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J Y Lapointe

Publications and source records attributed to J Y Lapointe.

At least 37 records · Page 2Linked to original sources

Volume regulation and ion transport in renal cells.

Volume regulatory mechanisms are reviewed for both short- and long-term adaptation of renal cells to anisotonic media. Within minutes after exposure to hypotonic solutions a common feature of many renal cells is the increase in plasma membrane potassium and chloride conductances. Although extrusion of intracellular potassium certainly contributes to a regulatory volume decrease, the role of chloride efflux itself is probably modest, given the relatively low intracellular chloride concentration. Indeed, other intracellular osmolytes such as taurine and other amino acids are transported out of the cell to achieve a regulatory volume decrease. On a longer-term basis cells from the renal medulla have to adapt to an extracellular milieu which can become extremely hypertonic under certain conditions. In tissue culture models, cells exposed to hypertonic media react by actively taking up small molecules, such as betaine, taurine and myo-inositol, and by synthesizing more sorbitol and glycerophosphocholine. For each of these osmolytes hypertonicity was shown to activate the transcription of a specific enzyme or transporter gene, the expression of which reaches a peak within 18 h of hypertonicity.

Animals↗

Kinetic separation and characterization of three sugar transport modes in Caco-2 cells.

The question of sugar transport heterogeneity in the human intestinal Caco-2 cell line was addressed using alpha-methyl-D-glucose (AMG) and 2-deoxy-D-glucose (DG) as substrate analogues for D-glucose, the transport inhibitors phlorizin (PZ) and phloretin (PT), and NaCl or choline chloride uptake media. The data are compatible with the existence of three distinct pathways that can be isolated kinetically according to specific characteristics: 1) an "AMG-strict" system, strictly Na+ dependent and specific for AMG [Michaelis-Menten constant value (K(m)) = 2.0 +/- 0.3 mM] but sensitive to both PZ and PT, with PZ being more potent than PT, 2) a "DG-strict" system, strictly Na+ independent and specific for both DG (K(m) = 5.2 +/- 0.5 mM) and PT; and 3) a "DG/AMG-mixed" system, strictly Na+ dependent, with loose specificities for the glucose analogues DG (K(m) = 0.81 +/- 0.07 mM) and AMG (K(m) = 8.1 +/- 0.8 mM), and the inhibitors PZ and PT, but with PT being more potent than PZ. Since SGLT-1 obtained by polymerase chain reaction from either Caco-2 cells or normal human jejunum demonstrated identical transport properties when expressed in Xenopus laevis oocytes, we conclude that the "AMG-strict" system represents the expression of human SGLT-1 activity in this cell line. Moreover, Western blot analysis revealed that SGLT-1 is located exclusively in the apical membrane. In contrast, neither the nature nor the membrane location of both the DG-strict and DG/AMG-mixed pathways could be resolved unambiguously. Still it has been demonstrated that expression of the latter system is constitutive to all Caco-2 cells and that its Na+ dependence is not the consequence of H(+)-dependent transport activity. Aside from the presence of the DG/AMG-mixed system, a salient feature of Caco-2 cells is that the GLUT-3 protein is located exclusively in the brush-border membrane. Due to these limitations, it is concluded that the Caco-2 cell line cannot be considered as equivalent to either fetal colonic cells or normal enterocytes.

Biological Transport↗

Inhibition of basolateral potassium conductance by taurine in the proximal convoluted tubule.

The effect of taurine on the electrophysiological properties of the basolateral membrane of the rabbit proximal convoluted tubule was examined. Short-duration isosmotic pulses of 40 mM taurine in the bath solution induced basolateral membrane depolarizations (delta Vbl) of 6.44 +/- 0.5 mV, which were reduced by 58% in absence of Na+. In presence of barium and quinine, delta Vbl values were reduced by 55% in Na(+)-containing bath solutions and were completely abolished in Na(+)-free solutions. Continuous addition of taurine into the bath solutions for a period of 5 min induced 1) a decrease in the partial conductance of the basolateral membrane to K+ (tK) from 0.39 to 0.23; 2) an increase in the partial conductance to Cl- (tcl) from 0.055 to 0.172; 3) a rapid and transient increase of the partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3) from 0.37 to 0.52, followed by a progressive decrease to 0.29; and 4) a depolarization of the basolateral membrane of 16 mV. The absolute membrane conductance mediated by the Na-HCO3 cotransporter was not initially affected by taurine, whereas that to K (GK) initially decreased by a maximal factor of 2 after 1 min, followed by a partial recovery after 5 min, and that to Cl (GCl) increased by a factor of 4. Addition of taurine after a hypotonicity-induced cell swelling produced an inhibition of GK comparable to the one observed under isotonic conditions. These results demonstrate the presence of an electrogenic Na-dependent transport of taurine and indicate that taurine inhibits GK.

Animals↗

Cell volume increases of physiologic amplitude activate basolateral K and CI conductances in the rabbit proximal convoluted tubule.

The effects of increases in cell volume (CV) of physiologic amplitude, induced either hypotonically or isotonically, were studied on the three major basolateral conductances of rabbit isolated proximal convoluted tubules. CV increases were produced by a 40 mosmol/kg H2O hypotonic shock or by the isotonic replacement of mannitol by 40 mM glucose or alanine. The hypotonic shock led to an increase in CV of 17 +/- 3% (N = 8), whereas additions of glucose and alanine led to increases in CV of 22.6 +/- 2.5% (N = 7) and 28.3 +/- 3.5 (N = 5), respectively. Under all of these conditions, the absolute conductance mediated by the NaHCO3 cotransporter did not vary appreciably. This allowed determination of the variations of the absolute conductances to potassium (GK) and chloride (GCl) from their measured partial conductances. All three protocols induced significant increases in GK by factors of 2.37 +/- 0.3, 1.43 +/- 0.16, and 1.69 +/- 0.40, and in GCI by factors of 3.32 +/- 0.57, 3.68 +/- 0.75, and 3.90 +/- 1.0 during the hypotonic, glucose, and alanine protocols, respectively. These increases in GK and GCl occurred with a delay compared with the variations in CV, indicating a more elaborate signaling mechanism than stretch-activation of channels that are known to activate channels within seconds. Intracellular pH increased from 7.19 +/- 0.03 to 7.23 +/- 0.03, 7.17 +/- 0.02 to 7.20 +/- 0.02, and 7.13 +/- 0.01 to 7.16 +/- 0.01 after the hypotonic shock and the glucose and alanine additions, respectively. The study presented here demonstrates that there is a close relationship between CV and GK and GCl, independent of the means used (hypotonically or isotonically) to increase CV in rabbit proximal convoluted tubules. CV activation of GK is proposed to account for part of the increase in GK reported previously during activation of transepithelial transport.

Animals↗

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes.

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na(+)-K(+)-ATPase activity from the dihydroouabain-sensitive current (IDHO) in the presence of increasing concentrations of tetraethylammonium (TEA+; 0, 5, 10, 20, 40 mM), a well-known blocker of K+ channels. The effects of TEA+ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (VM) and a saturable inhibitory component affecting an outward current easily detectable at positive VM. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA+ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA+ on K+ channels. Interestingly, this latter component disappears when the Na(+)-K(+)-ATPase is inhibited by 10 microM DHO. Conversely, TEA+ inhibits a component of IDHO with a KD of 25 +/- 4 mM at +50 mV. As the TEA(+)-sensitive current present in IDHO reversed at -75 mV, we hypothesized that it could come from an inhibition of K+ channels whose activity varies in parallel with the Na(+)-K(+)-ATPase activity. Supporting this hypothesis, the inward portion of this TEA(+)-sensitive current can be completely abolished by the addition of 1 mM Ba2+ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA(+)-sensitive K+ currents and indicates that, in the absence of effective K+ channel inhibitors, IDHO does not exclusively represent the Na(+)-K(+)-ATPase-generated current.

Animals↗

Thermodynamic determination of the Na+: glucose coupling ratio for the human SGLT1 cotransporter.

Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.

Animals↗

Evidence for apical sodium proton exchange in macula densa cells.

These studies were performed to determine if changes in luminal sodium chloride concentration ([NaCl]) might alter macula densa intracellular pH. Isolated thick ascending limbs with attached glomeruli were bathed in a 150 mM NaCl Ringer's solution and perfused in vitro with a 25 mM NaCl solution; N-methyl-D-glucamine cyclamate was used to substitute for NaCl. Macula densa cells were loaded with BCECF and intracellular pH was monitored using a microscope based-dual excitation photometer system. Control intracellular pH for all experiments in which tubules were initially perfused with 25 mM NaCl averaged 7.22 +/- 0.06; N = 28. Increasing luminal [NaCl] from 25 to 150 mM elevated macula densa pH by 0.15 +/- 0.03 (N = 6; P < 0.05) while increasing just luminal [Na] from 25 to 150 mM alkalinized macula densa cells by 0.17 +/- 0.05 (N = 6; P < 0.05). In addition, there was a highly significant linear relationship between luminal [Na] and intracellular pH between 25 and 150 mM NaCl. Other studies were performed to assess the effects of amiloride, an inhibitor of Na:H exchange, on macula densa intracellular pH. Addition of amiloride, to the 25 mM NaCl perfusate acidified macula densa cells by 0.09 +/- 0.03 (N = 6; P < 0.001) and significantly attenuated the increase in pH obtained when luminal [NaCl] was raised from 25 to 150 mM. Other studies evaluated the effects of inhibition of Na:2Cl:K cotransport on macula densa pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

Activation of Na:2Cl:K cotransport by luminal chloride in macula densa cells.

Changes in macula densa intracellular pH (pHi) were used to monitor the direction of flux mediated by the apical Na:2Cl:K cotransporter. At the macula densa, a decrease in luminal [Cl] ([Cl]1) from 60 to 1 mM produced cellular alkalinization secondary to a cascade of events involving a decrease in apical Na:2Cl:K cotransport, a fall in intracellular [Na] ([Na]i) and a stimulation of Na:H exchange. This is supported by the fact that 97% of the change in macula densa pHi with reduction in [Cl]1 was bumetanide-sensitive whereas 92% of this pH change was amiloride-sensitive. We found that, in the presence of 20 mM Na and 5 mM K, a [Cl]1 of 14.3 +/- 2.4 mM (N = 7) produced equilibrium of the apical cotransporter since the pHi obtained under this condition was identical to the pHi found after reducing the net ionic flux to zero with bumetanide. Using this value together with the expected stoichiometry for the bumetanide-sensitive cotransporter, it was estimated that the intracellular [Cl] ([Cl]i) at equilibrium (or in the presence of bumetanide) could be as low as 5 mM. Also, using a Hill number of 2 which is consistent with the present data, the affinity for [Cl]1 was found to be 32.5 mM. Under physiological luminal conditions prevailing at the end of the thick ascending limb (approximately 3.5 mM K, and approximately 25 to 30 mM NaCl), macula densa cells are probably operating close to equilibrium while maintaining a small net reabsorption of Na/K and Cl. Since macula densa cells appear capable of reducing [Cl]i to very low levels, a reabsorptive flux should continue to occur until [NaCl]1 is reduced to 18 mM.

Animals↗

Electrogenic amino acid exchange via the rBAT transporter.

A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Alanine↗

Basic properties and potential regulators of the apical K+ channel in macula densa cells.

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside-out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside-out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.

Animals↗

Coupling between transepithelial Na transport and basolateral K conductance in renal proximal tubule.

A common feature of sodium-reabsorbing epithelia is their ability to match salt entry to salt exit. It is recognized that a key strategy to perform this feat involves the coupling between basolateral sodium pump and potassium conductance (pump-leak coupling). In the renal proximal tubule this coupling is of major importance, as regions of this nephron segment are faced with ever-changing reabsorptive loads. An understanding of this coupling can be facilitated by critically examining those studies that have looked at the problem from the point of view of the whole cell (macroscopic studies) and of single channels (microscopic studies). An overview of such work suggests that the transduction mechanisms which are likely to effect pump-leak coupling in the renal proximal tubule involve cell volume, ATP, and pH (but not calcium). Although the relationship between ATP and potassium conductance may be relatively straightforward, the involvement of pH is likely to be only transient and that of volume remains controversial, occurring either directly though stretch-activated channels in amphibian preparations or indirectly through an as yet unidentified second messenger system in mammalian preparations.

Animals↗

Regulation of basolateral K channels in proximal tubule studied during continuous microperfusion.

Potassium channel activity of the basolateral membrane of the collagenase-treated rabbit proximal convoluted tubule (PCT) was studied during continuous luminal microperfusion. In cell-attached patches (high-K pipette) an inwardly rectifying potassium channel was observed with an inward slope conductance of 60.8 +/- 3.3 pS (n = 12) and outward slope conductance of 17.1 +/- 2.7 pS (n = 6). Stimulation of transcellular sodium transport with luminal glucose and alanine increased channel activity [measured as single-channel open probability (NPo)] from 0.19 +/- 0.11 to 0.44 +/- 0.09 (n = 8). This increase in channel activity was not likely to be mediated by either cell depolarization or cell swelling, because channel activity was voltage insensitive over physiological potentials and because the channel was not activated by stretch. However, channel activity was pH sensitive; reducing luminal pH from 7.4 to 6.5 reduced NPo from 0.63 +/- 0.24 to 0.26 +/- 0.16 (n = 5). Our work demonstrates the feasibility of patch clamping the basolateral membrane of microperfused nephron segments. This has allowed us to follow the activity of this potassium channel during an increase in sodium transport and show that its activity does increase during this maneuver. We conclude that: 1) it is possible to patch clamp the basolateral membrane of microperfused nephron segments, and 2) basolateral membrane of the rabbit PCT contains an inwardly rectifying, pH-sensitive potassium channel. The behavior of this channel on stimulation of transcellular sodium transport could explain the macroscopic increase in basolateral potassium conductance observed under similar conditions.

Alanine↗

Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule.

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

Basolateral membrane potassium channels in rabbit cortical thick ascending limb.

The nature of K exit across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) was investigated using the patch clamp technique. The basolateral membrane was exposed by mild collagenase treatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductance was 35.0 +/- 1.3 pS (n = 9) compared with an outward conductance of 7.0 +/- 0.9 pS (n = 5), and the current reversed at a pipette potential of -63.5 +/- 3.1 mV (n = 9). The channel is strongly voltage dependent, showing an e-fold increase in open probability per 18-mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channel current amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excised patch recordings were possible, which confirmed the presence of a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possible to obtain patch clamp recordings from the rabbit CTAL basolateral membrane using a very mild collagenase treatment, and 2) the exit of K across the basolateral membrane is mediated at least in part by the presence of voltage-sensitive K channels.

Animals↗

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules.

We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (Vbl) in rabbit proximal convoluted tubule (PCT, S1 segment). The present study is aimed at understanding the origin of the membrane repolarization following the initial effect of addition of luminal cotransported solutes. Luminal addition of 10-15 mM L-alanine produced a rapid and highly significant depolarization of Vbl (20.3 +/- 1.1 mV, n = 15) which was transient and associated with an increase in the fractional K+ conductance of the basolateral membrane (tK) from 8 to 29% (P less than 0.01, n = 6). Despite the significant increase in tK, the repolarization was only slightly reduced by the presence of basolateral Ba2+ (2 mM, n = 6) or quinine (0.5 mM, n = 5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4'isothiocyamostilbene-2,2'-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pHi) determined with the fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein (BCECF), averaged 7.39 +/- 0.02 in control solution (n = 9) and increased to 7.50 +/- 0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16 +/- 0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K+ conductance but also through a cellular acidification reducing the basolateral HCO3- exit through the electrogenic Na-3(HCO3) cotransport mechanism.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

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

Intracellular potassium activity (alpha Ki) was measured in control conditions in mid-cortical rabbit proximal convoluted tubule using two methods: (i) by determination of the K+ equilibrium potential (EK) using Ba(2+)-induced variations in the basolateral membrane potential (VBL) during transepithelial current injections and (ii) with double-barrel K-selective microelectrodes. Using the first method, the mean VBL was -48.5 +/- 3.2 mV (n = 16) and the mean EK was -78.4 +/- 4.1 mV corresponding to alpha Ki of 68.7 mM. With K-selective microelectrodes, VBL was -36.6 +/- 1.1 mV (n = 19), EK was -64.0 +/- 1.1 mV and alpha Ki averaged 40.6 +/- 1.7 mM. While these last EK and VBL values are significantly lower than the corresponding values obtained with the first method (P less than 0.001 and P less than 0.01, respectively), the electrochemical driving force for K transport across the basolateral membrane (microK = VBL-EK) 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 of VBL by the reference barrel of the double-barrel microelectrode. Such double-barrel microelectrodes were used to measure temporal changes in alpha Ki and microK in different experimental conditions where Na reabsorption rate (JNa) was reduced. alpha Ki was shown to increase by 12.2 +/- 2.7 (n = 5) and 14.1 +/- 4.4 mM (n = 5), respectively, when JNa was reduced by omitting in the luminal perfusate: (i) 5.5 mM glucose and 6 mM alanine and (ii) glucose, alanine, other Na-cotransported solutes and 110 mM Na. In terms of the electrochemical driving force for K exit across the basolateral membrane, microK, a decrease of 5.4 +/- 2.0 mV (P less than 0.05, n = 5) was measured when glucose and alanine were omitted in the luminal perfusate while microK remained unchanged when JNa 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.

Alanine↗

Basolateral ionic permeabilities of macula densa cells.

It has recently been shown that membrane ionic transport pathways of macula densa cells can be measured using conventional microelectrodes. To determine if conductances could be identified at the basolateral membrane of macula densa cells, cortical thick ascending limbs (CTAL) with attached glomeruli were continuously perfused with a 25 mM NaCl bicarbonate-free Ringer solution. Individual basolateral Na+, Cl-, NaCl, and K+ concentrations were altered by isosmotic replacement with N-methyl-D-glucamine and/or cyclamate. Reduction in basolateral [Na+] from 150 to 25 mM hyperpolarized basolateral membrane potential (Vbl) by 9.9 +/- 1.3 mV (n = 10; all data are corrected for changes in liquid junction potential at bath electrode). A decrease in bath [Cl-] from 150 to 25 mM depolarized Vbl by 20 +/- 2.4 mV (n = 13), whereas decreases in bath [NaCl] from 150 to 25 mM depolarized Vbl by 29 +/- 6.8 mV (n = 5). In the presence of 150 mM NaCl bathing solution, a stepwise increase in [K+] from 5 to 15 mM (by replacement of 10 mM NaCl with 10 mM KCl) depolarized Vbl by 3.3 +/- 1.1 mV (n = 8). After correction for individual transepithelial diffusion potentials, Cl conductance averaged 59 +/- 19% of the total basolateral conductance, whereas K+ (23 +/- 8%) and Na+ (17 +/- 10%) contributed significantly less to the overall basolateral conductance. These results indicate that membrane potential of macula densa cells may be very sensitive to alterations in intracellular Cl- activity and suggest that apical transport of NaCl through a furosemide-sensitive Na(+)-K(+)-2Cl- transporter may affect membrane potential in macula densa cells via a change in intracellular Cl- activity.

Action Potentials↗