Na+ absorption in these tubules may be rationalized in terms of a carbonic anhydrase-dependent CO2 hydration process. In addition, there may occur in these isolated proximal tubules an acetazolamide-insensitive moiety of HCO-/3 absorption comparable to that observed for proximal tubules in vivo. Finally, we provide evidence that net efflux of luminal acetate is due to metabolic energy-dependent processes other than CO2 hydration and may, under appropriate conditions, account for approximately one-fourth of net Na+ absorption.

Interactions among prostaglandin E2, antidiuretic hormone, and cyclic adenosine monophosphate in modulating Cl- absorption in single mouse medullary thick ascending limbs of Henle.

This paper describes the inhibitory effect of prostaglandin E2 (PGE2) on antidiuretic hormone (ADH)-stimulated net Cl- absorption and spontaneous transepithelial voltage (Ve) in single medullary thick ascending limbs of Henle (TALH, thick ascending limb; mTALH, medullary segment; cTALH, cortical segment) obtained from mouse kidney. The experimental data indicate that PGE2 reduced the ADH-dependent values of net Cl- absorption (JnetCl, eq cm-2 s-1) and Ve (mV) in a dose-dependent manner; that increasing concentrations of peritubular ADH reversed the PGE2-mediated reductions in the ADH-dependent moiety of Ve in the mouse mTALH; that PGE2 had no effect on cyclic AMP-stimulated increments in Ve in the mouse mTALH; and that PGE2 had no effect on Ve in the cTALH, where Ve is unaffected either by ADH or by cyclic AMP. These effects might be obtained because of a direct competition between ADH and PGE2 for receptor binding on basolateral membranes. Alternatively, PGE2 might have reduced the affinities between ADH-receptor units and a component(s) of the series of processes leading to adenyl cyclase activation. The latter argument requires that basolateral membranes of the mouse mTALH exhibit receptor reserve, i.e., at the minimum concentration of ADH required to enhance Ve and JnetCl maximally, a fraction of basolateral membrane ADH receptors were unoccupied. According to this view, increasing peritubular ADH concentrations might reverse the PGE2-mediated reduction in ADH-dependent salt transport by increasing the number of basolateral membrane receptors occupied by ADH.

The effect of antidiuretic hormone on solute flows in mammalian collecting tubules.

These experiments were intended to evaluate the antidiuretic hormone (ADH)-dependent reflection coefficients of urea, sucrose, and NaCl in cortical and outer medullary collecting tubules isolated from mammalian kidney. In one group of experiments, the ADH-dependent osmotic water flows, when the perfusing solutions contained hypotonic NaCl solutions, were indistinguishable from control observations when either urea or sucrose replaced, in part, NaCl in isotonic bathing solutions (cortical collecting tubules). Similarly, both in cortical and outer medullary collecting tubules exposed to ADH, there was zero net osmotic volume flow when a portion of the NaCl in the bathing and/or perfusing solutions was replaced by either sucrose or urea, so long as the perfusing and bathing solutions were isosmolal. Taken together, these observations suggest that the ADH-dependent reflection coefficients of NaCl, urea, and sucrose, in these tubules, were identical. Since the effective hydrodynamic radii of urea and sucrose are, respectively, 1.8 and 5.2 A, it is likely that sigma(i), for urea, sucrose, and NaCl, was unity. In support of this, the diffusion permeability coefficient (P(Di) cm sec(-1)) of urea was indistinguishable from zero. Since the limiting sites for urea penetration were the luminal interfaces of the tubules, these data are consistent with the view that ADH increases diffusional water flow across such interfaces.

Cellular constraints to diffusion. The effect of antidiuretic hormone on water flows in isolated mammalian collecting tubules.

These experiments were intended to evaluate the effects of antidiuretic hormone (ADH) on dissipative water transport in cortical collecting tubules isolated from rabbit kidney. In the absence of ADH, the osmotic (P(f), cm sec(-1)) and diffusional (P(DW) cm sec(-1)) water permeability coefficients were, respectively, 6+/-6 and 4.7+/-1.3 (SD). When ADH was added to the bathing solutions, P(f) and P(DW) rose to, respectively, 186+/-38 and 14.2+/-1.6 (SD). In the absence of ADH, the tubular cells were flat and the lateral intercellular spaces were closed when the perfusing and bathing solutions were, respectively, hypotonic and isotonic; in the presence of ADH, the cells swelled and the intercellular spaces dilated. These data suggest that ADH increased the water permeability of the luminal membranes of the tubules. It was possible that the ADH-dependent P(f)/P(DW) ratio was referable to the resistance of the epithelial cell layer (exclusive of luminal membranes) to water diffusion (R(DW), sec cm(-1)). Such a possibility required that R(DW) be approximately 650, i.e., approximately 25-fold greater than in an equivalent thickness of water. To test this view, it was assumed that R(Di) values for lipophilic solutes in lipid bilayer membranes and in luminal membranes were comparable. In lipid bilayer membranes, R(Di) was substantially less than 90 sec cm(-1) for pyridine, n-butanol, and 5-hydroxyindole. In renal tubules, R(Di) for these solutes ranged from 795 to 2480 with and without ADH. It was assumed that, in the tubules, R(Di) was referable to cellular constraints to diffusion; for these solutes, the latter were 12-25 times greater than in water. Accordingly, it is possible that the ADH-dependent P(f)/P(DW) ratio was also due to cellular constraints to diffusion.

CO2-stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. Evidence for synchronous Na +/H+ and Cl-/HCO3- exchange in apical plasma membranes.

These experiments evaluated salt transport processes in isolated cortical thick limbs of Henle (cTALH) obtained from mouse kidney. When the external solutions consisted of Krebs-Ringer bicarbonate (KRB), pH 7.4, and a 95% O2-5% CO2 gas phase, the spontaneous transepithelial voltage (Ve, mV, lumen-to-bath) was approximately mV; the net rate of Cl- absorption (JnetCl) was approximately 3,600 pmols s-1 cm-2; the net rate of osmotic solute absorption Jnetosm was twice JnetCl; and the net rate of total CO2 transport (JnetCO2) was indistinguishable from zero. Thus, net Cl- absorption was accompanied by the net absorption of a monovalent cation, presumably Na+, and net HCO3- absorption was negligible. This salt transport process was stimulated by (CO2 + HCO3-): omission of CO2 from the gas phase and HCO3- from external solutions reduced JnetCl, Jnetosm, and Ve by 50%. Furthermore, 10(-4) M luminal furosemide abolished JnetCl and Ve entirely. The lipophilic carbonic anhydrase inhibitor ethoxzolamide (10(-4) M, either luminal or peritubular) inhibited (CO2 + HCO3-)-stimulated JnetCl, Jnetosm, and Ve by approximately 50%; however, when the combination (CO2 + HCO3-) was absent, ethoxzolamide had no detectable effect on salt transport. Ve was reduced or abolished entirely by omission of either Na+ or Cl- from external solutions, by peritubular K+ removal, by 10(-3) M peritubular ouabain, and by 10(-4) M luminal SITS. However, Ve was unaffected by 10(-3) M peritubular SITS, or by the hydrophilic carbonic anhydrase inhibitor acetazolamide (2.2 x 10(-4) M, lumen plus bath). We interpret these data to indicate that (CO2 + HCO3-)-stimulated NaCl absorption in the cTALH involved two synchronous apical membrane antiport processes: one exchanging luminal Na+ for cellular H+; and the other exchanging luminal Cl- for cellular HCO3- or OH-, operating in parallel with a (CO2+ HCO3-)-independent apical membrane NaCl cotransport mechanism.

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption.

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.

Chloride transport in porous lipid bilayer membranes.

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. P(DCl) (cm.s(-1)), the diffusional permeability coefficient for Cl(-), estimated from unidirectional (36)Cl(-) fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, ohm(-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, P(DCl) was 1.36 x 10(-4) cm.s(-1) when Gm was 0.02 ohm(-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 P(DCl) computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for approximately 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 P(DNa)/P(DCl) 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.

The effect of valinomycin on the electrical properties of solutions of red cell lipids in n-decane.

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 (C(L)). The limiting resistance, as (C(L) (-1)) approached zero, was 3 x 10(8) ohm-cm(2). Resistance was also found to be linearly related to membrane thickness. The limiting resistance at zero thickness was again 1-3 x 10(8) ohm-cm(2). These data are interpreted to indicate that the DC resistance of thick lipid membranes comprises two surface resistances (R(S)) at each interface with the aqueous bathing solutions, and a bulk resistance (R(B)) 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 R(S) but had no effect on R(B). Under certain conditions, thick lipid membranes containing valinomycin behaved like highly selective K(+) electrodes.

An analysis of unstirred layers in series with "tight" and "porous" lipid bilayer membranes.

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 (P(DDi), cm sec(-1)) when the aqueous phase viscosity (eta) was increased with solutes having a unity reflection coefficient, such as sucrose or dextran. The effective thickness of the unstirred layers (alpha(t), cm) and the true, or membrane, permeability coefficients for diffusion of water and nonelectrolytes (P(mmi), cm sec(-1)) were computed from, respectively, the slope and intercept of the linear regression of 1/P(DDi) on eta. In both the native and porous membranes, alpha(t) was approximately 110 x 10(-4) cm. The ratio of P(f), the osmotic water permeability coefficient (cm sec(-1)) to P(mmH2O) 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 P(mmi) and P(DDi), indicated that the porous membranes accounted for 16, 25, and 66% of the total resistance to the diffusion of, respectively, H(2)O, urea, and glycerol, while the remainder was referable to the unstirred layers.

The interaction of polyene antibiotics with thin lipid membranes.

Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (R(m) congruent with 10(8) ohm-cm(2)) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (T(i)) indicate that these membranes are cation-selective (T(Na) congruent with 0.85; T(Cl) congruent with 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 approximately 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 R(m) to approximately 10(2) ohm-cm(2); concomitantly, T(Cl) became congruent with0.92. The slope of the line relating log R(m) and log antibiotic concentration was congruent with4.5. 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 R(m) 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 R(m) 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(+)).

The effect of valinomycin on the ionic permeability of thin lipid membranes.

Optically black membranes prepared from sheep red cell lipids have a high electrical resistance (1-3 x 10(8) ohm-cm(2)). The ionic transference numbers (T(i)) 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 T(Na)/T(K), 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 component of fluid absorption linked to passive ion flows in the superficial pars recta.

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.

Osmosis in cortical collecting tubules. A theoretical and experimental analysis of the osmotic transient phenomenon.

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: P(f) (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, P(f) 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 P(f) and P(DDw) (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 P(f).

Osmosis in cortical collecting tubules. ADH-independent osmotic flow rectification.

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)) P(f) (l-->b), computed from volume flows from hypotonic lumen to isotonic bath, was 20 +/- 4 x 10(-4) (SEM); the value of P(f) (b-->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 (14)C-urea fluxes from lumen to bath (P(DDurea) (l-->b)) and from bath to lumen (P(DDurea) (b-->l)). For hypotonic luminal solutions and isotonic bathing solutions, P(DDurea) (l-->b) was 0.045 +/- 0.004 x 10(-4) and was unaffected by ADH. The ADH-independent values of P(DDurea) (l-->b) and P(urea) (b-->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, P(DDurea) (l-->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 P(f) (b-->l) in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of P(f) (l-->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.

Volume reabsorption, transepithelial potential differences, and ionic permeability properties in mammalian superficial proximal straight tubules.

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 HCO(3) (-) (KRB) and the bath contained KRB plus 6% albumin, net volume reabsorption (J(v), nl min(-1) mm(-1) was -0.46 +/- 0.03 (SEM); V(e), the spontaneous transepithelial potential difference, was -1.13 +/- 0.05 mV, lumen negative. Both J(v), and V(e), were reduced to zero at 21 degrees C or with 10(-4) M ouabain, but J(v), was not HCO(3) (-) dependent. Net Na(+) reabsorption, measured as the difference between (22)Na(+) 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 (22)Na(+) 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 V(e), rose to +0.82 mV when tubules were perfused with 138.6 mM Cl(-) solutions, V(e) may become positive when tubular fluid Cl(-) concentrations rise during volume reabsorption. The permeability coefficients P(Na) and P(Cl) computed from tracer fluxes were, respectively, 0.23 x 10(-4) and 0.73 x 10(-4) cm s(-1). A P(Na)/P(Cl) 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 P(Na)/P(Cl) 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.

Coupling of solute and solvent flows in porous lipid bilayer membranes.

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.

The effect of valinomycin on potassium and sodium permeability of HK and LK sheep red cells.

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.

The formation and properties of thin lipid membranes from HK and LK sheep red cell lipids.

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 approximately 10(8) ohm-cm(2) and a capacitance of 0.38-0.4 microf/cm(2). 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, T(ion)) was computed from the steady-state membrane voltage and the activity ratio of the ions in the compartments bathing the membrane. T(Na) and T(K) were approximately equal ( approximately 0.8) and considerably greater than T(Cl) ( approximately 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 degrees 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 effect of amphotericin B on the water and nonelectrolyte permeability of thin lipid membranes.

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 (P(DDi)) were estimated from unidirectional tracer fluxes when net water flow (J(w)) was zero. Alternatively, an osmotic water permeability coefficient (P(f)) was computed from J(w) 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, P(f) was 22.9 +/- 4.6 microsec(-1) and P(DDHDH2O) was 10.8 +/- 2.4 microsec(-1). Furthermore, P(DDi) was < 0.05 microsec(-1) for urea, glycerol, ribose, arabinose, glucose, and sucrose, and sigma(i), 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, P(DDHDH2O) was 18.1 +/- 2.4 microsec(-1); P(f) was 549 +/- 143 microsec(-1) when glucose, sucrose, and raffinose were the aqueous solutes. Concomitantly, P(DDi) varied inversely, and sigma(i) 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.