Mireille Marsolais

Role of NHERF1, Cystic Fibrosis Transmembrane Conductance Regulator, and cAMP in the Regulation of Aquaporin 9

Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP.

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pHi) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K+ and Na+ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pHi depends strongly on the activity of a K+/H+ exchanger. In the absence of toxin, removal of K+ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pHi equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pHi. B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H+, K+ and Na+.

Segmental Expression of the Bradykinin Type 2 Receptor in Rat Efferent Ducts and Epididymis and Its Role in the Regulation of Aquaporin 9

Abstract Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

Hypertonicity Decreases Basolateral K+ and Cl− Conductances in Rabbit Proximal Convoluted Tubule

Abstract. Collapsed proximal convoluted tubules (PCT) shrink to reach a volume 20% lower than control and do not exhibit regulatory volume increase when submitted to abrupt 150 mOsm/kg hypertonic shock. The shrinking is accompanied by a rapid depolarization of the basolateral membrane potential (VBL) of 8.4 ± 0.5 mV, with respect to a control value of −54.5 ± 1.9 mV (n= 15). After a small and transient hyperpolarization, VBL further depolarizes to reach a steady depolarization of 19.5 ± 1.5 mV (n= 15) with respect to control. In the post-control period, VBL returns to −55.8 ± 1.5 mV. The basolateral partial conductance to K+ (tK) which is 0.17 ± 0.01 (n= 5) in control condition, decreases rapidly to nonmeasurable values during the hypertonic shock and returns to 0.23 ± 0.03 in the post-control period. The basolateral partial conductance to Cl− (tCl), which is 0.05 ± 0.02 (n= 5) in control, also decreases in hypertonicity to a nonmeasurable value and returns to 0.03 ± 0.01 in post control. The partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3), which is 0.48 ± 0.06 (n= 5) in control condition, remains the same at 0.44 ± 0.05 (n= 5) during the hypertonic period. Similarly, the membrane absolute conductance mediated by the Na-HCO3 cotransporter (GNa-HCO3) does not vary appreciably. Concomitant with cell shrinkage, intracellular pH (pHi) decreases from a control value of 7.26 ± 0.01 to 7.13 ± 0.02 (n= 12) and then remains constant. Return to control solution brings back pHi to 7.28 ± 0.03. From these results, we conclude that in collapsed PCT, a sustained decrease in cellular volume leads to cell acidification and to inhibition of K+ and Cl− conductances.

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca.

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 degrees C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.

Dibutyryl Cyclic Adenosine Monophosphate Stimulates the Sodium Pump in Rabbit Renal Cortical Tubules

The elevation in oxygen consumption (QO2) observed following addition of the sodium ionophore nystatin in suspensions of rabbit renal proximal tubules was significantly increased by 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The QO2 after subsequent addition of strophanthidin to block the sodium pump was unaffected by db-cAMP. However, 10 microM forskolin in the presence of 100 microM IBMX had no significant effect on the QO2 observed following addition of either nystatin or strophanthidin. Nevertheless, we can conclude that db-cAMP does stimulate the sodium pump activity independently of sodium transport mechanisms in the rabbit renal proximal tubule.

Effects of Mutations Within Surface-Exposed Loops in the Pore-Forming Domain of the Cry9Ca Insecticidal Toxin of Bacillus thuringiensis

The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.

Isolation of Single Mammalian Proximal Tubule Cells: Effects of Hypotonic Shocks on Cell Yield and Function

Nord et al. [Am J Physiol 1986; 250:F539-F550] proposed a method to give a high yield of proximal tubule cells by exposing a suspension of rabbit cortical tubules to a hypotonic shock in calcium-free media. The present study describes the effects of both amplitude and duration of the hypotonic treatment on some transport-related characteristics of individual cells as compared to the starting tubule suspension. The averaged cell yield increased by an order of magnitude when the osmolality of the hypotonic solution was varied in four steps from 200 (C200 cells) to 70 mosm/kg H2O (C70 cells) while the proportion of trypan blue-positive cells progressively decreased from 33% for C200 cells to 9.5% for C70 cells. An increase in duration of the hypotonic shock from 0.5 to 6 min did not change the cell yield of C200 cells while it significantly increased that of C70 cells by 61%. Basal and ouabain-sensitive oxygen consumption (QO2) increased by 57 and 155%, respectively, from C70 to C200 cells but was approximately one order of magnitude smaller than the QO2 measured for tubule suspension. Intracellular ATP content averaged 5.5 +/- 0.8 nmol/mg for the starting tubule suspension, 4.6 +/- 0.8 nmol/mg for C70 cells but only 1.3 +/- 0.1 nmol/mg for C200 cells. The maximal velocity for phloridzin-sensitive alpha-methyl glucose transport averaged 13.7 +/- 1.7 nmol min-1 mg-1 for C70 cells and only 6.3 +/- 1.3 nmol min-1 mg-1 for C200 cells which is approximately one order of magnitude smaller than what can be expected from a tubule presenting a good access to luminal membrane. We conclude from these results that, in the process of isolating individual cells from a polarized epithelium, membrane transport rates have decreased by one order of magnitude and this reduction is intensified by a large hypotonic shock. In comparison with C200 cells, the cells obtained with a large hypotonic shock give a high yield, a larger proportion of trypan blue-negative cells and their lower overall transport rate allows the cells to maintain a better electrochemical gradient for Na and a higher intracellular ATP level.