pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Fluoride transmembrane exchange in human erythrocytes measured with 19F NMR magnetization transfer

¹⁹F NMR spectra of sodium fluoride in suspensions of human erythrocytes were seen to yield separate resonances for the F^- populations inside and outside the cells. Selective saturation of the magnetization of the intracellular population gave rise to transfer of that saturation to the extracellular population. The extent of magnetization transfer was high and it was blocked by the capnophorin (band 3) anion exchange inhibitor 4,4-dini-trostilbene-2,2-disulfonic acid (DNDS). A series of magnetization-inversion transfer experiments was carried out for the range of intracellular fluoride concentrations of 11 mM to 136 mM and analysed using one-dimensional overdetermined exchange analysis. This yielded an estimate of the equilibrium exchange Michaelis constant and maximal velocity of 27 ± 3 mM and 180 ± 5 × 10^-16 mol cell^-1 s^-1, respectively. There was no alteration of exchange flux of fluoride at an intracellular concentration of 49 mM in the presence of 50 mM glucose; thus suggesting no interaction between glucose and anions in capnophorin-mediated exchange of solutes.     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Activating Effect of NO on Ca2+-Dependent K+ Channels in Smooth Muscle Cells of the Main Pulmonary Artery of the Rabbit

Using a patch-clamp technique in the whole-cell configuration, we studied the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on outward transmembrane ion current in isolated smooth muscle cells (SMC) of the main pulmonary artery of the rabbit. We also studied the characteristics of unitary high-conductance Ca²⁺-dependent K⁺ channels (K_Ca channels) in the SMC membrane in the cell-attached and outside-out configurations. Nitroglycerin in a 10 M concentration increased the amplitude and intensified oscillations of outward transmembrane current induced by step depolarization. In this case, the threshold of activation of the current (–40 mV) did not change. If the potential was +70 mV, the transmembrane current in the presence of NG increased, as compared with the control, by 32.6 ± 19.4% (n = 6), on average. Simultaneous addition of 10 M NG and 1 mM tetraethylammonium chloride (TEA), a blocker of K_Ca channels, to the external solution at the potential of +70 mV decreased the amplitude of outward transmembrane current with respect to the control by 25.2 ± 11% (n = 6) and suppressed oscillations of this current. In the series of experiments carried out in the outside-out configuration (concentration of K⁺ ions in the external solution was 5.9 mM), we calculated the conductance of a single K_Ca channel, which was approximately 150 pS. In the case where the potential was equal to +40 mV, 1 mM TEA suppressed completely the current through unitary K_Ca channels. In the series of experiments performed in the cell-attached configuration, 100 M NG to a considerable extent intensified the activity of unitary high-conductance K_Ca channels by increasing the probability of the channel open state (P ₀), on average, by 80 ± 1%, as compared with the control. In this case, NG did not influence the conductance of single K_Ca channels. We concluded that the NO donor NG increases the amplitude of outward transmembrane current in SMC of the rabbit main pulmonary artery by stimulation of the activity of TEA-sensitive high-conductance K_Ca channels. Our experiments carried out on single K_Ca channels demonstrated that the activating effect of NG on K_Ca channels is realized at the expense of an increase in the P ₀ of these channels, but not of a change in the conductance of single channels.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin

Summary The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01±0.01, 0.24±0.03 and 0.39±0.07, at 100 nmol/liter, 1 mol/liter or 10 mol/liter calcium activity, respectively. The single-channel slope conductance is 34±2 pS, if the potential difference across the patch (V ) is zero, and approaches 59±1 pS, ifV is –50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability=0.03±0.03). If 1 mol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34±0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06±0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59±4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mol/liter and by extracellular application of 1 mol/liter serotonin.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Ion transport across the early chick embryo: I. Electrical measurements,ionic fluxes and regional heterogeneity

The chick blastoderm at the stage of late gastrula is a flat disc formed by three cell layers and exhibiting epithelial properties. Blastoderms were cultured in miniature chambers and their electrophysiological characteristics were determined under Ussing conditions.Under open-circuit condition and identical physiological solutions on both sides, spontaneous transblastodermal potential difference (V _oc) of –7.5±3.3 mV (ventral side positive) was measured. Under short-circuit condition (transblastodermal V = 0 mV), the blastoderm generated short-circuit current (I _sc) of 21±8 A/cm², which was entirely dependent on extracellular sodium, sensitive to ouabain applied ventrally and independent of extracellular chloride. The net transblastodermal Na⁺ flux fully accounted for the measured I _sc, both under control conditions and with ouabain. The total transblastodermal resistance (R _tot) was 390±125 cm².Frequently, the V _oc, I _sc and R _tot showed spontaneous oscillations with a period of 4–5 min. Removal of endoderm and mesoderm did not significantly affect the electrical properties, indicating that the electrogenic sodium transport is generated by the ectoderm.The V _oc and I _sc measured in the area pellucida (–1.3±0.8 mV, 9.3±4.4 A/cm²) and extraembryonic area opaca (–7.8±1.1 mV, 31.2±12.7 A/cm²) were significantly different. Such a heterogeneous distribution of electrical properties can explain the presence in the blastoderm of extracellular electrical currents found by using a vibrating probe.This work was supported by the Swiss National Research Foundation (grant. 3.418-0.86 to P.K.) and by Roche Research Foundation (grant. to U.K.). We thank Drs. E. Raddatz and Y. de Ribaupierre for helpful discussions.     

Pressure- and parathyroid-hormone-dependent Ca2+ transport in rabbit connecting tubule: Role of the stretch-activated nonselective cation channel

To characterize the Ca²⁺ transport process across the apical membrane of the rabbit connecting tubule (CNT), we examined the effects of luminal pressure on parathyroid hormone (PTH)-dependent apical Ca²⁺ transport in this segment perfused in vitro. An increase of perfusion pressure (0.2 to 1.2 KPa) caused cytoplasmic free Ca²⁺ concentration ([Ca²⁺].) to increase by 42 ± 11 nm in Fura-2 loaded perfused CNT. The response was accentuated when 10 nm PTH was added to the bath (101 ± 30 nm, n = 6). Addition of 0.1 mm chlorphenylthio-cAMP (CPT-cAMP) to the bath also augmented the [Ca²⁺]; response to pressure from 36 ± 16 to 84 ± 26 nm (n = 3). Under steady perfusion pressure at 1.2 KPa, PTH (10 nm) increased [Ca²⁺]; by 31 ± 7 nm (n = 5), whereas it did only slightly by 6 ± 2 nm (n = 12) at 0.2 KPa. The pressure-dependent increase of [Ca²⁺]; was abolished by removing luminal Ca²⁺ (n = 3), and was not affected by 0.1 and 10 m nicardipine (n = 4) in the presence of 10 nm PTH. Cell-attached patch clamp studies on the apical membrane of everted CNT with pipettes filled with either 200 mm CaCl₂ or 140 mm NaCl revealed channel activities with conductances of 42 ± 2 pS (n = 4) or 173 ± 7 pS (n = 5), respectively. An application of negative pressure (–4.9 KPa) to the patch pipette augmented its mean number of open channels (NP ₀ ) from 0.005 ± 0.001 to 0.022 ± 0.005 in the Ca²⁺-filled pipette, and was further accelerated to 0.085 ± 0.014 (n = 3) by 0.1 mm CPT-cAMP. In the Na⁺-filled pipette, similar results were obtained (n = 3), and CPT-cAMP did not activate the stretch-activated channel in the absence of negative pressure (n = 3). These results suggest that a stretch-activated nonselective cation channel exists in the apical membrane of the CNT and that it is activated by PTH in the presence of hydrostatic pressure, allowing entry of Ca²⁺ transport from the apical membrane.We appreciate Ms. Hisayo Hosaka and Ms. Yuki Oyama for their technical assistance and Ms. Keiko Sakai for her secretarial work. This research was supported by grants from the Ministry of Education and Culture of Japan (No. 05670054) and from Yamanouchi Foundation for Research on Metabolic Disorders (1992–1993).     

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone

Summary We have chosen the MDCK cell line to investigate aldosterone action on H⁺ transport and its role in regulating cell membrane K⁺ conductance (G _m ^K ). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K⁺ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH _i ) andG _m ^K were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH _i andG _m ^K (Hill coefficient 4.4±0.4), indicating multiple H⁺ binding sites at a single K⁺ channel. Application of aldosterone increased pH _i within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO₂/HCO ₃ ^– buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO₂/ HCO ₃ ^– is limited by the increased activity of a pH _i -regulating HCO ₃ ^– extrusion system. SinceG _m ^K is stimulated half-maximally at the pH _i of 7.18±0.04, internal H⁺ ions could serve as an effective intracellular signal for the regulation of transepithelial K⁺ flux.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Resistance properties of the diluting segment ofAmphiuma kidney: Influence of potassium adaptation

Summary Chronic exposure to high potassium stimulates K⁺-secretory mechanisms in the diluting segment of the amphibian kidney (K⁺ adaptation). Since K⁺ net flux depends critically on the passive cell membrane permeabilities for K⁺ ions, cable analysis and K⁺-concentration step changes were applied in this nephron segment to assess the individual resistances of the epithelium and the K⁺ conductance of the luminal cell membrane. Experiments were performed in the isolated, doubly-perfused kidney of both control and K⁺-adaptedAmphiuma. In control animals transepithelial resistance was 290±27 cm², which decreased significantly to 199±17 cm² after K⁺ adaptation. The resistance in parallel of the luminal and peritubular cell membrane decreased from a control value of 157±14 to 108±6 cm² after chronic K⁺ treatment. This was paralleled by a decrease of the ratio of the luminal to peritubular cell membrane resistance from 2.5±0.1 to 1.9±0.1, respectively. Estimation of the individual cell membrane resistances reveals that the combined resistance of the luminal and peritubular cell membrane is in the same order of magnitude as the paracellular shunt resistance in diluting segments of both control and K⁺-adapted animals. The luminal cell membrane is K⁺ selective under both conditions, but the absolute luminal K⁺ conductance increases by some 60% with K⁺ adaptation. This leads to an increased back-leak of K⁺ from cell to lumen and may explain stimulated K⁺ net secretion found after chronic K⁺ loading.     

Intracellular potassium activity in mammalian proximal tubule: Effect of perturbations in transepithelial sodium transport

Summary Intracellular potassium activity (a _{K i} ) was measured in control conditions in mid-cortical rabbit proximal convoluted tubule using two methods: (i) by determination of the K⁺ equilibrium potential (E _K) using Ba²⁺-induced variations in the basolateral membrane potential (V _BL) during transepithelial current injections and (ii) with double-barrel K-selective microelectrodes. Using the first method, the meanV _BL was –48.5±3.2 mV (n=16) and the meanE _K was –78.4±4.1 mV corresponding to aa _{K i} of 68.7mm. With K-selective microelectrodes,V _BL was –36.6±1.1 mV (n=19),E _K was –64.0±1.1 mV anda _{K i} averaged 40.6±1.7mm. While these lastE _K andV _BL values are significantly lower than the corresponding values obtained with the first method (P<0.001 andP<0.01, respectively), the electrochemical driving force for K transport across the basolateral membrane ( _K =V _BL –E _K) is not significantly different for both techniques (30.1±3.3 mV for the first technique and 27.6±1.8 mV for ion-selective electrodes). This suggests an adequate functioning of the selective barrel but an underestimation ofV _BL by the reference barrel of the double-barrel microelectrode. Such double-barrel microelectrodes were used to measure temporal changes ina _{K i} and _K in different experimental conditions where Na reabsorption rate (J _Na) was reduced.a _{K i} was shown to increase by 12.2±2.7 (n=5) and 14.1±4.4mm (n=5), respectively, whenJ _Na was reduced by omitting in the luminal perfusate: (i) 5.5mm glucose and 6mm alanine and (ii) glucose, alanine, other Na-cotransported solutes and 110mm Na. In terms of the electrochemical driving force for K exit across the basolateral membrane, _K, a decrease of 5.4±2.0 mV (P<0.05,n=5) was measured when glucose and alanine were omitted in the luminal perfusate while _K remained unchanged whenJ _Na was more severely reduced (mean change =–1.7±2.1 mV, NS,n=5). In the latter case, this means that the electrochemical driving force for K efflux across the basolateral membrane has not changed while both the active influx through the Na–K pump and the passive efflux in steady state are certainly reduced. If the main pathway for K transport is through the basolateral K conductance, this implies that this conductance must have decreased in the same proportion as that of the reduction in the Na–K pump activity.     

Characteristics of L-Type Ca2+ Channels in the Membrane of Mesenteric Artery Myocytes from Genetically Hypertensive Rats

Using the standard voltage-clamp technique in the whole-cell mode, we studied the characteristics of barium currents (I _Ba; Ba²⁺ concentration in the external solution was 5 mM) carried through L-type Ca²⁺ channels in the membrane of myocytes of the resistive mesenteric artery from normotensive and genetically hypertensive rats (NR and GHR, respectively). To perforate the membrane, we used amphotericin B. The arbitrary density of I _Ba through the plasma membrane of GHR myocytes significantly exceeded this parameter in the NR group. For both animal groups, activation curves plotted as the dependence of the membrane conductance (G _Ba) on the membrane potential were not significantly different: the membrane potential for half activation (V _0.5) of I _Ba in the NR myocytes was equal to 1.0 ± 0.3 mV with slope factor k = 6.3 ± 0.4 mV, whereas in the GHR myocytes V _0.5 = -1.6 ± 0.2 mV and k = 6.2 ± 0.5 mV. The stationary inactivation curves for I _Ba differed significantly: in the NR myocytes, V _0.5 = -24.2 ± 0.4 mV and k = 8.3 ± 0.2 mV, whereas in the GHR myocytes such parameters were, respectively, -21.4 ± 0.4 and 8.7 ± 0.3 mV. The pattern of intersection of stationary activation and stationary inactivation curves for I _Ba was indicative of the existence of a window current, i.e., the non-inactivating component of I _Ba within the -40 to ±20 mV range; the phenomenon was clearly pronounced in the GHR myocytes. Differences in the arbitrary density of integral I _Ba and window current were observed. These differences can cause an increased tone of the blood vessels in hypertensive animals.     

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

Summary The inhibition of Ca^2–-ATPase, (Na⁺+K⁺)-ATPase and Na⁺/Ca²⁺ exchange by Cd²⁺ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven ⁴⁵Ca²⁺ uptake into inside-out membrane vesicles displayed a K _m for Ca²⁺ of 88±17 nm, and was extremely sensitive to Cd²⁺ with an IC₅₀ of 8.2±3.0 pM Cd²⁺, indicating an inhibition via the Ca²⁺ site. (Na⁺+K⁺)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd²⁺, displaying an IC₅₀ of 2.6±0.6 m Cd²⁺. Cd²⁺ ions apparently compete for the Mg²⁺ site of the (Na^– +K⁺)-ATPase. The Na⁺/Ca²⁺ exchanger was inhibited by Cd²⁺ with an IC₅₀ of 73±11 nm. Cd²⁺ is a competitive inhibitor of the exchanger via an interaction with the Ca²⁺ site (K _i = 11 nm). Bepridil, a Na⁺ site specific inhibitor of Na⁺/Ca²⁺ exchange, induced an additional inhibition, but did not change the K _i of Cd²⁺. Also, Cd²⁺ is exchanged against Ca²⁺, albeit to a lesser extent than Ca²⁺. The exchanger is only partly blocked by the binding of Cd²⁺. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na⁺/Ca²⁺ exchanger. We conclude that intracellular Cd²⁺ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca²⁺ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).     

Intracellular potential and K+ activity in rat kidney proximal tubular cells in acidosis and K+ depletion

Summary Techniques were developed for the measurement of intracellular potentials and potassium activities in rat proximal tubule cells using double barreled K⁺ liquid-ion-exchanger microelectrodes. After obtaining measurements of stable and reliable control values, the effects of K⁺ depletion and metabolic and respiratory acidosis on the intracellular potential and K⁺ activity in rat kidney proximal tubular cells were determined. At a peritubular membrane potential of –66.3±1.3 mV (mean±se), intracellular K⁺ activity was 65.9±2.0 mEq/liter in the control rats. In metabolic acidosis [70 mg NH₄ Cl/100 g body wt) the peritubular membrane potential was significantly reduced to –47.5±1.9 mV, and cellular K⁺ activity to 53.5±2.0 mEq/liter. In contrast, in respiratory acidosis (15% CO₂) the peritubular membrane potential was significantly lowered to –46.1±1.39 mV, but the cellular K⁺ activity was maintained at an almost unchanged level of 63.7±1.9 mEq/liter. In K⁺ depleted animals (6 weeks on low K⁺ diet), the peritubular membrane potential was significantly higher than in control animals, –74.8±2.1 mV, and cellular K⁺ activity was moderately but significantly reduced to 58.1±2.7 mEq/liter. Under all conditions studied, cellular K⁺ was above electrochemical equilibrium. Consequently, an active mechanism for cellular K⁺ accumulation must exist at one or both cell membranes. Furthermore, peritubular HCO₃ ^– appears to be an important factor in maintaining normal K⁺ distribution across the basolateral cell membrane.