Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

Active Ca2+ reabsorption in the proximal tubule of the rat kidney

Summary Using the stop flow microperfusion technique with simultaneous capillary perfusion the rate of active Ca²⁺ reabsorption was evaluated by measuring the static head electrochemical potential difference as well as the permeability of the tubular wall for Ca²⁺ ions. Under control conditions the active Ca²⁺ transport was calculated to be 3.35×10^–13 mol/cm·s. It declined toward zero if the ambient Na⁺ was replaced by choline or lithium. Parallel experiments in the golden hamster showed that active Ca²⁺ transport, vanished completely if active Na⁺ transport was blocked by ouabain (1 mM). These data indicate that the active Ca²⁺ reabsorption from the proximal tubule depends on the active reabsorption of Na²⁺ presumably via a Na⁺–Ca²⁺ countertransport at the contraluminal cell membrane. The static head electrochemical potential difference of Ca²⁺ is the same in late and early proximal tubules. It is also not affected by the presence of acetazolamide (10^–4 M) by the absence of bicarbonate or glycodiazine buffer or by the absence or presence of phosphate (2 mM).     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.     

Stimulation of NaCl reabsorption by antidiuretic hormone in the cortical thick ascending limb of Henle's loop of the mouse

The effect of antidiuretic hormone (ADH) on transepithelial Na⁺, Cl^–, Ca²⁺ and Mg²⁺ net fluxes (J_Na, J_Cl, J_Mg, J_Ca) was investigated in isolated perfused cortical thick ascending limb segments (cTAL) of the mouse nephron, using the microperfusion technique and the electron microprobe analysis to determine the ionic composition of the collected tubular fluid. Simultaneously, the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were recorded. Prior to the flux measurements cTAL segments were perfused for one hour. During this equilibration period PD_te decreased significantly from +19.9±1.6 to +14.9±1.l mV and R_te increased from 30.6±3.5 cm² to 38.8±2.4 cm² (n=7), reflecting a decline in NaCl transport. After ADH was added to the bath solution at 10^–10 mol.l^–1, PD_te increased from +14.4±1.1 to +18.0±1.5 mV, accompanied by a rise in J_Na and J_Cl from 205±11 to 273±19 and from 216±12 to 283±21 pmol.min^–1.mm^–1 (n=7), respectively. J_Ca and J_Mg also increased from 0.81±0.07 to 1.50±0.12 and from 0.43±0.11 to 0.76±0.08 pmol.min^–1.mm^–1 (n=7), respectively. All these effects were fully reversible after withdrawal of the hormone. In conclusion our data indicate that ADH stimulates divalent cation transport and NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse.     

Tizolemide-induced changes of passive transport components across the basolateral membrane of isolated frog skin

The effect on transepithelial Na transport of tizolemide was investigated in isolated frog skin (Rana temporaria). It was found that tizolemide (2–5 mM, serosal side) decreased transepithelial Na transport (measured as short circuit current and as net sodium flux) within 60 min to 25–40% of the control level resulting from reduction of the unidirectional sodium influx. Intracellular recording with microelectrodes revealed that these changes were associated with depolarization of the intracellular space to less than 40% of the control values (averaging –71.7±5.1 mV) which is a consequence of a decrease in conductance of the basolateral border to about 25% of the control values. The conductance of the apical border was only slightly reduced. It is suggested that tizolemde blocks the partial conductance of potassium at the basolateral border which secondarily diminishes trans-epithelial Na transport due to a decrease of the driving force for apical border Na entry. A certain degree of inhibition of the Na-K-ATPase by tizolemide cannot be excluded. When vasopressin (ADH) was added to frog skin after treatment with tizolemide, the response was markedly reduced compared to that of untreated control preparations. Under these conditions, the conductance of the basolateral border increased while the apical border remained little influenced by the hormone — opposite to the response of frog skins under control conditions. It is concluded that the mode of action of ADH is more complex than has been recognized hitherto and includes effects at the basolateral border.Dedicated to Professor H. Schwiegk on the occassion of his 75th birthday.Some of these results have been presented at the 8. Int. Congr. Néphrology, Athens 1981.     

Contraluminal sulfate transport in the proximal tubule of the rat kidney

In order to study contraluminal sulfate transport the influx rate of³⁵SO ₄ ^2– from the interstitium into cortical tubular cells has been determined. Preloading of the rat with sulfate augmented contraluminal³⁵SO ₄ ^2– influx; preperfusion with sulfate-free solutions diminished it. The contraluminal³⁵SO ₄ ^2– influx in sulfate-loaded animals followed two parameter kinetics (K _m 1.4 mmol/l,J _max 1.2 pmol·s^–1·cm^–1). The contraluminal³⁵SO ₄ ^2– influx (starting concentration 10 mol/l) did not change when the K⁺ concentration was varied between 4 and 40 mmol/l and the Ca²⁺ concentration from zero to 3 mmol/l. Omission of Na⁺ from the perfusates augmented contraluminal³⁵SO ₄ ^2– influx markedly. The increase is larger at pH 6 than at pH 7.4. Changes of pH affect contraluminal³⁵SO ₄ ^2– influx only when the solutions are Na⁺- and K⁺-free. Under these conditions the³⁵SO ₄ ^2– influx decreased when the ambient pH was raised from pH 6.0 to pH 8.0. Thiosulfate, selenate, molybdate, oxalate, phosphate, arsenate, and bicarbonate exerted competitive inhibition, while formate, 2-oxoglutarate and paraaminohippurate showed a biphasic response: inhibition at 50 mmol/l, no inhibition at 150 mmol/l. Chloride and bicarbonate inhibited³⁵SO ₄ ^2– influx at 10 mol/l³⁵SO ₄ ^2– , but augmented sulfate influx at 5 mmol/l³⁵SO ₄ ^2– concentration in rats not preloaded with sulfate. The data indicate the presence of a contraluminal sulfate transport system which is shared by a variety of inorganic and organic anions. The biphasic behaviour of some anions suggests parallel pathways leading to a cis-inhibition at small and trans-stimulation at high anion concentrations. Na⁺ and H⁺ may be cotransported or interact with the transport system at a modifier site.     

Postnatal expression of transport proteins involved in acid–base transport in mouse kidney

The kidney plays a major role in maintaining and controlling systemic acid–base homeostasis by reabsorbing bicarbonate and secreting protons and acid-equivalents, respectively. During postnatal kidney development and adaptation to changing diets, plasma bicarbonate levels are increasing, the capacity for urinary acidification maturates, and the final morphology and distribution of intercalated cells is achieved. In adult kidney, at least two types of intercalated cells (IC) are found along the collecting duct characterised either by the expression of AE1 (type A IC) or pendrin (non-type A IC) where non-type A IC are found only in the convoluted distal tubule, connecting tubule and cortical collecting duct. Here we investigated in mouse kidney the relative mRNA abundance, protein expression levels and distribution of several proteins involved in renal acid–base transport, namely, the Na⁺/HCO₃ ^– cotransporter NBC1 (SLC4A4), the Na⁺/H⁺-exchanger NHE3 (SLC9A3), two subunits of the vacuolar H⁺-ATPase [ATP6V0A4 (a4), ATP6V1B1 (B1)], the Cl^–/HCO₃ ^– exchangers AE1 (SLC4A1) and pendrin (SLC26A4). Relative mRNA abundance of all transport proteins was lowest at day 3 after birth and increased thereafter in parallel with protein levels. The numbers of type A and non-type A IC in the cortical collecting duct (CCD) increased from day 3 to days 18 and 24, whereas the number of IC in the CCD with apical staining for the vacuolar H⁺-ATPase subunits a4 and B1 decreased from day 3 to days 18 and 24, respectively. In addition, cells with characteristics of non-type A IC (pendrin expression, basolateral expression of vacuolar H⁺-ATPase subunits) were found in the inner and outer medulla 3 days after birth but were absent from the medulla of 24-day-old mice. Taken together, these results demonstrate massive changes in mRNA and protein expression levels of several acid–base transporters during postnatal kidney maturation and also show changes in intercalated cell phenotype in the medulla during these processes.