Model of electrical conductivity of skeletal muscle based on tissue structure

Recent experiments carried out in our laboratory with the four-electrode method showed that the electrical conductivity of skeletal muscle tissue depends on the frequency of the injected current and the distance between the current electrodes. A model is proposed in order to study these effects. The model takes into account the structure of the tissue on the scale of individual fibres. It discerns three main components with respect to electrical properties: (a) extracellular medium with electrical conductivity σ_e; (b) intracellular medium with electrical conductivity σ_i; (c) muscle fibre membrane with impedance Z_m. The model results show an apparent frequency dependence of the electrical conductivity of skeletal muscle tissue, as well as the way the conductivity is affected by the length the current is conducted.     

Physiological quality assessment of stored whole blood by means of electrical measurements

The physiological parameters of blood such as extracellular Na⁺, K⁺, Cl⁻, pH, 2,3-DPG and ATP and the complex electrical impedance were measured using whole blood samples from 31 male donors (21 donors form the training set and ten donors were used for testing), on the 0th, 10th and 21st day of blood bank storage. During storage, while the extracellular fluid resistance (R _e) and the intracellular fluid resistance (R _i) decreased progressively with time, the effective cell membrane capacitance (C_m) has increased. Blood bank storage resulted in a rise in K⁺ and a fall in Na⁺, Cl⁻, pH, 2,3-DPG and ATP. Accordingly, all electrical parameters correlated with Na⁺, K⁺, Cl⁻, pH and ATP, at varying levels. By applying the multi-regression analysis, it was demonstrated that R _i, R _e and especially C _m were appropriate for the assessment of Na⁺, K⁺, Cl⁻, pH and ATP until the 21st day of storage.     

Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle

To study the stimulating effect of adrenaline (ADR) on active Na⁺/K⁺ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K⁺ (aK_e) and intracellular Na⁺ (aNa_i) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10⁻⁶ M) resulted in a membrane hyperpolarization and transient decreasesaK_e andaNa_i which could be blocked by ouabain (3×10⁻⁴ M). In the h.p.p. muslce a continuous rise ofaNa_i induced by elevation ofaK_e to 5.2 mM could be stopped by ADR. In addition, the intracellular K⁺ activity (aK_i), the free intracellular Ca²⁺ concentration (pCa_i) and intracellular pH (pH_i) were monitored in rat soleus muscle. During ADRaK_i increased, pH_i remained constant and intracellular Ca²⁺ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na⁺/K⁺ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle. Parts of the results have been presented to the German Physiological Society (Ballanyi and Grafe 1987)     

Protons make tumor cells move like clockwork

Cancer accounts for 13% of the yearly total mortality worldwide. Most cancer deaths are the sequel of metastatic diseases rather than of primary tumor growth. Thus, the major challenge in tumor therapy is the tumor cells’ ability to metastasize. The extent to which a tumor metastasizes correlates with the tumor cells’ migratory activity. Tumor cell migration requires a coordinated formation and release of cell adhesion contacts, a controlled cytoskeletal dynamics, the digestion and reorganization of the extracellular matrix, and local ion and water transport across the plasma membrane. All of these operations depend on intracellular pH (pH_i) and extracellular pH (pH_e). Numerous H⁺, HCO₃⁻, and monocarboxylate transporters as well as different carbonic anhydrase isozymes have considerable impact on pH_i and pH_e which spotlights them as possible, potential targets for anticancer therapeutics. Especially in solid tumors whose vascularization is often not sufficient, tumor cells cope with hypoxia and the resulting glycolysis by overexpressing the Na⁺/H⁺ exchanger NHE1, monocarboxylate transporters MCT1 and/or MCT4, and the carbonic anhydrase CA IX. NHE1, MCT, and CA IX activity lead to an acidification of the extracellular space in order to maintain the cytosolic pH homeostasis stable. The present article gives a review on how this characteristic, acidic tumor micro- and nanoenvironment controls tumor cell migration.     

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes

We have studied the regulation of intracellular pH (pH_i), and HCO ₃ ⁻ -dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pH_i was 6.96 in both nominally CO₂/HCO ₃ ⁻ -free, HEPES-buffered saline (6.96 ±0.14;n=48) and in a saline containing 5% CO₂/24 mM HCO ₃ ⁻ (6.96±0.18;n=48) (at pH 7.4). Inhibition of the Na⁺/H⁺ exchange by amiloride (2 mM) caused a significant decrease of pH_i in nominally CO₂/HCO ₃ ⁻ -free saline. Addition of CO₂/HCO ₃ ⁻ in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na⁺ also caused a fall of pH_i, and addition of CO₂/HCO ₃ ⁻ in Na⁺-free saline evoked a further fall of pH_i, while the outward current was reduced or even reversed. The stilbene 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.3 mM) reduced the pH_i recovery from the CO₂/HCO ₃ ⁻ -evoked acidification, and blocked the prominent intracellular acidification upon removal of CO₂/HCO ₃ ⁻ . Removal of external Cl⁻ had little effect on these pH_i changes. Lowering the external pH from 7.4 to 6.6 in CO₂/HCO ₃ ⁻ -containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO₂/HCO ₃ ⁻ . The results suggest that the CO₂/HCO ₃ ⁻ -dependent current is partly due to a reversible bidirectional, electrogenic Na⁺-HCO ₃ ⁻ cotransporter, which helps to regulate pH_i in these cells. In addition, a prominent Na⁺/H⁺ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steadystate pH_i.     

Estimation of transmembrane potential of thymic lymphocytes during dexamethasone-induced apoptosis

The use of potential-sensitive fluorescent probes allowed us to estimate transmembrane potentials of the plasma (Δφ_p) and mitochondrial (Δφ_m) membranes of rat thymocytes, which were −58±3 mV and −169±7 mV, respectively. Dexamethasone-induced apoptosis led to a significant decrease in Δφ_p (by 55%) and Δφ_m (by 17%). This effects of dexamethasone was dose- and time-dependent. Changes in Δφ_m were greater and preceded those in Δφ_p. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 117–120, January, 1999     

Detubulation abolishes membrane potential stabilization in amphibian skeletal muscle

A recently reported stabilization (`splinting') of the resting membrane potential (E _m) observed in amphibian skeletal muscle fibres despite extracellular hyperosmotic challenge has been attributed to high resting ratios of membrane Cl⁻ to K⁺ permeability (P _Cl/P _K) combined with elevations of their intracellular Cl⁻ concentrations, [Cl⁻]_i, above electrochemical equilibrium by diuretic-sensitive cation--Cl⁻, Na--Cl (NCC) and/or Na--K--2Cl (NKCC), co-transporter activity. The present experiments localized this co-transporter activity by investigating the effects of established detubulation procedures on E _m splinting. They exposed fibres to introduction and subsequent withdrawal of 400 mM extracellular glycerol, high divalent cation concentrations, and cooling. An abolition of tubular access of extracellularly added lissamine rhodamine fluorescence, visualized by confocal microscopy, and of the action potential afterdepolarization together confirmed successful transverse (T-) tubular detachment. Fibre volumes, V, of such detubulated fibres, determined using recently introduced confocal microscope-scanning methods, retained the simple dependence upon 1/[extracellular osmolarity], without significant evidence of the regulatory volume increases described in other cell types, previously established in intact fibres. However detubulation abolished the E _m splinting shown by intact fibres. _m thus varied with extracellular osmolarity in detubulated fibres studied in standard, Cl⁻-containing, Ringer solutions and conformed to simple predictions from such changes in assuming that intracellular ion content was conserved and membrane potential change ΔE _m was principally determined by the K⁺ Nernst potential. Furthermore, cation--Cl⁻ co-transport block brought about by [Cl⁻]_o or [Na⁺]_o deprivation, or inclusion of bumetanide (10 μM) and chlorothiazide (10 μM) in the extracellular fluid gave similar results. When taken together with previous reports of significant Cl⁻ conductances in the surface membrane, these findings suggest a model that contrastingly suggests a T-tubular location for cation--Cl⁻ co-transporter activity or its regulation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Radial changes of extracellular potential amplitude and integral characteristics and the inverse problem in electroneurography

The possibility of solving the inverse problem in electroneurography, i.e. of estimating the main parameters specifying the activated fibre's functional state, using the amplitude and integral characteristics of the surface potentials generated by infinite homogeneous fibres, has been analysed. An analytical expression has been found for the amplitude of the negative phase A_nph of the single fibre extracellular action potential (SFEAP) as a function of the wavelength b, the fibre-electrode distance y and a scale factor A_o proportional to the intracellular action potential amplitude V_m, to the square of the fibre radius a and to the ratio of the axoplasm conductivity τ_a and volume conductor conductivity τ_e. For a large fibre-electrode distance, typical of surface recordings, an analytical expression of the integral of the negative phase I_nph of the SFEAP as a function of A_o, b, y and the propagation velocity v was also found. Simple methods are proposed for estimating v, the location of the electrical centre of the activated fibres' territory and the product of the number of activated fibres N, duration T_in of the intracellular action, potential and of the factor A_o. The estimation errors due to the temporal and spatial dispersion of the activated fibres were analysed as a function of the fibre-electrode distance and the territory shape.     

Membrane potentials in Rana temporaria muscle fibres in strongly hypertonic solutions

Conventional microelectrode methods were used to measure variations in resting membrane potentials, E _m, of intact amphibian skeletal muscle fibres over a wide range of increased extracellular tonicities produced by inclusion of varying extracellular concentrations of sucrose. Moderate increases in extracellular tonicity to up to 2.6× normal (2.6τ) under Cl⁻ free conditions produced negative shifts in E _m that followed expectations for the K⁺ Nernst equation (E _K) applied to a perfect osmometer containing a conserved intracellular K⁺ content despite any accompanying cell volume change. In contrast, E _m remained stable in fibres studied in otherwise similar Cl⁻ containing solutions, consistent with E _m stabilization despite negative shifts in E _K through inward cation-Cl⁻ co-transport activity. Short exposures to higher tonicities (>3τ) similarly produced negative shifts in E _m in Cl⁻ free but not Cl⁻ containing solutions. However, prolonged exposures to solutions of >3τ caused gradual net positive changes in E _m in both Cl⁻ containing and Cl⁻ free solutions suggesting that these changes were independent of cation-Cl⁻ transport. Indeed, there was no evidence of cation-Cl⁻ co-transport activity in strongly hypertonic solutions despite its predicted energetic favourability, suggesting its possible regulation by E _m in muscle. Additional findings implicated a failure to maintain greatly increased transmembrane [K⁺] gradients in these E _m changes. Thus: (1) halving or doubling [K⁺]_e produced negative or positive shifts␣in E _m, respectively in isotonic or moderately hypertonic (<2.7τ), but not strongly hypertonic (>3τ) solutions; (2) subsequent restoration of isotonic extracellular conditions produced further positive changes in E _m consistent with a dilution of the depleted [K⁺]_i by fibres regaining their original resting volumes; (3) quantitative modelling similarly predicted a gradual net efflux of K⁺ as the balance between active and passive [K⁺] fluxes altered due to increased transmembrane [K⁺] gradients in hypertonic and low [K⁺]_e solutions. However, the observed positive changes in E _m in the most strongly hypertonic solutions eventually exceeded these predictions suggesting additional limitations on␣Na⁺/K⁺-ATPase activity in strongly hypertonic solutions.James A. Fraser and Kai Yuen Wong have equally contributed to this paper.     

Conductivity and geometrical factors affecting volume measurements with an impedancimetric catheter

With a model based on Poisson’s equation, computations employing published data and measurements made in glass cylinders filled with saline, the influence of conductivity and geometry on the determination of volume was analysed with an impedancimetric catheter. Two distal electrodes inject a constant current while a set of central electrodes sense by sections the potential along the cylindrical chamber. It was concluded that each section ought to be independently calibrated, the distribution of the electrodes along the catheter and its relationship to the chamber radius being of paramount importance for a linear calibration. The ratio σ _r , of the external medium conductivity σ _e to the internal conductivity σ _i influences drastically both the linearity and the calibration of each section. If a section volume is linearly related to the total chamber volume, that section admittance is a total volume estimator.     

Secretin and VIP-stimulated adenylate cyclase from rat heart

The extrusion mechanism ofd-glucose by tubular cells was investigated in isolated rabbit kidneys perfused with normal or hyperglicemic blood, added with¹⁴C-inulin, by relating the values of the ratio between the intracellular and extracellular glucose concentration (G_i/G_e) in the cortical tissue to the respective plasma concentration G_p (=G_e). The apparent extracellular space was calculated in each experiment from the kinetics of the¹⁴C-inulin efflux from cortical slices excised at the end of the perfusion period (mean value = 20.7%±1.7 of the total vol.). The total intra and extracellular amount of glucose was measured in two contiguous slices excised from the cortex. The total water was expressed by the difference between the fresh and dry weight of the slice, the intracellular water by the difference between the total and extracellular water. From these volumes the respective apparent glucose concentrations could be found. The G_i/G_e values tend to 1, that is to a uniform intra and extracellular distribution of glucose, only at high plasma glucose levels. These results do not seem to be in agreement with ad-glucose transfer from the cell to the subepithelial spaces down a concentration gradient.     

Biomechanical and Microstructural Properties of Common Carotid Arteries from Fibulin-5 Null Mice

Alteration in the mechanical properties of arteries occurs with aging and disease, and arterial stiffening is a key risk factor for subsequent cardiovascular events. Arterial stiffening is associated with the loss of functional elastic fibers and increased collagen content in the wall of large arteries. Arterial mechanical properties are controlled largely by the turnover and reorganization of key structural proteins and cells, a process termed growth and remodeling. Fibulin-5 (fbln5) is a microfibrillar protein that binds tropoelastin, interacts with integrins, and localizes to elastin fibers; tropoelastin and microfibrillar proteins constitute functional elastic fibers. We performed biaxial mechanical testing and confocal imaging of common carotid arteries (CCAs) from fibulin-5 null mice (fbln5 ^−/−) and littermate controls (fbln5 ^+/+) to characterize the mechanical behavior and microstructural content of these arteries; mechanical testing data were fit to a four-fiber family constitutive model. We found that CCAs from fbln5 ^−/− mice exhibited lower in vivo axial stretch and lower in vivo stresses while maintaining a similar compliance over physiological pressures compared to littermate controls. Specifically, for fbln5 ^−/− the axial stretch λ = 1.41 ± 0.07, the circumferential stress σ _θ  = 101 ± 32 kPa, and the axial stress σ _z  = 74 ± 28 kPa; for fbln5 ^+/+ λ = 1.64 ± 0.03, σ _θ  = 194 ± 38 kPa, and σ _z  = 159 ± 29 kPa. Structurally, CCAs from fbln5 ^−/− mice lack distinct functional elastic fibers defined by the lamellar structure of alternating layers of smooth muscle cells and elastin sheets. These data suggest that structural differences in fbln5 ^−/− arteries correlate with significant differences in mechanical properties. Despite these significant differences fbln5 ^−/− CCAs exhibited nearly normal levels of cyclic strain over the cardiac cycle.     

Estimation of cardiomyocyte transmembrane potential with potential-sensitive fluorescent probes

Transmembrane potentials on the plasma (Δφ₄) and mitochondrial (Δφ_m) membrane of isolated rat cardiomyocytes were estimated using the potential-sensitive fluorescent probe DSM. The values were −93±4 and −196±11 mV, respectively. Sufan significantly decreased the reduction of Δφ_m induced by chemical hypoxia. The effects of antiarrhythmic drugs on changes in Δφ_p were studied using the fluorescent probe dis-C₃-(5). Lidocaine, novocainamide, richlocaine, and leocaine blocked depolarization of the myocyte plasma membrane induced by electrical stimulation and did not affect the Δφ_m. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 11, pp. 594–597, November, 1998     

Endothelial function in aorta segments of apolipoprotein E-deficient mice before development of atherosclerotic lesions

Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE^−/−) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca²⁺]_i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE^−/− and C57BL/6J (WT) mice were evoked by phenylephrine (3 × 10⁻⁹–3 × 10⁻⁵ M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca²⁺]_i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE^−/− mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca²⁺]_i in apoE^−/− strips with 10⁻⁶ M cyclopiazonic acid or 10⁻⁷ M Ca²⁺-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE^−/− and WT aorta segments. Basal [Ca²⁺]_i was significantly lower in apoE^−/− than in WT strips. Relaxation by ACh (3 × 10⁻⁹–10⁻⁵ M) was time- and dose-dependently related to [Ca²⁺]_i, but neither ACh-induced relaxation nor Ca²⁺ mobilization were diminished in apoE^−/− strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE^−/− mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca²⁺]_i. These findings further point to important differences between basal and stimulated eNOS activity.     

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na⁺–K⁺ pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na⁺]_i, [K⁺]_i, and [Cl⁻]_i, as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na⁺]_i increase, a [K⁺]_i decrease, and a membrane depolarization, and it also evoked an increase in [Cl⁻]_i. The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na⁺ or Cl⁻, NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na⁺–K⁺ pump was inhibited by omitting bath K⁺. Also, in K⁺-free solution, [Na⁺]_i increased and [K⁺]_i dropped, but [Cl⁻]_i slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl⁻ gradient.     

Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

Ryanodine receptor 1 (RyR1) is well-known to be expressed in systemic and pulmonary vascular smooth muscle cells (SMCs); however, its functional roles remain largely unknown. In the present study, we attempted to determine the potential importance of RyR1 in membrane depolarization-, neurotransmitter-, and hypoxia-induced Ca²⁺ release and contraction in pulmonary artery SMCs (PASMCs) using RyR1 homozygous and heterozygous gene deletion (RyR1^−/− and RyR1^+/−) mice. Our results indicate that spontaneous local Ca²⁺ release and caffeine-induced global Ca²⁺ release are significantly reduced in embryonic RyR1^−/− and adult RyR^+/− cells. An increase in [Ca²⁺]_i following membrane depolarization with high K⁺ is markedly attenuated in RyR1^−/− and RyR1^+/− PASMCs in normal Ca²⁺ or Ca²⁺-free extracellular solution. Similarly, muscle contraction evoked by membrane depolarization is reduced in RyR1^+/− pulmonary arteries in the presence or absence of extracellular Ca²⁺. Neurotransmitter receptor agonists and inositol 1,4,5-triphosphate elicit a much smaller increase in [Ca²⁺]_i in both RyR1^−/− and RyR1^+/− cells. We have also found that neurotransmitter-evoked muscle contraction is significantly inhibited in RyR1^+/− pulmonary arteries. Hypoxia-induced increase in [Ca²⁺]_i and contraction are largely blocked in RyR1^−/− and/or RyR1^+/− PASMCs. Collectively, our findings provide genetic evidence for the functional importance of RyR1 in spontaneous local Ca²⁺ release, and membrane depolarization-, neurotransmitter-, as well as hypoxia-induced global Ca²⁺ release and attendant contraction in PASMCs.     

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn²⁺ on whole-cell Ca²⁺-activated Cl⁻ currents (I _CLCA) in mouse inner medullary collecting duct cells (mIMCD-3). I _CLCA was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10–400 μM) to the bathing solution resulted in a dose-dependent increase in I _CLCA with little change in Cl⁻ selectivity or biophysical characteristics, whereas gadolinium chloride (30 μM) and lanthanum chloride (100 μM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn²⁺ (400 μM) stimulated an increase in intracellular Ca²⁺ to an elevated plateau. The Zn²⁺-stimulated [Ca²⁺]_i increase was inhibited by thapsigargin (200 nM), the IP₃ receptor antagonist 2-aminoethoxydiphenyl borate (10 μM) and removal of bath Ca²⁺. Pre-exposure to Zn²⁺ (400 μM) markedly attenuated the ATP (100 μM)-stimulated [Ca²⁺]_i increase. These data are consistent with the hypothesis that extracellular Zn²⁺ stimulates an increase in [Ca²⁺]_i by a release of calcium from thapsigargin/IP₃ sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca²⁺-sensing receptor, in IMCD cells is discussed.     

Streaming potentials during the confined compression creep test of normal and proteoglycan-depleted cartilage

The streaming potential response of cartilage in the confined compression creep configuration was assessed theoretically and measured experimentally in normal and proteoglycan-depleted tissue. The analytical solution, using the linear biphasic continuum model including electrokinetics and assuming homogeneous material properties, predicted that: (i) the peak streaming potentials is ΔV=k_e·Δσ, where k_e is the electrokinetic coefficient and Δσ is the change in compressive stress; (ii) the potential is maintained at 95 to 100% of the peak value of 0<t<0.10τ, where τ is the gel diffusion time constant; and (iii) during short times, 0<t<0.01 τ, 90% of the peak streaming potential occurs over a region extending 23% into the tissue sample. Experimentally, adult bovine cartilage disks, 0.5 mm thick, were subjected to step changes of compressive stress. The measured changes in potential indicated a linear response for changes in stress up to 0.10 MPa. The k_e of normal cartilage, estimated from the short time (0<t<2 sec) change in potential, was −1.65±1.25 mV/MPa. Digestion of cartilage by chondroitinase ABC resulted in an increased (less negative) k_e of −0.75±0.70 mV/MPa and a 33±29% depletion of anionic glycosaminoglycan, whereas digestion with trypsin resulted in a further increase in k_e to +1.64±0.95 mV/MPa and a 98±1% depletion of glycosaminoglycan. The streaming potential measurement may be a useful addition to the widely used confined compression creep test to assess cartilage material properties.     

Das Verhalten der intracellulären Magnesiumkonzentration im Myokard bei Insuffizienz,Hypoxie und Kammerflimmern

Zusammenfassung Die im Serum gemessene extracelluläre Magnesiumkonzentration beträgt bei Meerschweinchen, unabhängig vom Funktionszustand und der Schädigung des Herzens, 1,9–2,0 mval/l. Die intracelluläre Magnesiumkonzentration des Myokards, berechnet über den gemessenen extracellulären Inulin-, Volumen- und Konzentrationsanteil, beträgt im Mittel von 25 normalen, suffizienten Meerschweinchenherzen 16,6 mval/l. Der Quotient Mg _i /Mg _e ist somit 8,5. Bei 48 experimentell erzeugten Herzinsuffizienzen (Hypoxie, Überlastung, verschiedene Intoxikationen) wurde eine intracelluläre Magnesiumkonzentration im Myokard im Mittel von 12,2 mval/l gefunden. Das ist eine Abnahme von 26% des Normalwertes und ein Quotient Mg_i/Mg_e 6,3. Auch beim Kammerflimmern wurde zwar eine geringere, aber doch signifikante Abnahme auf Mg _i 14,4 mval/l ermittelt. Bei Asyphyxie nimmt die intracelluläre Magnesiumkonzentration, parallel der hypoxiebedingten Herzdilatation, beginnend nach 30 sec, bis auf einen Minimalwert von 10,9 mval/l nach 2,5 min Asphyxiedauer ab (Abnahme 34%; Mg_i/Mg_e 5,5). Die Bedeutung des Magnesiums für Funktion, Stoffwechsel und Struktur des Herzmuskels und der Wert für Prophylaxe und Therapie von Herzkrankheiten werden diskutiert.

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Summary The extracellular concentration of magnesium in guinea pigs was found to be 1,9–2,0 meq/l. It does not change, when heart failure occurs. In 25 normal guinea pig hearts without failure, the intracellular concentration of magnesium in the myocardium was 16,6 meq/l on the average (Mg_i/Mg_e 8,5). In 48 guinea pigs with experimentally induced heart failure, the intracellular magnesium concentration of the myocardium decreased up to a value of 12,2 meq/l (Mg_i/Mg_e 6,3). In ventricular fibrillation also, myocardial cells lost magnesium significantly (Mg _i =14,4 meq/l). In asphyxia intracellular magnesium concentration decreases after 30 sec with the lowest value of 10,9 meq/l being reached after 2,5 min of asphyxia. The loss of magnesium from the myocardial cells in asphyxia parallels in time the course of cardiac dilatation. The importance of magnesium in the function, metabolism and structure of heart muscle, and its value in prophylaxis and therapy of heart diseases is discussed.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Role of nonconserved charged residues of the AE2 transmembrane domain in regulation of anion exchange by pH

The ubiquitous AE2/SLC4A2 anion exchanger is acutely and independently regulated by intracellular (pH_i) and extracellular pH (pH_o), whereas the closely related AE1/SLC4A1 of the red cell and renal intercalated cell is relatively pH-insensitive. We have investigated the contribution of nonconserved charged residues within the C-terminal transmembrane domain (TMD) of AE2 to regulation by pH through mutation to the corresponding AE1 residues. AE2-mediated Cl⁻/Cl⁻ exchange was measured as 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid-sensitive ³⁶Cl⁻ efflux from Xenopus oocytes by varying pH_i at constant pH_o, and by varying pH_o at near-constant pH_i. All mutations of nonconserved charged residues of the AE2 TMD yielded functional protein, but mutations of some conserved charged residues (R789E, R1056A, R1134C) reduced or abolished function. Individual mutation of AE2 TMD residues R921, F922, P1077, and R1107 exhibited reduced pH_i sensitivity compared to wt AE2, whereas TMD mutants K1153R, R1155K, R1202L displayed enhanced sensitivity to acidic pH_i. In addition, pH_o sensitivity was significantly acid- shifted when nonconserved AE2 TMD residues E981, K982, and D1075 were individually converted to the corresponding AE1 residues. These results demonstrate that multiple conserved charged residues are important for basal transport function of AE2 and that certain nonconserved charged residues of the AE2 TMD are essential for wild-type regulation of anion exchange by pH_i and pH_o. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. K. Stewart and C. E. Kurschat have contributed equally to this work.