Localization of severa G-protein subunits to the apical and basolateral membranes of cortical tubular cells from the rat kidney

Dopamine was shown to affect Na⁺, K⁺-ATPase activity in basolateral membranes of the rat kidney via a pertussis toxin dependent mechanism. In order to examine if some form of pertussis toxin sensitive G-protein is present exclusively in the basolateral membrane of the rat renal cortex we examined the G-protein composition of both apical and basolateral membrane vesicles. Western blots showed an essentially uniform distribution of G_α total, G_αs and Gβ over the two membranes. G_o could not be detected with western blot technique in the vesicle preparations. By contrast, the distribution of ADP-ribosylation with the bacterial toxins pertussis toxin and cholera toxin depended on the amount of detergent in the assay and perhaps other factors, and thus could not be used to evaluate the relative amounts of G-protein subunits. Thus, in contrast to the situation in cultured renal cells, unequal distribution of receptor and G-protein substrates is apparently not paralleled by an unequal distribution of the detected forms of G-proteins under physiological conditions.     

Interleukin-8 secretion of cortical tubular epithelial cells is directed to the basolateral environment and is not enhanced by apical exposure to Escherichia coli

In upper urinary tract infections, tubular epithelial cells (TEC) may play a pivotal role in the initiation of the renal inflammatory response. They exert crucial immunological functions such as processing and presentation of foreign antigen, secretion of proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha) and chemokines (IL-8, MCP-1, ENA-78, and RANTES). Since monolayer cultures are a limited model for polarized tubular epithelial cells, we studied the side-dependent IL-8 secretion of TEC by using cell culture inserts as a basement membrane imitation. Primary cultures of proximal TEC were stimulated with differently fimbriated mutants of Escherichia coli, E. coli LPS, S-fimbria isolates, and IL-1alpha. IL-8 protein was measured by enzyme-linked immunosorbent assay, and IL-8-like biological activity was tested by measuring elastase release from polymorphonuclear cells in supernatants of the upper and lower compartments. IL-8 mRNA was compared by competitive PCR. IL-8 secretion by TEC into the basolateral environment was significantly higher than secretion into the apical compartment, representing the tubular lumen. However, stimulation of IL-8 secretion by TEC was restricted to IL-1alpha and was not inducible by E. coli mutants, S fimbriae, or lipopolysaccharide. With this in vitro model of polarized TEC, we show that luminal contact of TEC with uropathogenic E. coli does not result in enhanced IL-8 secretion. The basolaterally directed production of the neutrophil chemotactic factor IL-8 by TEC after stimulation with IL-1alpha might play an important role in the initiation of inflammatory cell influx into the renal parenchyma.     

Single channel recordings from basolateral and apical membranes of renal proximal tubules

A new method is described, which enables the recording of single ionic channels from the basolateral as well as the luminal membrane of renal proximal tubules with the patch-clamp technique. Segments of late proximal tubules of rabbit kidney are cannulated and perfused from one end. The other end is open and freely accessible to a patch pipette. The patch electrode can be moved against lateral cell membranes or can be inserted through the open end into the lumen and brought to contact with the brush-border membrane. Both, in the basolateral as well as in the luminal membrane, giga seals can be achieved. In both membranes, K⁺ selective channels could be identified.Part of this work was presented at the 59th Meeting of the Deutsche Physiologische Gesellschaft, March 1984 in Dortmund.     

Effects of ADH on the apical and basolateral membranes of toad urinary bladder epithelial cells

Short-circuited urinary bladders from Bufo marinus were supported on their apical surface by an agar mounting method and impaled with microelectrodes via their basolateral membrane. This arrangement provided stable and long-lasting impalements of epithelial cells and yielded reliable membrane potentials and voltage divider ratios (R _a/R _b), where R _a and R _b are apical and basolateral membrane resistances respectively. The membrane potential under short-circuit conditions (V _sc) was –51.4±2.2 mV (n=59), while under open-circuit conditions apical membrane potential (V _a) and basolateral membrane potential (V _b) were –31.0±2.4 and 59.5±2.4 mV, respectively. This yields a well-shaped potential profile across the toad urinary bladder, where V _a is inversely related to the rate of transport, I _sc. Antidiuretic hormone (ADH) produced a hyperpolarisation of V _sc and V _b but had no significant effect on V _a. In addition, R _a/R _b was significantly increased by ADH (4.6±0.5 to 10.2±3.6). Calculation of individual membrane resistances following the addition of amiloride showed that ADH produced a parallel decrease in R _a and R _b membrane resistance, with the observed increase in R _a/R _b being due to a greater percentage decrease in R _b than in R _a. The ability of ADH to effect parallel changes in apical and basolateral membrane conductance helps to maintain a constant cellular volume despite an increase in transepithelial transport.     

Transfer of base across the basolateral membrane of cortical tubules of rat kidney

The transfer of HCO ₃ ^– /OH^– across the peritubular membrane of rat cortical tubules was studied by measuring capillary pH during stopped-flow microperfusion of peritubular capillaries with electrolyte solutions containing 3 mM HCO ₃ ^– . The rate of alkalinization of these solutions was significantly delayed when 10^–4 M acetazolamide, 5×10^–4 M SITS or 2 mM Ba²⁺ were added, as well as when chloride was substituted by gluconate. Under these conditions, stationary capillary pH was slightly but significantly increased. In another series, the lumen of proximal tubules was perfused with alkaline solutions while pH was measured in adjacent capillaries. During perfusion, capillary pH rose to a level 0.4 units higher than in free-flow conditions, returning after filling the lumen with oil; the rate of capillary pH return to baseline is a measure of base extrusion from cells, in the absence of influx from the lumen. This rate is also significantly delayed by acetazolamide. The data show that peritubular base extrusion is dependent on carbonic anhydrase, on basolateral membrane voltage, and on interstitial chloride, and delayed by the anion exchange inhibitor SITS; they are compatible with both basolateral HCO ₃ ^– /Cl^– exchange and conductive bicarbonate transfer.Supported by APESP, CNP_q, FINEP and PNUD-UNESCOSupported by CNPq, BrasilSupported by Conicyt, Argentina.     

Phosphorylation of rat kidney proximal tubular brush border membranes

A possible correlation between cyclic-AMP dependent protein phosphorylation and altered sodium dependent transport of inorganic phosphate was analyzed in isolated rat renal proximal tubular brush border membrane vesicles. In transiently opened vesicles (opened by an osmotic shock), the addition of gamma-32P-ATP leads to 32P-incorporation into several membrane proteins. The simultaneous addition of cyclic-AMP leads to increased phosphorylation of several proteins (e.g. apparent molecular weights: 40 kD, 46 kD, 55 kD). The addition of ATP, GTP and ITP to the osmotic shock medium leads to an (non-specific) inhibition of the sodium gradient dependent phosphate uptake. No further inhibition of the sodium dependent phosphate transport was observed when membrane vesicles were phosphorylated by ATP in the presence of cyclic-AMP. These data show a lack of correlation between cyclic-AMP dependent protein phosphorylation and altered sodium gradient dependent phosphate transport. Thus, there is no experimental support for the involvement of cyclic-AMP dependent protein phosphorylation as one of the final events in the regulation of phosphate transport across the rat renal proximal tubular brush border membrane.     

Fluidity of brush border and basolateral membranes from human kidney cortex

The physical state of lipids in brush border and basolateral membrane vesicles prepared from normal human kidney cortex was investigated by fluorescence polarization and electron spin resonance. At physiologic temperature, lipids were significantly less ordered, i.e., more fluid, in basolateral than in brush border membranes. This difference was also observed using corresponding liposomes made from total lipid extracts. For both brush border and basolateral membranes, temperature-dependent experiments revealed the existence of a broad thermotropic transition extending approximately from 20 to 42 degrees C. These data are interpreted to indicate that plasma membranes from human kidney cortex function physiologically at the upper critical temperature of a transition that probably corresponds to a liquid crystalline-to-gel lipid phase separation.     

Asymmetry in the transport of lactate by basolateral and brush border membranes of rat kidney cortex

The uptake ofl(+)lactate into rat renal cortical brush border (BBV) and basolateral (BLV) membrane vesicles, isolated through differential centrifugation and free flow electrophoresis, were studied using a rapid filtration technique. In contrast to the lactate transport into the BBV, that into the BLV: 1) was found to proceed only towards equilibrium, 2) showed Na⁺-independent coupling of the influx ofl(+)lactate and the efflux ofl(+) but not to the efflux ofd(–)lactate, 3) was not inhibited byd(–)lactate, 2-thiolactate or 3-phenyl-lactate, but 4) was inhibited by 3-thiolactate and -hydroxybutyrate and 5) was accelerated by changes in inwardly directed ionic gradients or by increases in cation conductance both of which led to increased intravesicular positivity. The latter changes had the opposite effect on the uptake ofl(+)lactate by BBV. Thus, while thel(+)lactate transport system present in BBV showed the characteristics of Na-dependent electrogenic cotransport system, that in the BLV was consistent with a carrier mediated Na-independent, facilitated diffusion system.     

Localization of tubular uptake segment of filtered Cystatin C and Aprotinin in the rat kidney

Aim: The renal tubular uptake of ¹²⁵I‐Aprotinin (*Ap) is on average located more superficially than its filtration site, causing transfer of some of *Ap filtered in deep to more superficial cortical zones. ¹²⁵I‐Cystatin C (*Cy) showed less uptake in deep cortical zones than Ap, suggesting a longer and/or a more superficial tubular uptake site. To test that hypothesis and to quantify the outward transfer of the filtered polypeptides, we estimated the tubular uptake pattern of the tracers in perfusion fixed rat kidneys after intravenous injection of *Cy and *Ap. Methods: Autoradiographs were made from 10 μm thick slices of Microfil nephron casts from outer (OC), middle (MC) and inner (IC) cortical zones to quantify cortical border‐crossing *Ap transfer. Single nephron glomerular filtration rate (snGFR) was estimated as the zonal uptake of *Ap corrected for *Ap transfer, divided by its time‐integrated plasma concentration and the zonal number of glomeruli. Results: *Ap and *Cy uptake fell exponentially along the proximal convoluted tubule (PCT), indicating an uptake proportional to luminal concentration. Uptake in IC exceededx that in MC and OC nephrons. The per cent PCT length with *Cy uptake (67.2 ± 1.6) exceeded that of *Ap (54.6 ± 1.8). The zonal border‐crossing PCT length (29–34% of total PCT) from deep to more superficial cortical zones transferred 4–6% more *Cy than *Ap. Conclusion: Greater tubular uptake length of *Cy than of *Ap causes more cortical border‐crossing of *Cy. The zonal snGFR estimated from Aprotinin uptake corrected for border‐crossing agreed well with that obtained with the Hanssen ferrocyanide technique.     

High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen

Using the patch clamp technique, one type of K⁺ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K⁺ over Na⁺ (typically 30-70-fold) and had a conductance of 180, SD±39 pS (n=6), referred to a situation of 140 mmolar K⁺-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba²⁺ (5 mmol/l) and partially inhibited by Na⁺. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na⁺ competed with K⁺ for channel entrace from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca²⁺ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K⁺ secretion to Na⁺ absorption in the fine regulation of cation excretion in renal collecting ducts.     

The intracellular chloride activity of rat kidney proximal tubular cells

The intracellular Cl^? activity was determined in rat kidney proximal tubular cells in vivo, using single-barreled Cl^? sensitive microelectrodes filled with Corning no. 477913 liquid ion exchanger resin to measureV _Cl and using — in separate experiments — conventional KCl-filled microelectrodes to measure the membrane potential,V _m. After correction for interference from other anions onV _Cl the intracellular Cl^? activity averaged 13.1 mmol·l^?1 SD±4.5 mmol·l^?1 (n=96). This value is approximately two-fold higher than the intracellular equilibrium activity which can be calculated from the extracellular Cl^? activity of 90–103 mmol·l^?1 andV _m of ?71.2 mV, SD±4.9 mV (n=23) to amount to 6.3 to 6.7 mmol·l^?1. Since both cell membranes are permeable for Cl^? ions, as concluded from luminal and/or peritubular Cl^? substitution experiments, we conclude that the cellular Cl^? accumulation above equilibrium results from transcellular active Cl^? transport, the detailed mechanism of which is presently not known. From the slow decline of intracellular Cl^? concentration after substitution of luminal Cl^? by gluconate, however, we deduce that transcellular Cl^? absorption is of minor importance in surface tubules of rat kidney under free flow and that the major part of transtubular Cl^? flux is passive and paracellular.     

Induced development of proximal tubular NaKATPase,basolateral cell membranes and fluid reabsorption

We have previously demonstrated that adrenal corticoid hormones (ACH) induce NaKATPase activity in immature proximal tubular (PT) cells in rats. We have now determined the effect of betamethasone (beta) and aldosterone (aldo) on the size of PT basal and lateral cell membranes (BLM), PT fluid reabsorption (J v (a)) measured in vivo with the split drop technique and PT NaKATPase activity in 20-day-old (young) and 40-day-old (adult) rats. Serum levels of ACH were the same in young and in adult rats, but adrenalectomy caused a significantly larger fall in NaKATPase activity in young than in adult rats. BLM surface area (μm²/μm^-3 cell volume), J v (a) and NaKATPase were significantly lower in young than in adult rats. Three-day treatment with high doses of beta (60 μg· 100 g^-1 or aldo (40 μg· 100 g^-1) significantly increased BLM surface area, J v (a) and NaKATPase activity in young but not in adult rats. Three days after adrenalectomy, J v (a) was significantly depressed in adult rats. There was a significant correlation between the ACH-dependent changes in NaKATPase activity and J v (a). Short-term treatment (2–3 hours) with high doses of ACH did not significantly increase NaKATPase activity and J v (a) in young rats.     

Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles

Basolateral membrane vesicles were isolated by a Percoll density gradient centrifugation method from small intestinal and renal proximal tubular epithelial cells. Transport of sulphate across the basolateral membrane was analyzed by measuring the uptake of tracer sulphate.In both membrane preparations, preloading the vesicles with sulphate-or hydroxyl-anions stimulated tracer sulphate uptake (trans-stimulation); an inwardly directed sodium gradient did not stimulate sulphate influx whether in the absence or in the presence of sulphate- or hydroxyl-iontrans-stimulation. Under sulphate trans-stimulation conditions, DIDS (10^–4 mol/l) inhibited sulphate influx.In intestinal membranes, trans-stimulation of sulphate influx was obtained by preloading the vesicles with chloride, in renal membranes by preloading with bicarbonate. Under sulphate trans-stimulation conditions, in intestinal membranes, sulphate influx was strongly inhibited by chloride, in renal membranes, chloride inhibition was absent. Under bicarbonate trans-stimulation conditions, in renal membranes, sulphate transport was inhibited by lactate.It is concluded that small intestinal and renal proximal tubular basolateral membrane vesicles contain a transport mechanism for sulphate that cannot be energized by a sodium gradient. The transport system in small intestinal basolateral membranes seems to be different from that in renal membranes. It is suggested that the observed interaction between inorganic and organic anion transport in renal basolateral membranes is indirect.Abbreviations DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - MES N-morpholinoethanesulfonic acid - PAH p-aminohippuric acid - PMSF phenylmethylsulfonyl fluoride - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate - TMA tetramethylammonium - Tris tris(hydroxymethyl)aminomethane     

Differential expression of glycine receptor subunits in the rat basolateral and central amygdala

The amygdalar complex is a limbic structure that plays a key role in emotional processing and fear conditioning. Although inhibitory transmission in the amygdala is predominately GABA-ergic, neurons of the amygdala are also known to express glycine receptors. The subtype and function of these glycine receptors within the synaptic circuits of the amygdala are unknown. In this study, we have investigated the relative expression of the four major glycine receptor subunits (α1–3 and β) in the rat basolateral (BLA) and central amygdala (CeA), using real-time PCR and protein biochemistry. We demonstrate that α1, α2, α3, and β subunits are all expressed in the BLA and CeA with α2 being the predominant α-subunit in both nuclei. Electrophysiological recordings from BLA and CeA neurons in acute brain slices indicated that differences in relative expression of these subunits were correlated with the pharmacological properties of native glycine receptors expressed on these neurons. We conclude that glycine receptors assembled in BLA neurons are largely α1β-containing heteromultimers whereas receptors assembled in neurons of the central amygdala are primarily α2β-, α3β- or α1β-containing heteromultimers, with a minor component of α2 or α3 homomeric receptors also expressed.     

Release of urodilatin from perfused rat kidney and from cultured neonatal rat kidney cells

Release of rat urodilatin (rURO) from isolated perfused rat kidneys and neonatal rat kidney cells could be demonstrated by a specific competitive radioimmunoassay (rURO-RIA) using [¹²⁵I]rURO as the competitive antigen and an antiserum against the hypothetical rURO-N-terminus, Ala-Gly-Pro-Arg, as concluded from the amino acid sequence of the rat prohormone CDD/ANP-1-126. This antiserum did not react with synthetic rCDD/ANP-99-126, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), or human URO (hURO). rURO could be demonstrated in the urine of the perfused rat kidney after an equilibration period of 20 min. After an initial slight decrease in the second 20 min, rURO production remained at almost the same level during the perfusion time of 100 min. A total of 470 fmol · 10 min^–1 · g^–1 kidney rURO was produced within 80 min. rURO was also produced by neonatal rat kidney cells kept in serum-free Dulbecco's modified Eagles medium. The production of rURO depended on the cultivation time of the cells. It increased up to 3 days reaching 239 ±7.5 fmol-h^–1 · g^–1 protein, afterwards it decreased rapidly. The results obtained indicate that the rat kidney produces a peptide of the type A family of natriuretic peptides, which very likely represents the putative rURO.     

Determination of intracellular K+ activity in rat kidney proximal tubular cells

The intracellular K⁺ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K⁺-sensitive microelectrodes, which were filled with a liquid K⁺-exchanger resin that has also a small sensitivity to Na⁺. Both electrodes had tip diameters of 0.2 m or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was –76.3±8.1 mV (n=81) and the potential difference measured with the K⁺-sensitive microelectrode was –7.2±5.8 mV (n=32). Based on the activity of K⁺ in the extracellular fluid of 3 mmol/l the intracellular K⁺ activity was estimated to be 82 mmol/l. Assuming equal K⁺-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K⁺ concentration is 113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K⁺-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K⁺ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K⁺ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na⁺/K⁺-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.     

K+ channels in the basolateral membrane of rat cortical collecting duct

Impalement studies in isolated perfused cortical collecting ducts (CCD) of rats have shown that the basolateral membrane possesses a K⁺ conductive pathway. In the present study this pathway was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells with the conventional cell-free, cell-attached and the cell-attached nystatin method. Two K⁺ channels were found which were highly active on the cell with a conductance of 67±5 pS (n=18) and 148±4 pS (n=21) with 145 mmol/l K⁺ in the pipette. In excised patches the first channel had a conductance of 28±2 pS (n=15), whereas the second one had a conductance of 85±1 pS (n=53) at 0 mV clamp voltage with 145 mmol/l K⁺ on one side and 3.6 mmol/l K⁺ on the other side of the membrane. So far it has not been possible to characterize the smaller channel further. Excised, and with symmetrical K⁺ concentrations of 145 mmol/l, the intermediate channel had a linear conductance of 198±19 pS (n=5). After excision in the inside-out configuration the open probability (P _o) of this channel was low (0.18±0.05, n=13) whereas in the outside-out configuration this channel had a threefold higher P _o (0.57±0.04, n=12). Several inhibitors were tested in excised membranes. Ba²⁺ (1 mmol/l), tetraethylammonium (TEA⁺, 10 mmol/l) and verapamil (0.1 mmol/l) all blocked this channel reversibly. Furthermore P _o was reversibly reduced by 10 nmol/l charybdotoxin (outside-out). This K⁺ channel of the basolateral membrane was regulated by cellular pH. P _o was reduced to 26±3% at pH 6.5 (n=6) and increased to 216±18% at pH 8.5 (n=7) compared to pH 7.4. Half-maximal inhibition was reached at pH 7.0. The channel had its highest P _o at a Ca²⁺ activity of less than 10^–8 mol/l (n=13). Increasing the Ca²⁺ activity to 1 mmol/l on the cytosolic side of the membrane resulted in a reduction of P _o to 13±3% (n=11). Half-maximal inhibition was reached at a Ca²⁺ activity of 10^–5 mol/l. The high activity of both K⁺ channels of the basolateral membrane on the cell indicates that they may serve for K⁺ recirculation across the basolateral membrane.     

Membrane fluidity and enzyme activities in brush border and basolateral membranes of the dog kidney

The physical state of membrane lipids and relationships with the activity of Na+-K+-ATPase and alkaline phosphatase were studied in basolateral and brush border membranes of the dog kidney. Fluorescence polarization and electron spin resonance experiments demonstrate that basolateral membranes are much more fluid than brush border membranes. This can be accounted for by a difference in fluidity of the lipid part of the membranes. Broad (43-17 degrees C) thermotropic transitions are observed in liposomes made from total lipid extracts of brush border and basolateral membranes. Fluorescence data strongly suggest that thermotropic transitions also occur in intact membranes and that a change in membrane physical state may take place around the physiological temperature. A nonlinear Arrhenius plot for the Na+-K+-ATPase activity in basolateral membranes (breakpoint 21 degrees C) provides additional support for the existence of a lipid liquid leads to gel transition in antiluminal plasma membranes. A break in the Arrhenius plot of alkaline phosphatase activity is also observed but at a temperature significantly higher (26 degrees C) than that of the end of the thermotropic transition. "Room temperature" appears as a critical zone for lipid physical state and activities of both enzymes.