Uroplakins do not restrict CO2 transport through urothelium.

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (∼1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (∼7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (∼ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.

No facilitator required for membrane transport of hydrogen sulfide.

Hydrogen sulfide (H(2)S) has emerged as a new and important member in the group of gaseous signaling molecules. However, the molecular transport mechanism has not yet been identified. Because of structural similarities with H(2)O, it was hypothesized that aquaporins may facilitate H(2)S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and H(2)S flux. To measure H(2)O and H(2)S fluxes, respectively, sodium ion dilution and buffer acidification by proton release (H(2)S left arrow over right arrow H(+) + HS(-)) were recorded in the immediate membrane vicinity. Both sodium ion concentration and pH were measured by scanning ion-selective microelectrodes. A lower limit of lipid bilayer permeability to H(2)S, P(M,H(2)S) >or = 0.5 +/- 0.4 cm/s was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, P(M,H(2)S) remained unchanged. These results indicate that lipid membranes may well act as a barrier to water transport although they do not oppose a significant resistance to H(2)S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an H(2)S barrier indicates that H(2)S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels.

Cholesterol's decoupling effect on membrane partitioning and permeability revisited: is there anything beyond Fick's law of diffusion?

In general, Fick's law of diffusion describes membrane permeation of hydrophobic or amphiphilic molecules. In contrast to this, Thomae et al. recently identified the volume ratio between barrier and aqueous compartments as important additional determinants of membrane permeability (Pm) [A.V. Thomae, T. Koch, C. Panse, H. Wunderli-Allenspach, and S.D. Kramer, Comparing the lipid membrane affinity and permeation of drug-like acids: the intriguing effects of cholesterol and charged lipids, Pharm. Res. 24 (2007) 1457-1472.]. This new theory was supported by the striking observation that low concentrations of cholesterol increased Pm of salicylic acid. As Fick's law is of fundamental importance to all membrane transport processes, we reinvestigated this phenomenon. We measured the electrophoretic mobility of vesicles and used electrochemical scanning microscopy to study the adsorption of the SA anion to lipid vesicular bilayers and SA transport through planar lipid bilayers, respectively. As predicted by Fick's law, Pm of SA decreased continuously with increasing cholesterol content. Thomae et al. made the contrasting artifactual observation because their kinetic approach lacked the required time resolution and led to an underestimation of Pm by five orders of magnitude. We conclude that there is nothing beyond Fick's law of diffusion. It is still valid.

110 years of the Meyer-Overton rule: predicting membrane permeability of gases and other small compounds.

The transport of gaseous compounds across biological membranes is essential in all forms of life. Although it was generally accepted that gases freely penetrate the lipid matrix of biological membranes, a number of studies challenged this doctrine as they found biological membranes to have extremely low gas-permeability values. These observations led to the identification of several membrane-embedded "gas" channels, which facilitate the transport of biological active gases, such as carbon dioxide, nitric oxide, and ammonia. However, some of these findings are in contrast to the well-established solubility-diffusion model (also known as the Meyer-Overton rule), which predicts membrane permeabilities from the molecule's oil-water partition coefficient. Herein, we discuss recently reported violations of the Meyer-Overton rule for small molecules, including carboxylic acids and gases, and show that Meyer and Overton continue to rule.

Reconstruction of the shape of conductivity spectra using differential multi-frequency magnetic induction tomography.

Magnetic induction tomography (MIT) of biological tissue is used for the reconstruction of the complex conductivity distribution kappa inside the object under investigation. It is based on the perturbation of an alternating magnetic field caused by the object and can be used in all applications of electrical impedance tomography (EIT) such as functional lung monitoring and assessment of tissue fluids. In contrast to EIT, MIT does not require electrodes and magnetic fields can also penetrate non-conducting barriers such as the skull. As in EIT, the reconstruction of absolute conductivity values is very difficult because of the method's sensitivity to numerical errors and noise. To overcome this problem, image reconstruction in EIT is often done differentially. Analogously, this concept has been adopted for MIT. Two different methods for differential imaging are applicable. The first one is state-differential, for example when the conductivity change between inspiration and expiration in the lung regions is being detected. The second one is frequency-differential, which is of high interest in motionless organs like the brain, where a state-differential method cannot be applied. An equation for frequency-differential MIT was derived taking into consideration the frequency dependence of the sensitivity matrix. This formula is valid if we can assume that only small conductivity changes occur. In this way, the non-linear inverse problem of MIT can be approximated by a linear one (depending only on the frequency), similar to in EIT. Keeping this limitation in mind, the conductivity changes between one or more reference frequencies and several measurement frequencies were reconstructed, yielding normalized conductivity spectra. Due to the differential character of the method, these spectra do not provide absolute conductivities but preserve the shape of the spectrum. The validity of the method was tested with artificial data generated with a spherical perturbation within a conducting cylinder as well as for real measurement data. The measurement data were obtained from a potato immersed in saline. The resulting spectra were compared with reference measurements and the preservation of the shape of the spectra was analyzed.

Solution of the inverse problem of magnetic induction tomography (MIT) with multiple objects: analysis of detectability and statistical properties with respect to the reconstructed conducting region.

Magnetic induction tomography (MIT) is a technique to image the passive electrical properties (i.e. conductivity, permittivity, permeability) of biological tissues. The inverse eddy current problem is nonlinear and ill-posed, thus a Gauss-Newton one-step method in combination with four different regularization schemes is used to obtain stable solutions. Simulations with 16 excitation coils, 32 receiving coils and different spherical perturbations inside a homogeneous cylinder were computed. In order to compare the statistical properties of the reconstructed results a Monte Carlo study with a SNR of 40 dB and 20 dB was carried out. Simulated conductivity perturbations inside a homogeneous cylinder can be localized and resolved and the results prove the feasibility of difference imaging with MIT.

Carbon dioxide transport through membranes.

Several membrane channels, like aquaporin-1 (AQP1) and the RhAG protein of the rhesus complex, were hypothesized to be of physiological relevance for CO(2) transport. However, the underlying assumption that the lipid matrix imposes a significant barrier to CO(2) diffusion was never confirmed experimentally. Here we have monitored transmembrane CO(2) flux (J(CO2)) by imposing a CO(2) concentration gradient across planar lipid bilayers and detecting the resulting small pH shift in the immediate membrane vicinity. An analytical model, which accounts for the presence of both carbonic anhydrase and buffer molecules, was fitted to the experimental pH profiles using inverse problems techniques. At pH 7.4, the model revealed that J(CO2) was entirely rate-limited by near-membrane unstirred layers (USL), which act as diffusional barriers in series with the membrane. Membrane tightening by sphingomyelin and cholesterol did not alter J(CO2) confirming that membrane resistance was comparatively small. In contrast, a pH-induced shift of the CO(2) hydration-dehydration equilibrium resulted in a relative membrane contribution of about 15% to the total resistance (pH 9.6). Under these conditions, a membrane CO(2) permeability (3.2 +/- 1.6 cm/s) was estimated. It indicates that cellular CO(2) uptake (pH 7.4) is always USL-limited, because the USL size always exceeds 1 mum. Consequently, facilitation of CO(2) transport by AQP1, RhAG, or any other protein is highly unlikely. The conclusion was confirmed by the observation that CO(2) permeability of epithelial cell monolayers was always the same whether AQP1 was overexpressed in both the apical and basolateral membranes or not.