Biomedical subjects
Publications and source records attributed to W Nagel.
Hyposmotic basolateral perturbations (-30 mosmol/kg) in cultured renal layers (A6) increased basolateral membrane conductance more than 2-fold within 10 min; the increase was partly due to upregulation of K+ conductance, but other conductive pathways were also activated. The raise in apical membrane amiloride-sensitive Na+ conductance was less pronounced; it appears to be due to secondary effects.
Conductance of apical and basolateral membranes in short-circuited cultured renal distal cells (A6) was determined using microelectrodes. Epithelia were pre-incubated with 0.1 mumol/l dexamethasone in the presence of 4 mumol/l amiloride to prevent increase in apical Na+ entry. Omission of amiloride increased the Isc from 5.7 to 27.6 microA/cm2 due to the rise in apical membrane conductance from 21 to 595 microS/cm2. Apical fractional resistance decreased from 0.89 to 0.40 and cells depolarized from -52 to -4 mV. Basolateral membrane conductance, which was 320 microS/cm2 at partially inhibited transport, was not significantly altered during the first 2 min following establishment of high transport activity; it started to increase thereafter reaching a more than threefold higher value of 1324 microS/cm2 within 12 min. The gain cannot be explained by increase in partial K+ conductance. Disappearance of the conductance after reduction of basolateral Cl- or in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate indicates a Cl- conductance, which appears to be activated by depolarization.
The effect of the diterpene, forskolin, on pathways for conductive Cl- transport was analyzed using isolated skins of Bufo viridis. Forskolin did not stimulate the voltage-activated Cl- movement from mucosa to serosa; the Lorentzian component in the power density spectrum, which was present at serosa positive clamp potentials under control conditions, decreased significantly. The observation that stimulation of cytosolic adenosine 3'-5'-cyclic monophosphate (cAMP) by forskolin has no effect on the voltage-activated Cl- transport argues against control of this pathway by cAMP. Our data further demonstrate that the forskolin-activated Cl- conductive pathway is also permeable for NO3-. This pathway was studied in absence of mucosal Cl-, which eliminates Cl- movement through the voltage-activated pathway. With SO4(2-) and Cl- on the mucosal and serosal sides, respectively, this forskolin-induced pathway displayed a linear current-voltage relationship. The associated Lorentzians increased at serosa negative clamp potentials. Transepithelial current and plateau value of the Lorentzian were related by a quadratic function, which suggests voltage-independence of number and open-close probability of these conductance sites. Morphological sites for voltage-activated and forskolin-induced conductive Cl- transport remain to be identified.
Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.
Cultured Na(+)-transporting epithelia from amphibian renal distal tubule (A6) were impaled with microelectrodes and analyzed at short-circuit and after transepithelial voltage perturbation to evaluate the influence of voltage on apical and basolateral membrane conductances. For equivalent circuit analysis, amiloride was applied at each setting of transepithelial potential. At short-circuit, apical and basolateral membrane conductances averaged 88 and 497 microS/cm2, respectively (n = 10). Apical membrane conductance, essentially due to Na(+)-specific pathways, decreased after depolarization of the apical membrane. The drop was considerably larger than predicted by the Goldman-Hodgkin-Katz (GHK) constant-field equation. This suggests decrease in permeability of the apical Na+ channels upon depolarization. Basolateral membrane conductance, preferentially determined by K+ channels, increased after hyperpolarization of the basolateral membrane. This behavior is contrary to the prediction of the GHK constant field equation and reflects inward rectification of the K+ channels. The observed rectification patterns can be valuable for maintenance of cellular homeostasis.
Fluctuation in transepithelial current was analysed in skins of Rana esculenta and Bufo viridis. Perturbation of the tissues to serosa + 40 mV or + 80 mV activated a pathway for conductive Cl transport and led to the appearance of a Lorentzian component in the power density spectrum. The presence of Cl on the mucosal side was mandatory for the observation of Lorentzians. Corner frequencies, fc, ranged between 48 Hz and 102 Hz. The mean value was slightly larger at 40 mV than at 80 mV (88.3 +/- 4.4 versus 72.5 +/- 6.4 Hz). Plateau values, So, were proportional to the magnitude of the activated Cl current. Single-channel currents of 48.3 +/- 5.2 fA and 93.0 +/- 8.7 fA were calculated at 40 mV and 80 mV, respectively, with the assumption of equal open and closed probabilities of the channels. From these data, a voltage-dependent open-channel conductance of about 1.2 pS is obtained. Activation of Cl conductance was associated with an increase in channel density. Stimulation of Cl conductance by procaine did not affect So and fc despite considerable stimulation of transepithelial Cl current. This could indicate that non-gated channels participate in the voltage-sensitive Cl conductance. Mucosal application of inhibitors of Cl conductance (3',5-dichlorodiphenylaminocarboxy acid, MK-196) decreased So in a concentration-dependent manner, but had no effect on fc. The data could support the hypothesis that the voltage-sensitive transepithelial Cl transport is localized to a cellular compartment, which is most likely the mitochondria-rich cells.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of aldosterone (1 mumol/l for 4-6 h) on Na+ transport across toad skin (Bufo viridis) was studied in skins preincubated in vitro. Short-circuit current (Isc) was consistently and reproducibly elevated in skins from NaCl(100 and 200 mmol/l)-acclimated toads, where the baseline Isc was greatly reduced. The effect of aldosterone was tested in NaCl and NaNO3 Ringer's and also after oxytocin (50 mU/ml) in the latter conditions. Apical membrane conductance of the principal cells increased consistently after aldosterone in all skins and was linearly correlated with the Isc under all conditions. This confirms that the stimulation of Na+ transport originates from the effect of the aldosterone on apical Na+ channels. Basolateral membrane conductance was also significantly elevated compared with control pieces in those tissues that were preincubated with aldosterone for 4-6 h. The increase, however, did not correlate with the magnitude of the Isc. It is therefore concluded that aldosterone specifically stimulates, in addition to the apical effect, the basolateral membrane conductance. This stimulation appears to be direct and not a secondary response to the elevation of transepithelial transport rate.
The "in situ" bypass technique was adopted by our group in 1986. A total of 53 "in situ" bypasses were carried out until June 1989. The worst symptoms were incapacitating claudication, rest pain or ischemic necrosis, each in about one third of the patients. The site of the distal anastomosis was the popliteal artery below the knee in 40 instances, the peroneal artery in 7 and other infrapopliteal vessels in 6. Valve incisions were performed uneventfully with reusable instruments featuring a sharp-edged blade and an indicator for rotation control of the blade ("Insitutom RC"). Vein branches were located mostly by angiography, but residual arteriovenous fistula remained a vexing problem. In the beginning they were ligated under local anaesthesia. Later on they were dealt with successfully using transluminal embolization. Patients were last examined in July 1990 when the follow-up time ranged from 12 to 51 months. 4 patients had died and 5 were lost to follow-up. Patency rates were evaluated according to the life-table method. They were gratifying for femoro-popliteal bypasses with 87 percent after one year and 83 percent after two and three years. Femoro-tibial-peroneal bypasses performed below average, but their number was too small to draw conclusions.
The "in situ" bypass technique was adopted by our group in 1986. A total of 53 "in situ" bypasses were carried out until June 1989. The worst symptoms were incapacitating claudication, rest pain or ischemic necrosis, each in about one third of the patients. The site of the distal anastomosis was the popliteal artery below the knee in 40 instances, the peroneal artery in 7 and other infrapopliteal vessels in 6. Valve incisions were performed uneventfully with re-usable instruments featuring a sharp-edged blade and an indicator for rotation control of the blade ("Insitutom RG"). Vein branches were located mostly by angiography, but residual arterio-venous fistula remained a vexing problem. In the beginning they were ligated under local anaesthesia. Later on they were dealt with successfully using transluminal embolization. Patients were last examined in July 1990 when the follow-up time ranged from 12 to 51 months. 5 patients had died and 5 were lost to follow-up. Patency rates were evaluated according to the life-table method. They were gratifying for femoro-popliteal bypasses with 87% after one year and 83% after two and three years. Femoro-tibial-peroneal bypasses performed below average, but their number was too small to draw conclusions.
The problem of differentiating between patients suffering from osteoporosis and those with healthy bones is discussed on the basis of distribution of the age-specific and sex-specific mineral salt values in a healthy population and in a population with clinically manifest osteoporosis. By comparing the results of quantitative mineralometry and conventional x-ray films of the human skeleton in a histologically clarified group of persons with clinically manifest osteoporosis signs and symptoms, the diagnostic ranking of both methods is assessed and discussed.
Regardless of the weak immunological response against the low-Mr metallothioneins (MTs) the production of murine monoclonal antibodies (mAbs) to monomeric rat liver MT-I was successful. ELISA revealed two groups of mAbs which exhibited different specificities as examined on the native and the lysine-residue modified antigen (Ag). One "lysine-directed mAb" group, consisting of three mAbs, exhibited a specific immunoreactivity with the lysine-containing epitopes of MTs. Their role in the antigenicity of MTs was examined by modifying these residues using glutaraldehyde (GA). Titration with GA resulted in a progressive decline in Ag recognition in the immunoblot; this was completely leveled off when equimolar concentrations were reached. A similar response employing the GA-modified protein in the ELISA was noticed. The second group of mAb cross-reacted with various MTs of different origin, indicating that the common, lysine-free NH2-terminus is exclusively recognized. In direct ELISA of cross-linked MTs, the observed reactivities were much more pronounced. Iodoacetamide (IA) modification of the lysines confirmed the above observations of the GA-derived Ag. Notably, the immunoreactivity was not affected when the cysteine residues were IA-carboxymethylated, nor did the subsequent loss of metals diminish the immunological response in the immunoblot.
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The effect of Ba2+ (0.5 mM, corial side) upon the transport characteristics of the frog skin epithelium was investigated. It was observed that Ba2+ decreased the conductance of the preferably K+-permeable basolateral border to less than 30% of its control value. Furthermore, Ba2+ abolished the K+ electrode-like behaviour, existing at the basolateral membrane under conditions of zero transcellular current flow, for [K+] below 10--15 mM. Effects upon other parameters of transepithelial transport (electromotive forces and resistance of outer or basolateral border and shunt pathway, respectively) were small and might represent secondary events. It is concluded that Ba2+ inhibits passive fluxes of K+ across basolateral membranes of tight, Na+ transporting epithelia, similar to its influence upon membranes of nonpolar cells.
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Studies were done with isolated frog skin to determine the effects of 10(-4) M ouabain on the electrophysiological parameters of outer and inner barriers of the Na-transporting cells. Microelectrodes were used to impale the skins from the outer surface to determine the intracellular voltages (Vsco) under conditions of short-circuiting and under conditions where a voltage clamp was used to vary the transepithelial voltage, VT. From this, the electrical resistances of outer (Rfo) and inner (RI) barriers were estimated. In addition, the driving force for active transepithelial Na transport (ENa = E'1) was estimated from the values of VT when the Vo = 0 mV (Helman and Fisher. 1977. J. Gen. Physiol. 69: 571-604). Studies were done with skins bathed with the usual 2.4 meq/liter [K]i in the inner solution as well as with reduced [K]i of 0.5 and 0 meq/liter. Characteristically, the responses to ouabain could be described by an initial rapid phase (5-10 min) during which time the Ri was increased markedly and the E'1 was decreased from control values. Thereafter, during the slow phases of the response, the resistances of both outer and inner barriers increased continuously and markedly with time leading ultimately to essentially complete inhibition of the short-circuit current. Similar studies were done with skins exposed to 10(-4) M amiloride in the outer solution. Although estimates of Ri could not be obtained under these conditions, the effects on the Vsco and E'1 were similar to those observed for the Na-transporting skins. However, the magnitudes of the effects were less and relatively slower than observed for the Na-transporting skins. The results of these studies were analyzed within the context of a proposed electrical model that takes into account the observation that the magnitude of the voltage at the inner barrier appears to exceed the equilibrium potential for K especially when transepithelial Na transport is inhibited at the apical barrier of the cells.
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The effect of ADH upon the intracellular potential and the resistance of inner and outer borders of the transport pathway was investigated on isolated skins of Rana temporaria. Within 40 min after ADH (100--300 mU/ml), the intracellular potential under short-circuit conditions decreased to about 40% of the control value (--79 +/- 4 mV), concomitant with an increase in the short-circuit current to about 160% of the control value. Amiloride, applied when steady values under ADH had been reached, caused an immediate rise of the intracellular potential to values typical for control conditions. This confirms (i) the intracellular location of the microelectrode and the absence of impalement artifacts, and (ii) the ineffectiveness of ADH upon the electromotive forces of the inner border. ADH had no effect upon the intracellular potential after blockage of the Na entry by Amiloride. The equilibrium potential of the outer border was estimated to be about +20mV under the influence of ADH. As this value is considerably less positive than might be expected for the chemical potential of Na, a significant contribution of ions other than Na to the outer border conductance and equilibrium potential is implicated. The resistance of the outer border was more significantly decreased than that of the active transcellular pathway after ADH due to an increase in the inner border resistance, which exceeded that of the outer border after ADH. The effect of ADH upon the outer membrane characteristics would be underestimated by a factor of two, if the alterations of the electrical potential difference were not taken into consideration.
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