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Conyza spp. are among the most common weeds found in perennial crops, and resistance to several herbicides has already occurred worldwide. In the present study, pot and laboratory experiments were conducted to evaluate differences in glyphosate and triclopyr susceptibility between different biotypes of Conyza spp. Plant material was obtained from seeds collected in perennial crops in Greece. Seeds of four Conyza spp. biotypes were sown in separate pots and treated with glyphosate at the rosette stage, while seeds of six different Conyza spp. biotypes were sown and treated with triclopyr. Twenty-four hours after the herbicide treatment, the aboveground part of the weeds was manually collected and plant samples were prepared to determine the analytes with LC-MS/MS. In two C. bonariensis biotypes, the glyphosate concentration was 50% to 52% lower than for other biotypes; therefore, they could be considered as potentially resistant to glyphosate because the resistant biotype had a stronger and faster metabolism than the sensitive biotype, transforming glyphosate into non-toxic metabolites. The higher glyphosate concentration was detected in C. canadensis biotypes, which proved to be potentially susceptible to glyphosate. Three C. bonariensis biotypes and one C. albida biotype were less susceptible to triclopyr because analytes were detected in 63% to 82% higher concentrations than the two C. canadensis biotypesthat were susceptible to triclopyr. These results reveal significant differences between the three species, confirm the problem of herbicide resistance, and suggest that glyphosate and triclopyr efficacy in Conyza spp. is merely due to herbicide metabolism to other compounds.

Three commercially available dental silane primers, two single-phase prehydrolyzed and one two-component system were investigated for their composition, extent of hydrolysis, surface interactions, bond strength and interfacial topography when used on three representative porcelain alloys (Au-Pd, high-Pd, Ni-Cr). Five tests, 1H FT-NMR, FTIR, GPC, ESCA and EPMA, were used to determine the composition and the surface interaction profiles. Shear tests were performed to assess the bond strength values. According to the results, all the primers contain gamma-methacryloxypropyltrimethoxysilane (gamma-MPTS) at concentrations of 1.15-18.86 g/100 mL, two in ethanol and one in isopropanol. Traces of acetic acid were found in the prehydrolyzed primers. All the primers demonstrated partial hydrolysis of the methoxy groups. Prehydrolyzed primers exhibited a higher rate of hydrolysis and better orientation of hydrolyzed methoxy groups towards the Ni-Cr alloy surface. Due to the extent of surface oxidation, the Ni-Cr alloy provided more bonding sites for silanols than the other two types of alloys. The Ni-Cr alloy demonstrated the highest shear bond strength values on smooth surfaces regardless of the type of primer used. The results of the present study suggest that active prepolymerized primers may provide significant advantages over two-component systems in the repair of Ni-Cr porcelain fractures involving removal of the metal oxide layer.

Ordering temperatures and magnetizations versus applied fields have been measured for the hexagonal phases RAu3.6. The compound PrAu3.6 can be regarded as a Van Vleck paramagnet with appreciable exchange. The Sm, Tb and Dy compounds show antiferromagnetic behaviour. HoAu3.6 may have an ordering point below 5 K. GdAu3.6 has two ordering points. The Curie paramagnetic temperatures of all RAu3.6 compounds are negative with values between −12.5 K (Tb) and −0.7 K (Nd). The observed effective moments are almost the same as the rare earth trivalent ion moments.

Glaphenine, a nonsteroid analgesic compound, administered by gastric gavage in rats (800 mg/kg), induced nonoliguric reversible acute renal failure (ARF). Intratubular deposits were found in medullary collecting ducts. Intratubular hydrostatic pressure (Pt) increased from 11.7 +/- 0.7 to 30.0 +/- 0.9 mmHg. Renal failure was almost completely prevented by concomitant high water and solute diuresis, achieved by furosemide infusion in Wistar rats and by high salt intake in Brattleboro rats with diabetes insipidus. In the latter protected animals, Pt was only slightly elevated (17.0 +/- 0.5 mmHg). Urinary excretion of prostaglandin E2 (PGE2) dropped dramatically after glaphenine administration in Wistar rats; the fall was slight in Brattleboro rats in which PGE2 excretion was normally low. We conclude that tubular obstruction plays a prominent role in glaphenine-induced ARF in the rat. High water and solute diuresis prevented tubular obstruction. Reduced renal PGE2 synthesis is probably not involved in the pathophysiology of this ARF model, inasmuch as Brattleboro rats on a high salt intake were protected despite low basal urinary excretion of PGE2.

The effects of d -glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, d -glucose produces 10.5 mV ± 1.1 (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to 2.1 ± 0.1 mV . (3) The glucose-evoked depolarization, ΔV G , does not alter the intracellular K+ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na+ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li+ substituting for Na+) does not change the magnitude of ΔV G ; complete removal of sodium from the lumen lowers the value of ΔV G ( 3.2 ± 0.2 mV ) but the response is not abolished. This observation suggests that the d -glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.

The transepithelial potential difference (VTE) and transepithelial resistance (RTE) were determined along the length of the distal tubule of the amphibian Triturus alpestris. The site of impalements was determined at the end of each experiment by latex injection and microdissection. Two segments, differing by their distribution at the surface of the kidney, by their respective diameters and by their electrical properties could be identified: the early distal tubule (EDT) and the late distal tubule (LDT). VTE was invariably positive in the EDT; it increased from approximately + 5 mV to approximately + 22 mV within the first 30% of this segment and remained roughly constant distally to this site. RTE was estimated at 57 omega X cm2 in the EDT. The LDT exhibited essentially negative VTE figures with the exception of its very initial portion; the peak negativity at the end of the LDT was -35 mV. RTE was assessed from 8 measurements performed in the first 30% of the LDT; the tentative transepithelial resistance was 530 omega X cm2. Provisional evidence suggests that the transepithelial resistance of the terminal portion of the LDT may be substantially larger than the resistance measured in other parts of this segment.

Double-barreled, selective microelectrodes filled with liquid ion exchanger were used to determine proximal tubule cell pH (pHcell), luminal pH (pHlum), and peritubular capillary blood pH (pHbl.pt) in Necturus kidney in vivo. The average pHbl.pt of 16 animals was 7.64 +/- 0.3; pHcell was 7.36 +/- 0.02 (n = 50), and pHlum was 7.50 +/- 0.05 (n = 16). Because of the variability in pHbl.pt from one animal to another, we studied the blood/cell/lumen pH differences. We sequentially measured with a single microelectrode pHcell and pHlum, and then pHbl.pt in an adjacent peritubular capillary. In 25 such paired determinations, the average pHbl.pt - pHcell difference was 0.28 +/- 0.03, cell acid, and the pHbl.pt - pHlum difference was 0.14 +/- 0.02, lumen acid. The pHcell in this series was significantly more acid than the pHlum (by 0.14 +/- 0.02), but in a few instances the pH gradient across the apical cell membrane was inversed. All measurements were performed in the initial portion of the proximal tubule. We conclude that 1) proximal cell pH is acid with regard to peritubular blood pH, 2) the proximal tubule of Necturus kidney is capable of establishing a small transepithelial pH difference (lumen acid), and 3) pHcell is generally more acid then pHlum.

This study deals with the electrical responses of the peritubular membrane of the Necturus proximal tubule to 8 organic anions, in NaHCO3-free (trismaleate-buffered) and NaHCO3-containing solutions. The anions glutamate and gluconate brought about a small depolarization, but only in NaHCO3-free media. Benzene sulfonate did not alter significantly membrane p.d. The 5 other test-anions produced hyperpolarization. The magnitude of membrane depolarization elicited by high-K media was proportionally larger in the presence of the test-anions propionate, lactate, pyruvate, acetate and formate than with chloride: it is inferred that these anions increasedTK. The same 5 anions shifted in the negative direction the p.d. achieved at peak K-depolarization; according to a previous analysis (Anagnostopoulos, 1977), this observation suggests that their permeabilities (PA) are greater thanPCl, at least during the substitution. The association ofPA>PCl with an increase ofTK, upon exposure of the kidney to test-anions, is at best accounted for by a decrease ofPCl. The pattern of voltage attenuation along the epithelial cable during anionic substitutions is also consistent with an increase ofTK via a decrease ofPCl. In conclusion, the apparent sequence of relative anionic permeabilities, as obtained from the responses of the tissue to a single anion, irrespective of buffering procedures, is:Pacet,Plact,Ppyruv,Pprop,Pform>PCl≃Pgluc,Pglut. The test-anions propionate, lactate, pyruvate, acetate and formate tend to increaseTK, mainly by reducingPCl. The effect of glutamate and gluconate on physiologic ion permeabilities is too small to be specified with accuracy: it depends to some extent on the buffer used in the solutions.

K+-selective liquid ion-exchanger double-barreled microelectrodes were used to measure intracellular K+ activity (alpha Ki) of proximal tubular cells in Necturus kidney. SEveral methodological problems inherent in the construction and in vivo application of such microelectrodes were considered, in particular the identification of leaky impalements and the correct assessment of alpha Ki during reversible perfusion of peritubular capillaries from control to a test solution. Peritubular potassium concentration (CpK) could be altered by means of double-barreled micropipettes containing a physiologic and a high or low K solution. The control alpha Ki value averaged 57.9 +/- 8.5 mM (mean +/- SD, n = 38) and the basolateral membrane potential was -70.4 +/- 5.8 mV. Thus, the K+ equilibrium potential across the basolateral membrane (EK) was -81.1 +/- 3.4 mV. Increasing CpK from 3.0 to 30.0 mM raised alpha Ki by 7.0 +/- 1.5 mM, and lowering CpK to 0.3 mM decreased alpha Ki by 9.2 +/- 2.9 mM. Conclusions: 1) the steady-state K+ distribution across the basolateral membrane indicates that potassium is actively pumped into the cell. 2) The relative stability of alpha Ki in the face of large CpK variations underscores the effectiveness of cell homeostasis; passive mechanisms appear to contribute to this end.

To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl− activity (α Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V BL) and Cl-selective electrode PD (V BL Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate α Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl− transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased α Cl , whereas addition of the drug in luminal fluid slightly increased α Cl ; both results are consistent with basolateral membrane uphill Cl− transport from interstitium to the cell. TMA+ for Na+ substitutions in either luminal or peritubular perfusate had no effect on α Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO 3 − outflux), resulted in an increase of α Cl , presumably related to enhanced Cl− cell influx: we infer that Cl− is exchanged against HCO 3 − at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl− concentration above equilibrium values: intracellular CO2 hydration gives rise to cell HCO 3 − concentrations above equilibrium. The passive exit of HCO 3 − at the basolateral membrane energizes an uphill entry of Cl− into the cell. The resulting increase of α Cl , above equilibrium, generates downhill Cl− diffusion from cell to lumen. As a result, luminal Cl− concentration also increases.

R 5 Pd 2 compounds are antiferromagnetic with Neel temperatures between 7 K (Tm) and 38 K (Dy). The magnetic moment of the rare earth is that of the trivalent ion. Palladium seems to have zero moment. The Neel and paramagnetic points deviate slightly from De Gennes' law.

Eleven different secosteroids or steroids (10(-10) to 10(-8) M) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibian Necturus kidney while recording basolateral cell membrane potential Vm. Seven of these molecules (1,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, 5,6-trans-25(OH)D3, 19-diol-cholesterol, estradiol and testosterone) resulted in changes of Vm (delta Vm) occurring in a few seconds, the largest delta Vm being observed with 1,25(OH)2D3, +6.5 +/- 0.75 mV (n = 19); these seven (seco)steroids, but not the four inactive sterols (vitamin D3, cholesterol, 1 alpha D3 and aldosterone) possess a hydroxyl group on at least one carbon of the C17 to C25 lateral chain of the sterol ring. The delta Vm effect was present in Na+-free or Cl-free media, but it was abolished in HCO3-free media. Depolarization of cell membrane potential by addition of glucose, 11 mM, in luminal perfusion fluid abolished the 1,25(OH)2D3-evoked delta Vm effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K+ conductances, suppressed the 1,25(OH)2D3-evoked delta Vm effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca2+-dependent K+ channels also abolished the 1,25(OH)2D3-evoked depolarization. Such observations are consistent with the presence of Ca2+-dependent K+ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)2D3 and probably by other (seco)steroids.

The electrical properties of the proximal tubule of the in vivo Necturus kidney were investigated by injecting current (as rectangular waves) into the lumen or into the epithelium of single tubules and by studying the resulting changes of transepithelial (VL) and/or cell membrane potential (VC) at various distances from the source. In some experiments paired measurements of VL and VC were performed at two abscissas x and x'. The luminal length constant of about 1,030 micrometer was shown to provide a good estimate of the transepithelial resistance, specific resistance (RTE = 420 omega.cm2) and/or per unit length (rTE = 1.3 x 10(4) omega.cm). The apparent intraepithelial length constant was subject to distortions arising from concomitant current spread in the lumen. The resistances of luminal membrane (rL), basolateral membrane (rB), and shunt pathway (rS) were estimated by two independent methods at 3.5 x 10(4), 1.2 x 10(4), and 1.7 x 10(4) omega.cm, respectively. The corresponding specific resistances were close to 1,200, 600, and 600 omega.cm2. There are two main conclusions of this study. (a) The resistances of cell membranes and shunt pathway are of the same order of magnitude. The figure of the shunt resistance is at variance with the notion that the proximal tubule of Necturus is a leaky epithelium. (b) A rigorous assessment of the conductive properties of concentric cylindrical double cables (such as renal tubules) requires that electrical interactions arising from one cable to another be taken into account. Appropriate equations were developed to deal with this problem.

Eight cations were reversibly and isosmotically substituted for sodium (45 mM) in peritubular circulation of the proximal tubule of the doubly perfused Necturus kidney. Li, choline and tetramethylammonium produced small shifts in peritubular potential difference with no change in conductance. Tris- and tetraethylammonium depolarized the membrane by about 10 mV without affecting its conductance. Rb and Cs produced a decrease of membrane permeability to potassium. Potassium for sodium substitution depolarized the membrane (3.1 mV) and increased its conductance. It is concluded that none among the cations tested may be considered as readily less permeant than sodium across the peritubular membrane.

Double-barreled microelectrodes selective to either potassium or sodium were used to determine the transepithelial potential difference (VTE) and the intraluminal activity of potassium (alpha LuK) or sodium (alpha LuNa) in the early distal tubule (EDT) of Triturus waltlii in vivo; luminal activities were compared with the corresponding plasma ion activities, alpha PtK and alpha PtNa. The transepithelial equilibrium potentials for potassium (EK(TE] and sodium (ENa(TE] were computed from the respective transmural chemical distributions: they were used to assess the transepithelial electrochemical potential differences [(V-EK)TE and (V-ENa)TE]. By dividing the raw data into three groups of 30% total tubular length (0-30, 31-60, 61-90%), the following results were obtained. 1) VTE increases from +15 to +20 mV (lumen positive) between the first and second portion of the EDT but remains constant thereafter. 2) The alpha LuK/alpha PtK ratio decreases steadily along the EDT from 1.92 to 1.66 and then to 1.32. 3) The values of alpha LuNa/alpha PtNa in the same three subdivisions are 0.79, 0.44, and 0.45. 4) The (V-EK)TE difference is largely positive along the whole EDT: +32, +33, and +27 mV. 5) The (V-ENa)TE difference declines from +9 mV (first portion) to values statistically not different from zero in the last two thirds of the EDT.(ABSTRACT TRUNCATED AT 250 WORDS)

We applied the technique of renal cell fusion (8) to the proximal convoluted tubule ofNecturus kidney. Giant fused cells from this segment exhibit stable cell membrane potential, Vm, for several tens of minutes. These cells display a number of electrophysiological properties characteristic of both the apical and basolateral membranes of the proximal tubule. These include 1) significant K+ conductance, 2) no detectable Cl− conductance, 3) apical Na-glucose and Na-amino-acid electrogenic carriers, 4) basolateral ouabainsensitive Na−K pump activity and 5) basolateral Na/HCO3 cotransport.

1. Double-barrelled (selective vs. conventional) microelectrodes were used to assess the steady-state activities (a) of the ions Cl-, K+, Na+ and H+ in peritubular blood capillaries (abld) and in cell (acell) and lumen (alum) of the late distal tubule (l.d.t.) of Necturus. 2. a(cell)cl, a(lum)cl and a(bld)cl were 5.5 +/- 0.3, 11.8 +/- 1.0 and 70.5 +/- 0.1 mM, respectively. They were used to compute the chemical potentials for Cl- across the three diffusive barriers of the tissue. Basolateral and apical membrane potentials were -74.3 +/- 1.1 and -60.1 +/- 2.0 mV, respectively (cell negative); the lumen was thus negative with respect to blood, by 13.6 +/- 1.5 mV. The electrochemical potential difference (e.p.d.) for Cl- of 42 mV across the apical membrane opposes Cl- absorption, implying active apical Cl- uptake, since Cl- is known to be absorbed in the l.d.t. Basolateral Cl- exit is favoured by an e.p.d. of 10 mV. 3. a(cell)K, a(lum)K and a(bld)K were 65.8 +/- 0.8, 2.5 +/- 0.1 and 2.5 +/- 0.1 mm, respectively. The electrochemical distribution of K+ indicates that K+ absorption, if present, proceeds against an adverse apical e.p.d. of 18 mV. Basolateral K+ distribution is close to its electrochemical equilibrium, suggesting high K+ permeability at this membrane. 4. a(cell)Na was 9.0 +/- 0.4 mM, a(bld)Na 71.0 +/- 0.3 mM, and a(lum)Na was approximated at about 9 mM. Diffusive Na+ entry from lumen to cell is favoured by an e.p.d. close to 65 mV. Basolateral Na+ exit must be active, since it proceeds against an e.p.d. of 130 mV. 5. Cell, luminal and blood pH were 7.14 +/- 0.03, 6.52 +/- 0.08 and 7.37 +/- 0.04, respectively. The luminal electrochemical potential of H+ is higher than that of cell (by 91 mV) and blood (by 34 mV) indicating that proton secretion into the lumen must be active. 6. The e.p.d. of each ion across the epithelium opposes, by its orientation, the established direction of net transepithelial ion transport, suggesting that the shunt pathway may serve only for back-diffusion.

Single proximal convoluted tubules (PCT) of Necturus kidney were impaled with three microelectrodes in the sequence M1, M2, M3. M1 was used for injecting short DC current pulses, M2 for recording peritubular membrane potential, V, and M3 for injecting longer DC current steps and thereby shifting V to a new baseline potential, V'. We define the p.d. changes at M2 due to M1-induced pulses as delta V and delta V' (for baselines V and V', respectively). Our objective was to test whether delta V' was equal to delta V. The main finding is that when V depolarized by 10 to 80 mV delta V'/delta V remained close to 1.00. Care was taken to ensure that this apparent stability of the pulse ratio was not due to opposite changes of apical and basolateral membrane conductances (g(A) and g(B) respectively), to changes of the sum g(A)+g(B) compensated for by changes of the cell-to-cell junctional conductance, g(j), or to a distortion of the delta V'/delta V ratio as a function of interelectrode distance, masking voltage-dependent changes of cell membrane conductances. Hyperpolarization of V produced gradual electrical uncoupling between cells as V' became increasingly negative, by a mechanism yet to be determined.

The significance of the "leaky" tight junction might be understood better if cells of the epithelial monolayer possessed mechanisms to regulate molecular flow through the junction. To test this possibility, Necturus gallbladder, a representative leaky epithelium, was studied before, during, and after mucosal exposure to plant cytokinins and two other microfilament-active drugs, cytochalasin B and phalloidin. Concomitant with morphological changes in microfilaments, cytokinins induced rapid reversible increases in transepithelial resistance and potential difference (PD) and decreases in NaCl dilution potentials, with no change in the ratio of relative cell membrane resistances. Cytochalasin B (0.2-1.2 microM) and phalloidin (0.6-12.7 microM) caused similar changes in transepithelial resistance and PD. When the intramembranous structure of tight junctions was studied by freeze fracture, peak cytokinin-induced increments in transepithelial resistance were associated with more disorder in the strand meshwork resulting in a small increase in tight junction depth, but there was no evidence of de novo strand assembly. These studies suggest that permeability of the tight junction of Necturus gallbladder is subject to rapid reversible modulation, possibly under cytoskeletal control.

The effect of foreign anions on transepithelial potential difference and transepithelial input conductance was studied in the isolated perfused Necturus kidney. Two microelectrodes (recording and current-injecting) were inserted into the lumen of single proximal tubules and the peritubular perfusate was shifted reversibly for 30-60 sec from a physiologic Ringer's solution to a test solution in which chloride was replaced isosmotically by a foreign anion. The permeability sequence, obtained by potential measurements, was: lactate less than glutamate less than gluconate less than pyruvate less than benzene sulfonate less than or equal to acetate less than or equal to F less than propionate less than BrO3 less than formate less than ClO3 less than Cl than ClO4 less than I less than or equal to Br less than NO3 less than SCN. Transepithelial conductance decreased when the tissue was perfused with anions less permeable than chloride but the conductance sequence was different from the permeability sequence. Such discrepancies were more pronounced during perfusion with hyperpolarizing anions; ClO4 and I- (both more permeable than chloride) produced an important decrease in transepithelial conductance, followed by incomplete reversibility when the perfusion was shifted again to chloride Ringer's. The results are best explained by the presence of weak positive fixed charges, governing anion permeation, at the shunt pathway of the proximal tubule. An analysis of the data allows tentative estimates of shape and size of the sites.

Publisher Summary This chapter provides an overview on the technique of isolation and perfusion of individual nephron segments. This technique provides basic information on the net transport rates of salt and water on the transport potentials and on the electrical and hydraulic conductance of virtually all nephron segments. One of the general principles that emerges from a comparison of the individual data, is the paramount role that Na + /K + ATPase plays in all renal absorption processes. This is demonstrated by the correlation between the density of the salt fluxes and the density of the enzyme in different nephron segments. The chapter presents that progress has been made in the analysis of transport properties of the individual cell membranes of proximal tubules by measuring intracellular Na + , K + , Cl - , and H + ion concentrations. It also explains the analysis of cotransport systems for Na + and organic substances in the brush border membrane. It describes the new anion transport systems and their coupling stoichiometry.

This study was undertaken to determine the passive electrophysiological properties of the diffusive barriers of the late distal tubule (LDT) in Necturus. The transepithelial resistance (RT) determined by cable analysis was 1,130 omega.cm2, which puts the LDT in the class of "tight" epithelia. Using two different methods, we did not find significant cell-to-cell electrical coupling. The fractional apical resistance was 0.93, and it did not vary with distance from the current-injecting electrode. Relative permeabilities of K+, Na+, and Cl- during peritubular ion concentration changes were assessed by circuit analysis. The conclusions are as follows. The basolateral cell membrane is highly permeable to K+; its apparent K+ transference number is 0.78. Basolateral chloride transference was very small. Sodium removal from peritubular fluid produced depolarization, suggesting carrier-mediated electrogenic Na+ transport. The high fractional resistance of the apical cell membrane prevented assessment of apical transference numbers. However, Cl- removal from luminal fluid produced cell hyperpolarization; the underlying mechanism has not been established with certainty. The paracellular pathway does not discriminate between Na+, Cl-, and some of their substitutes; it is poorly permeable to gluconate and prefers K+ to Na+.

IN epithelia classified as “leaky”, net transport efficiency of NaCl and water may be determined not only by cellular metabolic activity, but also by tight junction permeability and the conjugate physical driving forces acting between lumen and intercellular space. The significance of paracellular ion movement across leaky epithelia would become more apparent if a cytoplasmic mechanism for regulation of tight junctional permeability could be defined. The structure–function observations reported here suggest that the microfilament system may have such a function. Rapid and reversible increases in shunt path electrical resistance were observed in Necturus gallbladder exposed to plant cytokinins. In electron micrographs zeiotic transformation of the luminal membrane was associated with disruption of microfilaments and in freeze fracture, with reorientation and disorder of tight junctional elements.

We studied the effects of millimolar Ba2+ concentrations on the properties of the basolateral membrane of the proximal tubule in Necturus kidney. Ba2+ was added in the peritubular perfusate by means of capillary microperfusion experiments. Basolateral membrane p.d. and conductance were continuously monitored in single tubules (their lumen was filled with oil) during reversible application of Ba2+ in peritubular fluid. The effect was membrane depolarization by 14 mV and a decrease of membrane conductance to 63% of control values. This association strongly suggests that the main effect of Ba2+ is a decrease of the partial conductance to K+ at the basolateral membrane. However, in other experiments, performed also in oil-filled tubules, Ba2+ was shown to decrease intracellular K+ activity, recorded continously by means of double-barreled (selective vs non selective) K+ microelectrodes. This and other, indirect evidence support the hypothesis that Ba2+ elicits also a small increase of the partial conductance to sodium at the basolateral membrane.

In the perfused Necturus kidney, the ratio of transepithelial sodium-to-chloride permeabilities (PNa/PCl) was estimated from dilution potentials at 1:7. The ratio of transepithelial permeabilities PHCO3/cl was estimated from bi-ionic potentials at 1:4 and that of Plact/PCl at 1:12. The permeability sequence, therefore, is PCl greater than PHCO3 greater than than PNa or PnaCl greater than PNaHCO3. The latter is similar to the situation in the rat. However, because of the low Na permeability, passive NaCl absorption by solvent drag is probably less important in Necturus than in the rat. The measurement of transepithelial input conductance during replacement of extracellular chloride by either bicarbonate or lactate shows that the former reduces the transmural conductance to 31% of control values as compared with 40% for the latter. Such discrepancies between permeability and conductance sequences are consistent either with an interference of the test anion with chloride permeation or with the presence of positive fixed changes within the shunt pathway.

Published values of transepithelial potential differences (VTE) in the proximal tubule of Necturus vary from approximately 0 to approximately -15 mV. In view of this disparity, we reinvestigated VTE. Our measured VTE was on the average -1.0 mV in early convolutions and +0.6 mV in terminal proximal segments. In the course of this study, we considered five distinct causes of artifacts. 1) Tip and pre-tip potentials: their occurrence was minimized by using Ringer's filled microelectrodes. 2) Interstitial tip localizations: the position of the tip was ascertained by the shift in potential, resulting in response to peritubular perfusion with gluconate solutions. 3) Leaky impalements: VTE responses to gluconate, input resistance determinations and the presence of positive VTE's rule out the leak hypothesis. 4) Zeroline shifts between pre- and postimpalement stages, and 5) Spontaneous VTE drifts in the positive direction, due to gradual passage of the tip from cell to lumen, or in the negative direction, resulting from tip contact with the lower cellular layer. All five causes of artifacts may be involved in the controversy regarding past VTE estimates.

Transepithelial potential difference (p.d.) was measured in the proximal tubule of Necturus kidney in vivo, by means of microelectrodes filled either with a 3M KClion or with a Ringer's solution for amphibians. The average transepithelial p.d., measured with KCl-tips, was: -1.4 +/- 2.4 mV (early convolutions), -0.1 +/- 2.0 mV (middle convolutions) and +0.1 +/- 2.4 mV (straight segment). The corresponding values obtained with Ringer's-filled microelectrodes were -2.3 +/- 1.8 mV, -1.3 +/- 1.1 mV and +0.1 +/- 1.2 mV, respectively. Tip localization into the lumen was ascertained by luminal injection of either oil (KCl electrode measurements) or artificial solutions which produced a measurable shift of transepithelial p.d. (determinations obtained with Ringer's-tips). Transepithelial p.d. in split-drops (mean reabsorptive half time 27.1 +/- 2.5 min) was -1.8 +/- 1.1 mV. The magnitude of transepithelial p.d. is discussed with respect to an equivalent electrical circuit; it is shown that high transepithelial p.d.'s are inconsistent with the known values of relative conductances of cell membranes in series and shunt pathway, respectively.

The effects of 0.5 mM 4-acetamido-4′-isothiocyano-stilbene-2,2′ disulfonic acid on the electrical properties of the peritubular membrane were studied in the proximal tubule of the perfused Necturus kidney. The addition of stilbene isothiocyanate disulfonic acid in peritubular perfusate resulted in an average 4.5 mV hyperpolarization with no detectable changes of peritubular membrane input conductance. The depolarization elicited by high-K media was enhanced by 18% in the presence of stilbene isothiocyanate disulfonic acid, an observation indicating that the inhibitor increased the contribution of potassium to membrane potential, presumably by decreasing anionic permeabilities. The hyperpolarizing effect of stilbene isothiocyanate disulfonic acid was abolished when peritubular bicarbonate was removed from the medium and isoosmotically replaced by chloride. These data suggest that (a) intracellular bicarbonate activity is higher than that predicted for passive distribution, (b) stilbene isothiocyanate disulfonic acid decreases P HCO3 , thus hyperpolarizing the membrane, (c) chloride distribution appears to be passive when bicarbonate is removed from the peritubular perfusate. The state of Cl distribution when extracellular bicarbonate is at physiologic concentration cannot be assessed from the present data.