Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

Chloride movement across the basolateral membrane of proximal tubule cells

Summary Electrophysiologic and tracer experiments have shown that Cl^– entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na⁺, and that Cl^– entry is electrically silent. The mechanism of Cl^– exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl^– ions across the basolateral membrane, the relative conductance of Cl^– to K⁺ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl^– and K⁺ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl^– conductance was about 10% of K⁺ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl^– activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl^– activity was virtually unaffected by the PD changes. We conclude that Cl^– leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na⁺ or K⁺.     

Cyclic AMP and intracellular ionic activities innecturus gallbladder

Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E _m ) and intracellular sodium, potassium, and chloride activities (a _Na ⁱ ,a _K ⁱ ,a _Cl ⁱ ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E _Tr ) was also measured. In the presence of cAMP,a _Cl ⁱ fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa _Na ⁱ decreased anda _K ⁱ increased,E _m was unchanged andE _Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina _K ⁱ was abolished when Cl^–-free incubation media were used. Addition of the Ca⁺⁺-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE _m ,E _Tr , ora _Cl ⁱ . When A23187 was added to the mucosal medium,E _m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl^– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca⁺⁺ concentration.     

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane

Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H⁺ (pH_i) and Cl^– (aCl_i) activities with double-barreled ion-selective microelectrodes. In a HCO ₃ ^– -free solution of pH 7.4 (HEPES/Tris buffered), pH_i was 7.25 andaCl_i was 33mm. By an exposure to a HCO ₃ ^– (25mm HCO ₃ ^– /5% CO₂, pH 7.4) solution for 15 min,aCl_i was decreased to 25mm and pH_i was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO ₃ ^– concentration [HCO ₃ ^– ]_i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO ₃ ^– -free solution reversed the changes inaCl_i and pH_i. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCl_i, pH_i, and [HCO ₃ ^– ]_i caused by admission/withdrawal of the HCO ₃ ^– , solution and decreased the buffer value. Replacement of all Cl^– with gluconate in the HCO ₃ ^– solution increased pH_i, and readmission of Cl^– decreased pH_i. The rates of these changes in pH_i were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO ₃ ^– solution, a stimulation of intracellular HCO ₃ ^– production by exposing the tissue to 25mm NH ₄ ⁺ increasedaCl_i significantly. While in the HCO ₃ ^– -free solution or in the HCO ₃ ^– , solution containing DIDS, exposure to NH ₄ ⁺ had little effect onaCl_i. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl^–/HCO ₃ ^– exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl^–/HCO ₃ ^– exchange is discussed.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).     

Dependence of cell membrane conductances on bathing solution HCO 3 − /CO2 inNecturus gallbladder

Summary The effects of bathing solution HCO ₃ ^– /CO₂ concentrations on baseline cell membrane voltages and resistances were measured inNecturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO ₃ ^– /CO₂ Ringer's solutions (2.4mm HCO ₃ ^– /air or 1mm HEPES/air) or a high HCO ₃ ^– /CO₂ Ringer's (10mm HCO ₃ ^– /1% CO₂). The principal finding of these studies was that the apical membrane fractional resistance (fR _a) was higher in tissues bathed in the 10mm HCO ₃ ^– /CO₂ Ringer's, averaging 0.87±0.06, whereasfR _a averaged 0.63±0.07 and 0.48±0.08 in 2.4mm HCO ₃ ^– and 1mm HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical (R _a) and basolateral membrane (R _b) resistances in tissues bathed in 10mm HCO ₃ ^– /1% CO₂ Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO ₃ ^– /CO₂ Ringer's, the higher value offR _a was found to be due to both an increase inR _a and a decrease inR _b. The higher values offR _a and lower values ofR _b confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K⁺ and Cl^– substitutions. The results of these studies suggest that an electrodiffusive Cl^– transport mechanism exists at the basolateral membrane of tissues bathed in a 10mm HCO ₃ ^– /1% CO₂ Ringer's, which can explain in part the fall inR _b. The above observations are discussed in terms of a stimulatory effect of solution [HCO ₃ ^– /PCO₂ on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^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 _i ^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 _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^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 _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^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 _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule

Summary The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (V _bl=–69 mV), a higher relative potassium conductance (t _K=0.6), lower intracellular Na⁺ activity (A _Na=18.4mm), and higher intracellular K⁺ activity (A _K=67.8mm). No evidence for a conductive sodium-dependent or sodium-independent HCO ₃ ^– pathway could be demonstrated. The basolateral Na–K pump is inhibited by 10^–4 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results inA _Na=64.8mm,A _K=18.5mm, andV _bl=–28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K⁺ conductance, as evaluated by increasing bath K⁺ to 17mm, is dependent upon the restingV _bl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na⁺ and K⁺ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.     

Transcellular Chloride Pathways in Ambystoma Proximal Tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl⁻ absorption linked to Na⁺ were investigated in isolated perfused tubules using Cl⁻-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl⁻ (a _i ^Cl ) is 11.3 ± 0.5 mm, the basolateral (V ₁ ), apical (V ₂ ), and transepithelial (V ₃ ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na⁺ absorption is decreased by removal of organic substrates from the lumen, a _i ^Cl falls by 1.3 ± 0.3 mm and V ₂ hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na⁺ from the lumen causes a _i ^Cl to fall further by 2.3 ± 0.4 mm and V ₂ to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a _i ^Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a _i ^Cl . The effects of luminal HCO⁻ ₃ removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO₃ or Cl-OH. The effects of basolateral HCO⁻ ₃ removal, of basolateral Na⁺ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO₃ exchange. Several lines of evidence favor conductive Cl⁻ transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a _i ^Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO⁻ ₃-free Ringer, the equilibrium potential for Cl⁻ does not differ from the membrane potential V ₂ . Finally, the logarithmic changes in a _i ^Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl⁻ channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998     

Electrogenic bicarbonate secretion in the turtle bladder: Apical membrane conductance characteristics

Summary We have recently shown that stimulation of electrogenic HCO ₃ ^– secretion is accompanied by a simultaneous increase in short-circuit current (I _sc, equivalent to HCO ₃ ^– secretion rate under these conditions), apical membrane capacitance (C _a , proportional to membrane area), and apical membrane conductance (G _a , proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase inG _a and the possibility that the rate of electrogenic HCO ₃ ^– secretion is regulated by changes inG _a . Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO ₃ ^– secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO ₃ ^– secretion with cAMP. The major conclusions are: (i) a measurable apical Cl^– conductance exists in control hemibladders; (ii) the transport-associated increase inG _a includes a Cl^–-conductive component; (iii)G _a also appears to reflect a HCO ₃ ^– conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl^– and HCO ₃ ^– are similar; (v) 9-AA reducesG _a andI _sc appear cAMP-stimulated hemibladders; and (vi) alterations inI _sc appear to be mediated by changes inG _a .     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.     

Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps

Summary The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K⁺)=72mm and (Cl^–)=15.8mm. Cl^– but not K⁺ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (P _Na/P _K) was 0.044, and the chloride to potassium permeability ratio (P _Cl/P _K) was 1.17. Since K⁺ was not in electrochemical equilibrium, intracellular (K⁺) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K⁺ and Cl^– the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na⁺. An equivalent electrical circuit for Na⁺ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.     

Bicarbonate-dependent chloride absorption in small intestine: Ion fluxes and intracellular chloride activities

Summary Proximal, stripped segments of small intestine from the urodeleAmphiuma were short-circuited in media containing Na⁺, Cl^– and HCO ₃ ^– . Under these conditions there was a large net absorption of Cl^–, a small net absorption of Na⁺ and a residual flux (J _Net ^R ) consistent with HCO ₃ ^– secretion. Net Cl^– absorption correlated with the short-circuit current (I _sc); net Na⁺ absorption correlated negatively withJ _Net ^R . Acetazolamide eliminated theI _sc, lowered Cl^– absorption by 50%, and reduced net Na⁺ absorption without alteringJ _Net ^R . Benzolamide inhibited theI _sc without alteringJ _Net ^R . Benzolamide inhibited theI _sc more rapidly when applied on the mucosal surface. Replacement of Na⁺ or HCO ₃ ^– (and CO₂) in the media eliminated theI _sc, net Cl^– absorption and the residual flux. Likewise, inclusion of the stilbene SITS in the serosal media eliminated theI _sc, net Cl^– absorption and the residual flux. The cytoplasmic activity of Cl^– (a _ci ^a ) was determined with single and double-barreled microelectrodes. Thea _ci ^a of villus absorptive cells in normal media was 21.0mm and in excess of that expected on the basis of electrochemical equilibrium of Cl^– at the mucosal membrane. Active Cl^– accumulation was also observed in the presence of acetazolamide but was eliminated upon replacement of media Na⁺ with choline. The mucosal membrane potential was depolarized upon replacement of media Na⁺. It is concluded that Cl^– is actively absorbed into intestinal cells ofAmphiuma by an electrogenic process located in the mucosal membrane. Depending on the level of intracellular HCO ₃ ^– , accumulated Cl^– may diffuse passively back into the mucosal media or undergo exchange with bath HCO ₃ ^– at the serosal membrane.     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Modes of Cl− transport across the mucosal and serosal membranes of urodele intestinal cells

Summary The characteristics of Cl^– movement across luminal and basolateral membranes ofAmphiuma intestinal absorptive cells were studied using Cl^–-sensitive microelectrodes and tracer³⁶Cl techniques. Intracellular Cl^– activity (a _Cl ⁱ ) was unchanged when serosal Cl^– was replaced; when luminal Cl^– was replaced cell Cl^– was rapidly lost. Accordingly, the steady statea _Cl ⁱ could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86mM,a _Cl ⁱ rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl^– transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl^– absorptive flux, attained a maximum whena _Cl ⁱ reached an apparent value of 17mm, indicating the presence of a saturable, serosal transport step. The stilbeneinsensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellulara _Cl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account.a _Cl ⁱ was unaffected by Na replacement, removal of medium K, or elevation of medium HCO ₃ ^– . Mucosae labeled with³⁶Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl^– efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl^– entry appears to be passive, but an electrogenic uptake cannot be discounted.     

Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.