Biomedical subjects

R Laprade

Publications and source records attributed to R Laprade.

At least 55 records · Page 3Linked to original sources

PubMed1994

Voltage gating of porins from Haemophilus influenzae type b.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (M(r) 37,782; 341 amino acids). Porins were purified from Hib strains representative of the three outer membrane protein subtypes 1H, 2L and 6U, reconstituted into artificial planar bilayers, and tested for their voltage dependency. At membrane potentials of 50-80 mV, individual Hib 2L and 6U porin channels showed a high probability of undergoing a reversible change to one of several lower conducting substates. Such behaviour was not observed for Hib 1H porin with transmembrane potentials up to 80 mV. The voltage dependence of Hib 2L and 6U porins was asymmetric: it occurred at only one polarity. The asymmetry was also observed for membranes with numerous porins incorporated, suggesting that Hib porin inserted asymmetrically into the bilayer. At macroscopic levels the voltage gating reduced the conductance by 25-50%, implying that the channels closed only partially. Hib 2L porin differs from Hib 1H porin by the substitution Arg166Gln and Hib 6U porin differs from Hib 1H porin by substitutions at ten amino acids including the change Arg166Leu. We conclude that substitutions at Arg166 residue, which is localized to surface-exposed loop number four, are associated with a lowered threshold potential for the voltage gating of Hib porin. This surface-exposed loop may play some role in the conformational changes that occur during voltage gating.

Haemophilus influenzae↗

PubMed1994

cAMP stimulates proximal convoluted tubule Na(+)-K(+)-ATPase activity.

The effect of adenosine 3',5'-cyclic monophosphate (cAMP) was examined on the electrophysiological properties of nonperfused proximal convoluted tubule in vitro. In 5 mM bath K+, the basolateral membrane potential (Vbl) was -66 +/- 1 mV (n = 26). Low bath K+ (0.1 mM) led to a transient hyperpolarization of Vbl followed by a sustained decrease to reach -48.6 +/- 5.0 mV. Return to 5 mM bath K+ produced a rapid and transient Vbl hyperpolarization of 24.6 +/- 1.4 mV (n = 5). This hyperpolarization was completely blocked by 100 microM strophanthidin (n = 4), demonstrating that the hyperpolarization was caused by reactivation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Addition of 1 microM forskolin (forsk) + 100 microM 8-(4-chlorophenylthio)-cAMP (cp-cAMP) significantly increased this hyperpolarization to 30.8 +/- 10 mV (P < 0.005, n = 5). In a separate series of experiments, addition of 1 microM forsk + 100 microM 3-isobutyl-1-methylxanthine increased this hyperpolarization from 21.7 +/- 2.8 to 27.1 +/- 1.6 mV (P < 0.05, n = 5), which excludes any nonspecific effect of cp-cAMP. Forsk + cp-cAMP decreased the apparent partial conductance to Cl- (tCl) from 0.049 +/- 0.003 to 0.031 +/- 0.007 (P < 0.06, n = 6), decreased that to K+ (tK) from 0.56 +/- 0.05 to 0.43 +/- 0.03 (P < 0.05, n = 6), slightly decreased that mediated by the Na-HCO3 cotransporter (tNaHCO3) from 0.26 +/- 0.03 to 0.21 +/- 0.05, and had no effect on the absolute conductance mediated by the Na-HCO3 cotransporter. Forsk + cp-cAMP had no effect on tK when determined using bath K+ steps from 15 to 45 mM (tK = 0.84 +/- 0.02, n = 5) instead of K+ steps from 5 to 15 mM as previously done, and did not affect the value of tK measured in the presence of strophanthidin (tK = 0.41 +/- 0.03, n = 5). These results demonstrate that the decrease of tK by forsk + cp-cAMP observed using K+ steps from 5 to 15 mM is due to modulation by these agents of the stimulated hyperpolarizing Na(+)-K(+)-ATPase current produced by the bath K+ steps. Consequently, the increased Vbl initial recovery from low bath potassium observed when intracellular cAMP is increased could not be the result of modulation of passive basolateral membrane properties and represents a stimulation of the pump current. The present work thus demonstrates that the Na(+)-K(+)-ATPase is stimulated by cAMP.

Animals↗

PubMed1994

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↗

PubMed1993

Evidence against a proton pump in rabbit proximal convoluted tubule.

H+/OH- transport in the absence of bicarbonate was studied in the rabbit proximal convoluted tubule (PCT) perfused in vitro using measurements of membrane potential and intracellular pH (pHi). Blockade of apical Na/H exchange led to a cell acidification of 0.64 +/- 0.1 pH units from a control pHi of 7.27 +/- 0.04. A bafilomycin-insensitive recovery of pHi of 0.05 +/- 0.02 pH units occurred, but pHi did not exceed electrochemical equilibrium. A larger, sustained acidification of 0.87 +/- 0.07 from an initial control pHi of 7.25 +/- 0.05 induced by bilateral Na removal left pHi substantially below electrochemical equilibrium. These results suggest the absence of Na-independent active proton extrusion. We also examined the possibility that a passive electrogenic proton leak may exist. The removal of luminal glucose and alanine led to a basolateral membrane hyperpolarization of 31.3 +/- 0.5 mV, which was associated with a cell acidification of 0.15 +/- 0.02 pH units. These responses were reversed by addition of luminal glucose and alanine but not by depolarization by basolateral barium, suggesting that luminal glucose and alanine effects on pHi were due to changes other than cell potential. We conclude that, in the absence of bicarbonate, all active proton extrusion in the rabbit PCT is dependent on active Na transport and that a proton leak is negligible.

Animals↗

PubMed1993

Basolateral K+, Cl-, and HCO3- conductances and cell volume regulation in rabbit PCT.

The relationship between changes in cellular volume, intracellular pH (pHi), basolateral membrane potential (VBL), and membrane partial basolateral conductances to K+ (tK) and Cl- (tCl) and mediated by the Na-HCO3 cotransporter (tNaHCO3) was determined in the collapsed proximal convoluted tubule (PCT) submitted to a 125-mosmol/kg hypotonic shock. The shock that produces a rapid swelling followed by partial volume regulation was accompanied by a rapid and transient VBL hyperpolarization of 10.0 +/- 1.5 mV and a second gradual hyperpolarization of 5.0 +/- 0.7 mV with respect to a control value of -44.0 +/- 4.6 mV.tK was 0.12 +/- 0.03 in control, increased transiently to 0.15 +/- 0.03, and then gradually increased to reach 0.32 +/- 0.06 at the end of hypotonic shock. In contrast, tCl was 0.03 +/- 0.01 in control, increased rapidly to a maximum of 0.16 +/- 0.01, and then decreased slowly to 0.08 +/- 0.02. During the same period, tNaHCO3 decreased rapidly from 0.41 +/- 0.04 to a minimum of 0.11 +/- 0.02 and slowly reincreased to reach 0.16 +/- 0.01.pHi increased transiently from 7.09 +/- 0.03 in control to 7.24 +/- 0.05 to come back gradually to 7.15 +/- 0.05 at the end of the hypotonic period. The membrane absolute conductance mediated by the Na-HCO3 cotransporter was found to increase only slightly in hypotonic conditions, whereas that to K+ and Cl-, GK and GCl, increased by at least factors of 8 and 17, respectively, with the increase of GCl being much faster than that of GK. In addition, the temporal variations in GCl followed closely those of the cellular water efflux. We conclude that the hypotonic swelling leads to important increases in the conductive pathways for K+ and Cl- and that the Cl- conductance pathway appears to be the rate limiting step in triggering and supporting regulatory volume decrease.

Animals↗

PubMed1993

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↗

PubMed1993

Metabolic cost of bafilomycin-sensitive H+ pump in intact dog, rabbit, and hamster proximal tubules.

Bafilomycin A1 is a specific inhibitor of the brush-border membrane-bound H(+)-adenosinetriphosphatase (H(+)-ATPase) of the kidney cortex with no effect on the mitochondrial ATP synthetase or on the basolateral Na(+)-K(+)-ATPase activities. Bafilomycin A1 is thus a useful tool to estimate the contribution of the activity of the H(+)-ATPase to the cellular ATP turnover in a suspension of proximal tubules containing largely S1 and S2 segments. In dog proximal tubules incubated under control conditions, we found that 81% of the respiration is directly related to ATP synthesis, i.e., is sensitive to oligomycin (phosphorylative respiration). Of this amount, 29% is inhibited by 5 x 10(-7) M bafilomycin A1 alone and 90-95% by the combination of bafilomycin plus ouabain. These results indicate that the H(+)-ATPase activity is a significant energy-requiring process in dog proximal tubules. If bafilomycin is added after a 5- to 7-min preincubation with 1 mM ouabain, then the bafilomycin-sensitive ATP turnover is larger, reaching 44% of total phosphorylation. This may suggest that the H+ pump is stimulated by the indirect inhibition of the Na+/H+ exchanger produced by the exposure of tubules to ouabain. The contribution of the bafilomycin-sensitive H+ pump to the cell ATP turnover is also increased by acidification of the extracellular medium. In rabbit and hamster proximal tubules, the bafilomycin-sensitive ATP requirement involves only 5 and 10%, respectively, of the total ATP turnover. These results demonstrate that the metabolic cost of proton secretion by the membrane-bound H(+)-ATPase in suspensions of proximal tubules may be considerable but varies significantly from species to species.

Adenosine Triphosphate↗

PubMed1993

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↗

PubMed1992

Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems.

The size of the renal sodium/phosphate symporter was estimated with the radiation inactivation technique in isolated bovine brush border membrane vesicles and after reconstitution in proteoliposomes. The functional unit of the native phosphate carrier had a radiation inactivation size of 172 +/- 17 kDa. Identical values were obtained for the reconstituted carrier whether it was irradiated before or after the formation of the proteoliposomes (161 +/- 9 and 159 +/- 11 kDa, respectively). The sodium-independent uptake of phosphate was not affected significantly by radiation doses up to 10 Mrad. This activity is therefore not due to the reconstitution of a large phosphate-binding protein such as alkaline phosphatase. Furthermore, bromotetramisole, a specific inhibitor of phosphate binding to this enzyme, had no significant effect on the uptake of phosphate by the proteoliposomes.

Animals↗

PubMed1992

Substrate-induced modulation of ATP turnover in dog and rabbit proximal tubules.

In dog proximal tubules in suspension, the addition of glucose increased significantly the ouabain-sensitive fraction of respiration, a response suppressed by phlorizin. The addition of alpha-methyl-D-glucoside (alpha-MG) had a modest effect and 3-O-methyl-D-glucoside (3-O-MG) had no effect. The different stimulation of the Na+,K(+)-ATPase activity elicited for each hexose could be explained by a different increment of net transepithelial flux of sodium induced by the sodium: hexose cotransport. This flux is a direct function of the transport characteristics of both luminal and antiluminal membranes of proximal cells for these sugars: glucose is rapidly transported by both membranes (allowing a large transepithelial flux of glucose: sodium) while alpha-MG is poorly transported by the basolateral, and 3-O-MG by the luminal, membrane of the dog proximal tubule (allowing a small transepithelial flux of hexoses and sodium). However the overall tubular respiration of dog proximal tubules was not increased by glucose addition because the increment in the ouabain-sensitive fraction was accompanied by a reciprocal decrement in an ouabain-insensitive but oligomycin- or N',N' dicyclohexylcarbodiimide (DCCD)-sensitive (or in the bafilomycin-sensitive) component of respiration. This component reflects the activity of a large BBM-bound H(+)-ATPase found in this species. The intracellular pH of dog proximal tubules in suspension was measured using the proton-sensitive fluorescent probe 2',7'-bis-2-(carboxyethyl)-5, (and 6)-carboxyfluorescein. Glucose application significantly alkalinized the cells. In contrast, other substrates such as lactate or acetate simultaneously acidified the cells and increased the ouabain-insensitive phosphorylative respiration of dog tubules. These observations suggest that a modulation of the activities of both the sodium and most probably the proton pump is elicited by substrate availability in suspensions of proximal tubules.

Adenosine Triphosphate↗

PubMed1992

Potassium conductance regulation by pH during volume regulation in rabbit proximal convoluted tubules.

When rabbit proximal convoluted tubules were microperfused in the presence of bicarbonate, a 90 mosmol hypotonic shock hyperpolarized the basolateral membrane by 5.5 +/- 1.4 mV, increased basolateral potassium selectivity (tK) from 0.30 +/- 0.02 to 0.45 +/- 0.02, and reduced the basolateral membrane resistance from 4,887 +/- 821 to 2,836 +/- 602 omega.cm. These data show that the hypotonic shock increased absolute basolateral potassium conductance. The same hypotonic shock elevated intracellular pH from 7.18 +/- 0.04 to 7.31 +/- 0.04. When bath pH was increased by 0.2 pH units (by reduction of CO2), intracellular pH rose by 0.13 +/- 0.01. In separate experiments this maneuver hyperpolarized the basolateral membrane by 5.0 +/- 0.8 mV and augmented basolateral tK from 0.58 +/- 0.06 to 0.68 +/- 0.04, suggesting that the basolateral potassium conductance is sensitive to pH changes of a magnitude similar to that evoked by a hypotonic shock. In the nominal absence of bicarbonate or presence of 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) in the bath, the hypotonic shock caused a transient intracellular acidification, suggesting involvement of basolateral bicarbonate transport in the hypotonic shock-induced alkalinization. In the absence of bicarbonate, the hypotonic shock did not increase basolateral tK or induce hyperpolarization of the basolateral membrane. We conclude that the increase in potassium conductance observed during hypotonic shock is at least partly mediated by a bicarbonate-dependent, SITS-sensitive intracellular alkalinization.

Alkalosis↗

PubMed1992

Involvement and source of calcium in volume regulatory decrease of collapsed proximal convoluted tubule.

We examined the role of Ca2+ in the volume regulatory decrease (VRD) of rabbit collapsed proximal tubules. Reduction of bath osmolality by 125 mosmol/kgH2O led to an initial cell swelling of 62.3 +/- 7.5% followed by a partial regulatory phase bringing cell volume to a value of 13.3 +/- 2.9% above control (n = 5). This swelling was accompanied by a transient intracellular Ca2+ ([Ca2+]i) increase from 174 +/- 33 to 306 +/- 67 nM (P < 0.05, n = 8). In the same condition, but in absence of extracellular Ca2+ ([Ca2+]e) [1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], VRD following hypotonic shock was identical to that observed in presence of [Ca2+]e (n = 5), and [Ca2+]i increased transiently from 136 +/- 29 to 161 +/- 31 nM (P < 0.05, n = 5). Addition of 100 microM 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an agent known to inhibit Ca2+ release from intracellular stores, did not affect the initial cell swelling (63.4 +/- 4.2%), and VRD occurred to the same extent (25.0 +/- 7.1%, n = 4), although at a lower rate. In these conditions, [Ca2+]i, which was 113 +/- 30 nM in the isotonic solution, decreased progressively to 81 +/- 20 nM over the 5-min hypotonic period (n = 5). Mere preincubation with 100 microM TMB-8 before hypotonic shock led to a VRD identical to that observed in presence of Ca2+ and absence of TMB-8 while still blocking the Ca2+ release, with cell Ca2+ decreasing progressively from 179 +/- 32 to 87 +/- 21 nM (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter.

A simple and rapid procedure was developed for the reconstitution of Na(+)-dependent phosphate-transport activity from bovine kidney brush-border membranes. The phosphate transporter appears to be particularly sensitive to extraction conditions. To prevent its inactivation, the phosphate carrier was solubilized in a buffer containing its substrates, Na+ and phosphate, CHAPS, dithiothreitol, brush-border membrane lipids and glycerol. The uptake of phosphate by reconstituted vesicles was strongly stimulated by the presence of a transmembrane Na+ gradient. This stimulation was abolished when the Na+ gradient was dissipated by monensin. The affinity of the carrier for phosphate was similar in proteoliposomes and in brush-border membrane vesicles (apparent Kt = 40 microM). The transporter was also stimulated by the presence of a high concentration of phosphate on the trans side of the membrane. The reconstituted transport activity was inhibited by arsenate, a known inhibitor of phosphate transport. However, the bovine phosphate carrier, intact or reconstituted, was much less sensitive to inhibition by phosphonoformic and phosphonoacetic acids than were those of other species studied so far. SDS/PAGE revealed that only a small number of brush-border membrane proteins were incorporated into the proteoliposomes. This reconstitution procedure should be useful for the purification and identification of the carrier protein.

Animals↗

PubMed1991

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.

Bacillus subtilis↗

PubMed1991

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↗

PubMed1991

Volume regulation and intracellular calcium in the rabbit proximal convoluted tubule.

The hypothesis that an increase of calcium leads to activation of calcium-activated ionic conductances during cell swelling was examined in the isolated perfused proximal convoluted tubule of the rabbit. Reduction of bath and luminal osmolality by 90 mosmol/kgH2O caused the cells to swell by 23.6 +/- 1.5% (n = 5) and intracellular calcium to rise from 227 +/- 35 to 347 +/- 60 nM (n = 6). Both these increases were transient, with volume decreasing to 5.5 +/- 1.2% above control and intracellular calcium concentration decreasing to 272 +/- 46 nM after 5-9 min. The addition of glucose and alanine to the tubule lumen to increase transcellular sodium transport caused a sustained increase in cell volume of 15.6 +/- 3.4% (n = 4). In parallel experiments, no significant increase in intracellular calcium concentration was observed. Addition of 1 microM of the calcium ionophore, ionomycin, reversibly increased intracellular calcium by 224 +/- 60 nM from a control value of 301 +/- 29 nM (n = 7) and reversibly depolarized the basolateral membrane by 3.6 +/- 0.9 mV (n = 5). However, there was no initial increase in the apparent transference number for potassium or chloride and no significant change in cell volume. We conclude from these observations that the sustained increase in basolateral potassium conductance observed when cells are swollen by hypotonicity or increased sodium transport (J. S. Beck and D. J. Potts. J. Physiol. Lond. 425: 369-378, 1990) is not due to a calcium-activated potassium conductance.

Alanine↗

PubMed1991

Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule.

The effect of alterations in sodium transport on cell ATP content and pH in the isolated perfused proximal convoluted tubule (PCT) of the rabbit was examined. Stimulating sodium transport by the addition of luminal glucose and alanine decreased cell ATP from 4.44 +/- 0.93 to 2.69 +/- 0.62 mM (n = 4), increased intracellular pH by 0.13 +/- 0.02 (n = 7), and increased cell volume by 0.10 +/- 0.02 nl/mm (n = 4). Blocking the sodium pump with 10(-4) M strophanthidin in tubules in which sodium transport had been stimulated increased cell ATP from 2.04 +/- 0.24 to 2.42 +/- 0.32 mM (n = 6). In parallel experiments the same dose of strophanthidin depolarized the basolateral membrane from -52.6 +/- 1.9 to -6.4 +/- 1.6 mV, depolarized the transepithelial potential from -3.2 +/- 0.3 to -0.1 +/- 0.1 mV, and reduced the basolateral membrane potassium transference number from 0.47 to 0.26 indicating a reduction in basolateral potassium conductance. Since strophanthidin caused a cell alkalinization of 0.15 +/- 0.03, this latter effect cannot be due to changes of intracellular pH. Strophanthidin caused no change in cell volume over the period studied, suggesting that stretch-activated potassium channels are not involved either. Instead, potassium conductance inhibition may be the result of the closure of ATP-sensitive potassium channels. These same channels might thus be partly responsible for the increase in potassium conductance commonly observed during stimulation of sodium transport.

Adenosine Triphosphate↗

PubMed1986

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes.

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.

Bacterial Outer Membrane Proteins↗