The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin-2 (AQP2)-bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP-independent Ca 2+ increases nor by AVP-induced Ca 2+ increases. However, clamping cytosolic Ca 2+ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2-bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca 2+ in the AQP2 shuttle.
To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176–30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/PAB = 1/PA + 1/PB (Eq. l), where PAB is the permeability of a bilayer composed of leaflets A and B, PA is the permeability of leaflet A, and PB is the permeability of leaflet B. Using for the MDCK leaflet–specific liposomes gives an estimated value for the osmotic water permeability (Pf) of 4.6 × 10−4 cm/s (at 25°C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711–1718). At 36°C, Pf was 3.44 ± 0.35 × 10−3 cm/s for symmetrical inner leaflet membranes and 3.40 ± 0.34 × 10−4 cm/s for symmetrical exofacial membranes. From , the estimated permeability of an asymmetric membrane is 6.2 × 10−4 cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 ± 0.7 × 10−4 cm/s, which is within 10% of the calculated value. Direct experimental measurement of Pf for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of .
The effects of the organic calcium channel blocker verapamil and the beta-receptor blocker propranolol on dipole (phi(d)) and surface (phi(s)) potentials of bilayer lipid membranes were studied. The boundary potentials (phi(b)= phi(d) + phi(s)) of black lipid membranes, monitored by conductance measurements in the presence of nonactin and by capacitive current measurements were compared with phi(s) calculated from the electrophoretic mobility of lipid vesicles. It was shown that the increase of boundary potential, induced by the adsorption of the positively charged propranolol, was caused solely by an increase in surface potential. Although phi(s) also increases due to the adsorption of verapamil, phi(b) diminishes. A sharp decrease of the dipole potential was shown to be responsible for this effect. From Langmuir adsorption isotherm the dissociation constant Kd of verapamil was estimated. The uncharged form of verapamil (Kd=(0.061+/-0.01) mM at pH 10.5) has a tenfold higher affinity to a neutral bilayer membrane than the positively charged form. The alteration of membrane dipole potential due to verapamil adsorption may have important implications for both membrane translocation and partitioning of small or hydrophobic ions and charged groups of membrane proteins.
Cytochrome c was immobilized on screen-printed thick-film gold electrodes by a self-assembly approach using mixed monolayers of mercaptoundecanoic acid and mercaptoundecanol. Cyclic voltammetry revealed quasi-reversible electrochemical behavior of the covalently fixed protein with a formal potential of +10 mV vs. Ag/AgCl. Polarized at +150 mV vs. Ag/AgCl the electrode was found to be sensitive to superoxide radicals in the range 300-1200 nmol L(-1). Compared with metal needle electrodes sensitivity and reproducibility could be improved and combined with the easiness of preparation. This allows the fabrication of disposable sensors for nanomolar superoxide concentrations. By changing the electrode potential the sensor can be switched from response to superoxide radicals to hydrogen peroxide-another reactive oxygen species. H(2)O(2) sensitivity can be provided in the range 10-1000 micromol L(-1) which makes the electrode suitable for oxidative stress studies.
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