Dependent Depolarization Research Articles

Tailoring the Cu(100) Work Function by Substituted Benzenethiolate Self-Assembled Monolayers

The structure and electronic interface properties of five differently substituted benzenethiol based self-assembled monolayers (SAMs) on Cu(100) have been studied by means of low energy electron diffraction, thermal desorption spectroscopy, X-ray absorption spectroscopy (NEXAFS), and UV photoelectron spectroscopy. Because highly ordered SAMs are formed of which lateral density had been precisely determined, effective molecular dipole moments were derived from the measured work function shifts. These values are compared with gas phase dipole moments computed by quantum chemical calculations for the individual thiol molecules considering the molecular orientation determined from NEXAFS data. Furthermore, this comparison yields clear evidence for a coverage dependent depolarization effect of the adsorbed molecules within the SAMs.

Fluorescence Anisotropy of Molecular Rotors

We present polarization-resolved fluorescence measurements of fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), 9-(2,2-dicyanovinyl)julolidine (DCVJ), and a meso-substituted boron dipyrromethene (BODIPY-C(12)). The photophysical properties of these molecules are highly dependent on the viscosity of the surrounding solvent. The relationship between their quantum yields and the viscosity of the surrounding medium is given by an equation first described and presented by Förster and Hoffmann and can be used to determine the microviscosity of the environment around a fluorophore. Herein we evaluate the applicability of molecular rotors as probes of apparent viscosity on a microscopic scale based on their viscosity dependent fluorescence depolarization. We develop a theoretical framework, combining the Förster-Hoffmann equation with the Perrin equation and compare the dynamic ranges and usable working regimes for these dyes in terms of utilising fluorescence anisotropy as a measure of viscosity. We present polarization-resolved fluorescence spectra and steady-state fluorescence anisotropy imaging data for measurements of intracellular viscosity. We find that the dynamic range for fluorescence anisotropy for CCVJ and DCVJ is significantly lower than that of BODIPY-C(12) in the viscosity range 0.6<η<600 cP. Moreover, using steady-state anisotropy measurements to probe microviscosity in the low (<3 cP) viscosity regime, the molecular rotors can offer a better dynamic range in anisotropy compared with a rigid dye as a probe of microviscosity, and a higher total working dynamic range in terms of viscosity.

Characterization of Saharan dust, marine aerosols and mixtures of biomass-burning aerosols and dust by means of multi-wavelength depolarization and Raman lidar measurements during SAMUM 2

The particle linear depolarization ratio δp of Saharan dust, marine aerosols and mixtures of biomass-burning aerosols from southern West Africa and Saharan dust was determined at three wavelengths with three lidar systems during the SAharan Mineral dUst experiMent 2 at the airport of Praia, Cape Verde, between 22 January and 9 February 2008. The lidar ratio Sp of these major types of tropospheric aerosols was analysed at two wavelengths. For Saharan dust, we find wavelength dependent mean particle linear depolarization ratios δp of 0.24–0.27 at 355 nm, 0.29–0.31 at 532 nm and 0.36–0.40 at 710 nm, and wavelength independent mean lidar ratios Sp of 48–70 sr. Mixtures of biomass-burning aerosols and dust show wavelength independent values of δp and Sp between 0.12–0.23 and 57–98 sr, respectively. The mean values of marine aerosols range independent of wavelength for δp from 0.01 to 0.03 and for Sp from 14 to 24 sr.

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl−/1HCO3− exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.

Field and temperature dependent charge transport characteristics in regio-regular Poly(3-octylthiophene-2,5-diyl) studied by Muon Spin relaxation

Spin dynamic of regio-regular Poly(3-octylthiophene-2,5-diyl) has been investigated with longitudinal field (LF) muon-spin-relaxation (μSR) techniques. The LF dependent muon-spin depolarization rate indicates the occurrence of dimensional crossover from one-dimensional intra-chain spin diffusion to three-dimensional inter-chain spin diffusion at 50 K.

Intra- and inter-chain polaron diffusion in regio-random polythiophene studied by muon spin relaxation

Longitudinal field (LF) muon-spin-relaxation measurements have been performed for polythiophene based polymers of regio-random poly(3-hexylthiophene-2,5-diyl) to elucidate the intra- and inter-chain hopping mechanisms. The LF dependent muon-spin depolarization rate indicates the occurrence of pronounced shift in the relative dominance of charge transport mechanism from intra-chain diffusion to inter-chain diffusion at 50 K which is higher than the 25 K observed previously for the regio-regular case.

Synaptic activation of kainate receptors gates presynaptic CB1 signaling at GABAergic synapses

Glutamate can control inhibitory synaptic transmission through activation of presynaptic kainate receptors. We found that glutamate released by train stimulation of Schaffer collaterals could lead to either short-term depression or short-term facilitation of inhibitory synaptic transmission in mouse CA1 pyramidal neurons, depending on the presence of cannabinoid type 1 (CB(1)) receptors on GABAergic afferents. The train-induced depression of inhibition (t-Di) required the mobilization of 2-arachidonoylglycerol through postsynaptic activation of metabotropic glutamate receptors and [Ca(2+)] rise. GluK1 (GluR5)-dependent depolarization of GABAergic terminals enabled t-Di by facilitating presynaptic CB(1) signaling. Thus, concerted activation of presynaptic CB(1) receptors and kainate receptors mediates short-term depression of inhibitory synaptic transmission. In contrast, in inhibitory connections expressing GluK1, but not CB(1), receptors, train stimulation of Schaffer collaterals led to short-term facilitation. Thus, activation of kainate receptors by synaptically released glutamate gates presynaptic CB(1) signaling, which in turn controls the direction of short-term heterosynaptic plasticity.

μSR study of electron radical dynamics in regio-regular polythiophene

We have carried out longitudinal field (LF) muon-spin-relaxation (μSR) measurements in polythiophene based polymers of Poly(3-hexylthiophene-2,5-diyl) with regio-regular structure to elucidate the intra- and inter-chain hopping mechanisms. The LF dependent muon-spin depolarization rate indicates the occurrence of dimensional crossover from 1 dimensional intra-chain spin diffusion to 3 dimensional inter-chain spin diffusion at 25 K.

In Vitro Cardiac Safety Profiling of a Novel Benzyl-Ethylamine Compound

When developing novel compounds for any clinical indication, the possibility of untoward cardiovascular effects must be addressed. To evaluate the cardiac electrophysiologic effects of A-674563 ((S)-1-benzyl-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine), a novel benzyl-ethylamine, we profiled the compound in a series of cellular and tissue assays. In canine Purkinje fibers (30 min exposure; 2 sec BCL), A-674563 elicited concentration dependent depolarization with shouldering of the terminal repolarization phase (20 μM) and increased abnormal automaticity (60 μM). In papillary muscles, concentration dependent depolarization was also seen, but the effect was much less potent; 60 μM induced shouldering of the terminal phase of repolarization. Contractility was assessed using percent changes in fractional shortening of sarcomere length (FS) in rabbit left ventricular myocytes. A-674563 reduced FS in a concentration dependent manner; 10% at 2 μM and 47% at 20 μM. Effects on ionic currents were further evaluated using heterologously expressed cardiac ion channel cell lines. A-674563 inhibited Cav1.2, expressed in HEK cells, with an IC50 of 20 μM, suggesting that the decreased contractility seen in native cells is due to L-type calcium channel block. The compound reduced Nav1.5 (HEK cells) by 50% at 20 μM and inhibited Kir2.1 (tSA201 cells) with an IC50 of 35 μM. Block of these channels would be expected to reduce the upstroke velocity, depolarize the cellular membrane and lead to abnormal automaticity as was seen in the tissue assays. Although A-674563 caused minimal prolongation of action potential duration in tissues up to 60 μM, it inhibited hERG (HEK cells) with an IC50 of 0.7 μM. This potent block was most likely offset by the concomitant block of multiple cardiac ion channels. In conclusion, A-674563 affects multiple cardiac ion channels to elicit depolarization and increased automaticity in native cardiac tissues.

Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormone.

Central orexin/hypocretin neurones are critical for sustaining consciousness: their firing stimulates wakefulness and their destruction causes narcolepsy. We explored whether the activity of orexin cells is modulated by thyrotropin-releasing hormone (TRH), an endogenous stimulant of wakefulness and locomotor activity whose mechanism of action is not fully understood. Living orexin neurones were identified by targeted expression of green fluorescent protein (GFP) in acute brain slices of transgenic mice. Using whole-cell patch-clamp recordings, we found that TRH robustly increased the action potential firing rate of these neurones. TRH-induced excitation persisted under conditions of synaptic isolation, and involved a Na(+)-dependent depolarization and activation of a mixed cation current in the orexin cell membrane. By double-label immunohistochemistry, we found close appositions between TRH-immunoreactive nerve terminals and orexin-A-immunoreactive cell bodies. These results identify a new physiological modulator of orexin cell firing, and suggest that orexin cell excitation may contribute to the arousal-enhancing actions of TRH.

K + -dependent paradoxical membrane depolarization and Na + overload, major and reversible contributors to weakness by ion channel leaks

Normal resting potential (P1) of myofibers follows the Nernst equation, exhibiting about -85 mV at a normal extracellular K(+) concentration ([K(+)](o)) of 4 mM. Hyperpolarization occurs with decreased [K(+)](o), although at [K(+)](o) < 1.0 mM, myofibers paradoxically depolarize to a second stable potential of -60 mV (P2). In rat myofiber bundles, P2 also was found at more physiological [K(+)](o) and was associated with inexcitability. To increase the relative frequency of P2 to 50%, [K(+)](o) needed to be lowered to 1.5 mM. In the presence of the ionophore gramicidin, [K(+)](o) reduction to only 2.5 mM yielded the same effect. Acetazolamide normalized this increased frequency of P2 fibers. The findings mimic hypokalemic periodic paralysis (HypoPP), a channelopathy characterized by hypokalemia-induced weakness. Of myofibers from 7 HypoPP patients, up to 25% were in P2 at a [K(+)](o) of 4 mM, in accordance with their permanent weakness, and up to 99% were in P2 at a [K(+)](o) of 1.5 mM, in accordance with their paralytic attacks. Of 36 HypoPP patients, 25 had permanent weakness and myoplasmic intracellular Na(+) ([Na(+)](i)) overload (up to 24 mM) as shown by in vivo (23)Na-MRI. Acetazolamide normalized [Na(+)](i) and increased muscle strength. HypoPP myofibers showed a nonselective cation leak of 12-19.5 microS/cm(2), which may explain the Na(+) overload. The leak sensitizes myofibers to reduced serum K(+), and the resulting membrane depolarization causes the weakness. We postulate that the principle of paradoxical depolarization and loss of function upon [K(+)](o) reduction may apply to other tissues, such as heart or brain, when they become leaky (e.g., because of ischemia).

High-resolution radio continuum survey of M 33

Aims. We study the magnetic field structure, strength, and energy density in the Scd galaxy M 33. Methods. Using the linearly polarized intensity and polarization angle data at 3.6, 6.2 and 20 cm, we determine variations of Faraday rotation and depolarization across M 33. We fit a 3D model of the regular magnetic field to the observed azimuthal distribution of polarization angles. We also analyze the spatial variation of depolarization across the galaxy. Results. Faraday rotation, measured between 3.6 and 6.2 cm at an angular resolution of 3 � (0.7 kpc), shows more variation in the south than in the north of the galaxy. About 10% of the nonthermal emission from M 33 at 3.6 cm is polarized. High degrees of polarization of the synchrotron emission (>20%) and strong regular magnetic fields in the sky plane (� 6.6 μG) exist in-between two northern spiral arms. We estimate the average total and regular magnetic field strengths in M 33 as � 6.4 and 2.5 μG, respectively. Under the assumption that the disk of M 33 is flat, the regular magnetic field consists of horizontal and vertical components: however the inferred vertical field may be partly due to a galactic warp. The horizontal field is represented by an axisymmetric (m = 0) mode from 1 to 3 kpc radius and a superposition of axisymmetric and bisymmetric (m = 0 + 1) modes from 3 to 5 kpc radius. Conclusions. An excess of differential Faraday rotation in the southern half together with strong Faraday dispersion in the southern spiral arms seem to be responsible for the north-south asymmetry in the observed wavelength dependent depolarization. The presence of an axisymmetric m = 0 mode of the regular magnetic field in each ring suggests that a galactic dynamo is operating in M 33. The pitch angles of the spiral regular magnetic field are generally smaller than the pitch angles of the optical spiral arms but are twice as big as simple estimates based on the mean-field dynamo theory and M 33’s rotation curve. Generation of interstellar magnetic fields from turbulent gas motion in M 33 is indicated by the equipartition of turbulent and magnetic energy densities.

Phosphate Is Essential for Inhibition of the Mitochondrial Permeability Transition Pore by Cyclosporin A and by Cyclophilin D Ablation

Energized mouse liver mitochondria displayed the same calcium retention capacity (a sensitive measure of the propensity of the permeability transition pore (PTP) to open) irrespective of whether phosphate, arsenate, or vanadate was the permeating anion. Unexpectedly, however, phosphate was specifically required for PTP desensitization by cyclosporin A (CsA) or by genetic inactivation of cyclophilin D (CyP-D). Indeed, when phosphate was replaced by arsenate, vanadate, or bicarbonate, the inhibitory effects of CsA and of CyP-D ablation on the PTP disappeared. After loading with the same amount of Ca(2+) in the presence of arsenate or vanadate but in the absence of phosphate, the sensitivity of the PTP to a variety of inducers was identical in mitochondria from wild-type mice, CyP-D-null mice, and wild-type mice treated with CsA. These findings call for a reassessment of conclusions on the role of the PTP in cell death that are based on the effects of CsA or of CyP-D ablation.

Intracellular Sodium Increase Induced by External Calcium Removal in Human Sperm

In human sperm, removal of external calcium produces a fast Na(+)-dependent depolarization that is presumably due to sodium permeation through calcium channels. Calcium restoration produces a ouabain-sensitive hyperpolarization that brings the membrane potential to values frequently more negative than resting. In this work, we show evidence indicating that external calcium removal induces an increase in the intracellular sodium ([Na(+)](i)) and that this phenomenon is related to the Na(+)-dependent depolarization. Calcium restoration blocked the [Na(+)](i) increase and then produced a slow decrease that was inhibited by ouabain. The [Na(+)](i) increase was inhibited by nanomolar-micromolar calcium or by millimolar magnesium, which has been previously shown to inhibit the Na(+)-dependent depolarization. This evidence supports the hypothesis that, in zero-calcium medium, a calcium channel that would contribute to resting intracellular calcium levels allows sodium permeation, producing depolarization and a significant [Na(+)](i) increase. Sodium loading would stimulate the Na(+),K(+)-ATPase, the activity of which contributes to the sperm hyperpolarization observed upon calcium restoration.

Role of CD37SMIP a Novel Engineered Small Modular Immunopharmaceutical in the Treatment of CLL.

CD37 is a lineage-specific B-cell antigen highly expressed on both normal and transformed B-cells. The significant B-cell selective CD37 expression makes it a valuable target for therapy against B cell malignancies including chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), and non-Hodgkins lymphoma (B-NHL). A novel CD37-specific small modular immunopharmaceutical (CD37SMIP) was engineered to contain a single chain variable region (scFv) linked to a human IgG1 hinge, CH2 and CH3 domains. CD37SMIP induces potent apoptosis in the presence of a crosslinker and antibody dependent cellular cytotoxicity (ADCC) against primary CLL cells. Significant in vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model that is dependent upon NK cell function. The induced cytotoxicity in primary CLL cells was independent of activation of caspase cascade, and consistent with this, the pan-caspase inhibitor z-VAD-fmk failed to rescue CD37 SMIP drug induced cytotoxicity. In an attempt to define the CD37 mediated early signaling events and their role in cytotoxicity, we investigated protein tyrosine phosphorylation as a potential early activation event responsible for CD37 SMIP drug mediated cytotoxicity. Primary B CLL cells were stimulated with increasing concentration of CD37 SMIP with or without crosslinker. Western blot analysis of cellular lysates with anti-phosphotyrosine antibody revealed several tyrosine phosphorylated proteins including a predominant ∼65KDa protein in response to the cross-linking of CD37 SMIP within 10 minutes. Further, pre-treatment with the tyrosine kinase inhibitor herbimycin prevented tyrosine phosphorylation of the predominant 65KDa protein and inhibited CD37 SMIP induced apoptosis in a dose dependent manner. Attempts to identify the tyrosine phosphorylated proteins, using MALDI-TOF mass spectrometry, and to define the potential role of these target proteins in the apoptotic pathway are ongoing. To further examine the molecular mechanism whereby CD37 SMIP triggers cell death, we investigated the involvement of mitochondrial pathways including changes in mitochondrial potential (Δψm) and generation of reactive oxygen species. Treatment with CD37 SMIP induces a time dependent mitochondrial membrane depolarization and increased production of ROS. Further, primary B CLL cells cultured with CD37SMIP induced cleavage and mitochondrial translocation of Bax and upregulation of Bim in a time dependent manner indicating a potential role for Bim and Bax pro apoptotic proteins in CD37 induced apoptosis. These findings provide strong justification for CD37 as a therapeutic target aimed at modulation of pro apoptotic proteins and introduce small modular immunopharmaceuticals as a novel class of targeted therapies for B-cell malignancies. (SMIP trademark is owned by Trubion Pharmaceuticals).

Intrinsic membrane properties and synaptic response characteristics of neurons in the rat’s external cortex of the inferior colliculus

The inferior colliculus (IC) can be divided into three anatomical subdivisions: the central nucleus (ICc), the dorsal cortex (ICd) and the external cortex (ICx). ICx receives its primary auditory inputs from ICc and auditory cerebral cortical areas, and non-auditory inputs from regions of motor and other sensory systems. This wide array of projections makes the ICx a distinct structure within the auditory brainstem. The purpose of the current study was to comprehensively characterize the neuronal population of ICx, by intrinsic and synaptic response properties. Visual whole-cell patch clamp recordings were taken from ICx neurons ( N=129) from rats between postnatal days 8 to 12. Neurons displayed various types of firing patterns in response to current injection, including regular, adapting, pauser and bursting. The regular cells constitute the majority (66%), followed by adapting (18%), pauser (13%) and bursting cells (2%). In response to hyperpolarizing current injection, many neurons illustrated a pronounced sag in the membrane potential, representing a hyperpolarization-activated current (I h ). Some neurons (25%) displayed a Ca 2+-dependent rebound depolarization following negative current injection. In response to depolarizing current injection, 70% of ICx neurons displayed a Ca 2+-mediated potential expressed as Ca 2+ spikes/humps, uncovered when Na + and K + currents were removed. Also, spikes displayed an undershoot which was in part mediated by Ca 2+. Stimulation of the ICc elicited graded synaptic responses, which displayed a combination of excitatory and/or inhibitory potentials, with excitation being predominant across firing patterns. Neurons displayed temporal summation in response to repetitive stimulation at 20 Hz and higher. The results indicate a relatively modest diversity in firing pattern and in intrinsic membrane properties, making this subnucleus distinct from its counterparts within the IC. The data show that ICx receives major excitatory input from ICc, supporting its role in integrating signals from brainstem and directing information to higher brain centers.

Membrane Potential-regulated Transcription of the Resting K+ Conductance TASK-3 via the Calcineurin Pathway

The 2P domain K(+) channel TASK-3 is highly expressed in cerebellar granule neurons where it has been proposed to underlie the K(+) leak conductance, IKso. In a previous work we showed that expression of TASK-3 increases in cerebellar granule neurons as they mature in culture. Here we show that within the cerebellum, levels of TASK-3 mRNA increase as granule neurons migrate to their adult positions and receive excitatory mossy fiber input. To understand the mechanism of this increase in TASK-3 expression we used an in vitro model culturing the neurons in either depolarizing conditions mimicking neuronal activity (25K, 25 mm KCl) or in conditions which approach deafferentation (5K, 5 mm KCl). An important increase in TASK-3 mRNA is uniquely observed in 25K and is specific since other background K(+) channel levels remain unchanged or decrease. The rise in TASK-3 mRNA leads to an increase in TASK-3 protein and the IKso conductance resulting in hyperpolarization. Blocking L-type calcium channels or their downstream effector calcineurin, abrogates TASK-3 expression and IKso, leading to hyperexcitability. This is the first study demonstrating that depolarization-induced Ca(2+) entry can directly regulate cellular excitability by dynamically regulating the transcription of a resting K(+) conductance. The appearance of this conductance may play an important role in the transition of depolarized immature neurons to their mature hyperpolarized state during neuronal development.

Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Potential Difference Measurements of Ocular Surface Na+Absorption Analyzed Using an Electrokinetic Model

Corneal and conjunctival epithelia are capable of transcellular Na+ absorption and Cl- secretion, which drives water movement across these tissues. A recent study demonstrated with a new open-circuit potential difference (PD) technique that Cl- moves across the ocular surface in mice through Ca2+- and cAMP-sensitive Cl- channels, the latter pathway being the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). The purpose of the present study was to identify transporting mechanisms involved in Na+ absorption and to develop a mathematical model of ocular surface ion transport to quantify the relative magnitudes of and electrochemical coupling among transporting processes. PDs across the fluid-bathed ocular surface were measured in anesthetized wild-type and CF mice in response to Na+, Cl-, and K+ ion substitution and transporter agonists, inhibitors, and substrates. An electrokinetic model of the ocular surface epithelium was developed to simulate PD measurements, which involved computation of membrane potentials and cell [Na+], [K+], [Cl-] and volume from transporter activities and extracellular ion concentrations. Na+ replacement produced a 6 +/- 2-mV depolarization that was blocked by amiloride (K(i) 0.8 microM) and benzamil (Ki 0.2 microM). The Na+-dependent depolarization by amiloride was significantly greater in CF mice (19 +/- 3 mV). In wild-type mice, D-, but not L-glucose produced a phloridzin-sensitive, 4.1-mV hyperpolarization in the presence of Na+ and amiloride, with a Km for D-glucose of 2.5 mM. Glycine and L-arginine also produced Na+-dependent hyperpolarizations. The epithelial transport model accurately reproduced experimental PD measurements. PD measurements coupled with model computations defined quantitatively the roles of Na+ and Cl- transport processes in ocular surface ion and fluid secretion, and indicated that CFTR-dependent changes in apparent epithelial Na+ channel (ENaC) activity could be accounted for by electrochemical coupling, without requiring ENaC-CFTR interactions. The data and modeling also predicted significant enhancement of ocular surface fluid secretion by ENaC inhibitors and CFTR activators as possible therapies for dry eye syndromes.

The magnetic field of M 31 from multi-wavelength radio polarization observations

The configuration of the regular magnetic field in M31 is deduced from radio polarization observations at the wavelengths �� 6, 11 and 20cm. By fitting the observed azimuthal distribution of polarization angles, we find that the regular magnetic field, averaged over scales 1-3kpc, is almost perfectly axisymmetric in the radial range 8 to 14kpc, and follows a spiral pattern with pitch angles of p ≃ −19 ◦ to p ≃ −8 ◦ . In the ring between 6 and 8kpc a perturbation of the dominant axisymmetric mode may be present, having the azimuthal wave number m = 2. A systematic analysis of the observed depolarization allows us to identify the main mechanism for wavelength dependent depolarization - Faraday rotation measure gradients arising in a magneto-ionic screen above the synchrotron disk. Modelling of the depolarization leads to constraints on the relative scale heights of the thermal and synchrotron emitting layers in M31; the thermal layer is found to be up to three times thicker than the synchrotron disk. The regular magnetic field must be coherent over a vertical scale at least similar to the scale height of the thermal layer, estimated to be hth ≃ 1kpc. Faraday effects offer a powerful method to detect thick magneto-ionic disks or halosaround spiral galaxies.