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)
Confluent monolayers of the cultured renal distal tubule cell line (A6) were impaled with microelectrodes under short-circuit conditions. Specific membrane conductances were calculated from equivalent circuit equations. Transport properties of the apical and basolateral membranes were investigated during control conditions and short-term increases in basolateral potassium concentration [K+] from 2.5 to 20 mmol/l, with or without 0.5 mmol/l Ba2+ at the basolateral side. As in most other epithelia, the apical membrane represents the major resistive barrier. Transcellular, apical and basolateral membrane conductances (gc, go and gi respectively), obtained from 22 acceptable microelectrode studies, averaged 61, 80 and 292 microS/cm2, respectively. There was a highly significant correlation between short-circuit current (Isc) and go, whereas gi was unrelated to Isc. The Isc, which averaged 4.1 microA/cm2, was almost completely blocked by amiloride. This was associated with fast hyperpolarization; the intracellular potential (Vsc) increased from -69 to -83 mV and the fractional apical resistance rose to nearly 100%. Using the values of Vsc during amiloride at normal and high [K+], an apparent transference number for K+ at the basolateral membrane of 0.72 can be calculated. This value corresponds with the decrease in gi to about 25% of the control values after blocking the K+ channels with Ba2+. The nature of the remaining conductance is presently unclear. The cellular current decreased during high [K+] and Ba2+, in part resulting from reduction of the electrochemical gradient for apical Na+ uptake due to the depolarization.(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.
The effect of isoproterenol on apical and basolateral membrane conductance in principal cells of short-circuited frog skin was analyzed using microelectrodes. Isoproterenol (10(-6) mol/l) increased the apical membrane conductance in addition to stimulating Cl- conductive pathways outside the principal cells. The effect on apical Na+ channels explains the increase in amiloride sensitive short-circuit current. Basolateral membrane conductance increased only slightly. Steady-state I/V relationships of the basolateral membrane indicate that the inward rectification of basolateral membrane K+ channels was not altered.
Electrophysiological characteristics of transepithelial Cl-specific conductance (gCl) and intracellular element concentrations were analyzed in frog skins before and during voltage perturbation to serosa +100 mV, both under control conditions and after mucosal application of procaine. Under control conditions, gCl was often minimal and almost insensitive to voltage perturbation. Procaine stimulated gCl in many cases considerably and further activation resulted then from voltage perturbation. Microelectrode determinations indicated that conductive pathways parallel to the principal cells account for the procaine-induced increase in gCl. The responses in gCl were not related to the density of mitochondria-rich (MR) cells. Electron microprobe analysis of intracellular electrolyte concentrations showed that procaine increased the Cl content of MR cells significantly. Gain of Cl was primarily due to uptake across the basolateral membrane, as indicated by the small accumulation of Br after unilateral mucosal application. Voltage perturbation to serosa +100 mV in the presence of Br on the mucosal side led in procaine-stimulated tissues to an increase of the ratio of Br/Cl content in the majority of MR cells. It was much less than predicted for conductive transcellular anion transport. Also, intracellular Cl concentrations of MR cells were far above those expected for a highly Cl-permeable basolateral membrane. The data, although indicating finite Cl/Br transport across MR cells, are incompatible with the idea that the voltage-activated conductive Cl transport occurs though these cells. Alternatively, we suggest passage across highly Cl-specific sites of a paracellular pathway.
The effect of Ba2+ on basolateral membrane conductance (gi) in isolated frog skins was analysed. Response patterns were different in tissues with high and low spontaneous intracellular potential. At high (negative) potentials, serosal Ba2+ inhibited gi as is expected of a potent K+ channel blocker, whereas in tissues with low potential, gi remained unchanged or even increased after Ba2+. The direction of change in gi was also dependent on the magnitude of gi under control conditions. Decrease of gi was only observed at high gi in the control period. In contrast, gi increased if control values of gi were below 0.5 mS/cm2. In tissues with spontaneously low intracellular potential, an inhibitory effect of Ba2+ on gi could be induced by hyperpolarization of the basolateral membrane with transepithelial voltage perturbation. Under these conditions, voltage-dependent, inward rectifying K+ channels are activated, which are Ba2(+)-sensitive. Furthermore, hyperpolarization of the basolateral membrane potential (Vi) during Ba2+ rapidly decreased gi. These results suggest that Ba2+, in addition to blocking K+ channels, activates (presumably unspecific) basolateral membrane channels. This dual effect, which is obvious in tissues with low spontaneous gi, might similarly exist in tissues with high control gi. Identification, however, is virtually impossible due to the large decrease in potassium conductance.
The role of mitochondria-rich cells (MR cells) in transepithelial Na transport was investigated by determining electrolyte concentrations and Rb uptake in individual cells of frog skin epithelium using electron microprobe analysis. Measurements were performed under control conditions and after blocking the transepithelial Na transport with amiloride. Under control conditions, Na and Cl concentrations of MR cells scattered much more than those of principal cells and ranged from a few up to more than 30 mmol/kg wet weight. Rb uptake from the basal side into individual MR cells also showed a large variation and was, on the average, much less pronounced than into the principal cells. In principal cells, amiloride reduced the Na concentration and Rb accumulation. In contrast, no effect was observed upon electrolyte concentration and Rb uptake of MR cells. Rb uptake was correlated to the Na concentration of MR cells both under control conditions and after amiloride. It is concluded that, in contrast to the principal cells, MR cells are not involved in amiloride-sensitive transepithelial Na transport and that their Na/K-pump activity is very low.
The elucidation of the molecular side of copper transport in biological systems is a promising task. In this context the transfer of ingested copper into the portal blood plasma was examined. Intralumenal addition of 200 microM copper caused the release of Cu-thionein into the venous effluent. This Cu-thionein became detectable after prior perfusion of the porcine small bowel using a modified isotonic phosphate-buffered saline (Pi/NaCl) medium. The protein was characterized by gel chromatography, luminescence, electronic absorption and immunological identification. ELISA and immunoblotting employing a murine monoclonal antibody to rat liver metallothionein-I proved to be most convenient. Using buffer-loaded sacs of porcine jejunum into which Cu2+, Zn2+ and Cd2+ were added, the release of metallothionein into the serosal fluid was successfully seen by ELISA. The observed excretion of metallothionein into the portal compartment may be a genuine metal transport system for many biochemically active metals.
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.
The phenomenon of an elevated copper concentration in melanoma tumors was examined. It was demonstrated that 50-60% of total tissue copper is associated with metallothionein. The amino acid composition, electronic absorption and fluorescence were identical to that of the many known vertebrate Cu-thioneins. The immunological identification of melanoma tissue metallothionein was successful. The elevated Cu-thionein concentration in melanoma tumor tissue is not yet understood. It appears to be a common concept that in most tumors transient changes of the copper status parallel the metallothionein levels.