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Biomedical subjects

B Lindemann

Publications and source records attributed to B Lindemann.

At least 55 records · Page 3Linked to original sources
PubMed1987

An amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder.

Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 omega cm2. The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5 mM Na+, and serosal Na+ is substituted by K+, Cs+, or N-methyl-D-glucamine, the basolateral membrane expresses what appears to be a large Na+ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca2+. In Cl-free SO2-(4) Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60 mM K+. This pathway can be blocked by serosal amiloride (Ki = 13.1 microM) or serosal Na+ ions (Ki approximately 10 to 20 mM). It also conducts Li+ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec-1 at 0 mV.

Amiloride↗
PubMed1987

Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: II. Side-chain modifications.

The overall on- and off-rate constants for blockage of epithelial Na channels by amiloride analogs were estimated by noise analysis of the stationary Na current traversing frog skin epithelium. The (2-position) side chain structure of amiloride was varied in order to obtain structure/rate constant relationships. Hydrophobic chain elongations (benzamil and related compounds of high blocking potency) increase the stability of the blocking complex (lowered off-rate), explained by attachment of the added phenyl moiety to a hydrophobic area near the site of side chain interaction with the channel protein. Some other chain modifications show that the on-rate, which is smaller than a diffusion-limited rate, varies with side chain structure. In several cases this effect is not attributable to steric hindrance on encounter, and implies that the side chain interacts briefly with the channel protein (encounter complex) before the main blocking position of the molecule is attained. The encounter complex must be labile since the overall rate constants of blockage are not concentration-dependent. In two cases, changes at the 2-position side chain and at other ring ligands, with known effects on the blocking rate constants, could be combined in one analog. The rate constants of blocking by the resulting compounds indicate that the structural changes have additive effects in terms of activation energies. Along with other observations (voltage dependence of the rate constants and competition with the transported Na ion), these results suggest a blocking process of at least two steps. It appears that initially the 2-position side chain invades the outward-facing channel entrance, establishing a labile complex. Then the molecule is either released completely (no block) or the 6-ligand of the pyrazine ring gains access to its receptor counterpart, thus establishing the blocking complex, the lifetime of which is strongly determined by the electronegativity of the 6-ligand.

Amiloride↗
PubMed1987

Patch-clamp study of isolated taste receptor cells of the frog.

Taste discs were dissected from the tongue of R. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110 mM KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was -65.2 mV and the slope resistance 150 to 750 M omega. Pulse-depolarization from a holding voltage of -80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at -25 mV and was half-maximal at -8 mV. Steady-state inactivation was half-maximal at -67 mV and complete at -50 mV. Peak Na current averaged -0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5 mM) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5 mM) and 4-amino-pyridine (1 mM) did not block, but 5 mM Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted the IK(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10 mM Ca caused a reversible 5- to 10-mV depolarization in the current-clamp mode. Quinine (0.1 mM, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5 mM in the external solution or 0.5 microM in the pipette) caused reversible depolarization (to -40 to -20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1987

Action potentials in epithelial taste receptor cells induced by mucosal calcium.

Chemosensory cells in the taste bud of the tongue of Necturus generate action potentials in response to electrical stimulation through a microelectrode, as recently described by Roper (Science, 220: 1311-1312, 1983). We report that the epithelial receptor cells also respond to 10 mM CaCl2, applied to the mucosal surface, with a depolarization which elicits action potentials when a threshold of -50 mV is reached. Since CaCl2 is one of the taste stimuli in amphibia, the firing of action potentials by chemoreceptor cells may be part of the signal chain in gustatory reception of Ca ions.

Action Potentials↗
PubMed1987

Properties of an anion-selective channel from rat colonic enterocyte plasma membranes reconstituted into planar phospholipid bilayers.

Vesicles derived from epithelial cells of the colonic mucosa of the rat were fused to planar phospholipid bilayer membranes, revealing spontaneously switching anion-conducting channels of 50 pS conductance (at -30 mV with 200 mM Cl- each side). The equilibrium selectivity series was I- (1.7)/Br- (1.3)/Cl- (1.0)/F- (0.4)/HCO3- (0.4)/Na (less than 0.11). Only one dominant open-state conductance could be resolved, which responded linearly to Cl- concentrations up to 600 mM. The single-channel current-voltage curve was weakly rectifying with symmetrical solutions. When 50 mV were exceeded at the high-conductance branch of the curve, switching was arrested in the closed state. At more moderate voltages (+/- 40 mV) kinetics were dominated by one open state of about 35-msec lifetime and two closed states of about 2 and 9-msec lifetime. Of these, the more stable closed state occurred less often. At these voltages one additional closed state of significantly longer lifetime (greater than 0.5 sec) was observed.

Animals↗
PubMed1985

Angiostrongylosis in a greyhound.

Angiostrongylus vasorum infection was diagnosed at necropsy of a Greyhound imported from Ireland. Granulomatous masses and pulmonary arterial thrombosis were associated with adult and larval stages of metastrongyle nematodes. Hemorrhages, widespread in subcutaneous tissue and muscle masses and at serosal surfaces, were suggestive of the onset of the bleeding disorder that has been seen in angiostrongylosis in Europe. The mollusks that can serve as intermediate hosts are known to be common in the United States, and therefore establishment of this parasite from imported racing or breeding stock is possible.

Angiostrongylus↗
PubMed1985

Voltage dependence of the blocking rate constants of amiloride at apical Na channels.

The overall rate constants of blockage of apical Na channels by amiloride, previously determined by noise analysis, were obtained in macroscopic relaxation experiments with toad urinary bladders exposed to a mucosal Na activity of 60 mM. By the use of step voltage perturbations and by admittance analysis we show that the on-rate constant of blockage increases, and the off-rate constant decreases when the outer membrane surface is made more positive. In the frame-work of a plug-type blocking model the results imply that the cationic amidino group of amiloride senses about 10% of the membrane voltage while invading the channel entrace and slightly more than 10% while leaving the entrance.

Amiloride↗
PubMed1985

Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: I. Pyrazine-ring modifications.

The overall on- and off-rate constants for blocking epithelial Na channels by amiloride analogs were estimated by noise analysis of frog skin epithelium. The substituents at position-5 and -6 of the pyrazine ring of amiloride were varied in order to obtain the structure/rate constant relationship. (1) The off-rate constant increases with halo-substitutions at position-6 in the order Cl less than Br less than l less than F less than H. Substitution of Cl by H lowers the standard free energy of activation of the off-step by 2.3 kcal mol-1. The on-rate constant is not affected. Apparently the substituent at ring position-6 controls the duration of attachment in the blocking position. pKa considerations show that the duration is longer when the 6-substituent is more negatively polarized. We suggest that this substituent binds to the receptor by virtue of its electronegativity. (2) In contrast, replacement of the adjacent 5-amino group (electron donor) by H or Cl affects both the on-rate and the off-rate. The dual effect may be explained by a decrease of the electronic charge at more remote parts of the molecule (on-rate decrease), as well as at the 6-position (off-rate increase). Apparently the 5-amino group stabilizes the blocking position by increasing the electron density on the 6-ligand.

Amiloride↗
PubMed1985

Voltage dependence of Na channel blockage by amiloride: relaxation effects in admittance spectra.

Amiloride, present in the mucosal solution, causes the appearance of a distinct additional dispersion in the admittance spectrum of the apical membrane of toad urinary bladder. The parameters of this dispersion (characteristic frequency, amplitude) change with amiloride concentration and with membrane voltage. They allow the calculation of the overall rate constants for Na channel blockage by the positively charged form of amiloride, and the voltage dependence of these rate constants. The on-rate of blockage increases and the off-rate decreases when the membrane surface to which cationic amiloride has access, is made more positive. This result is suggestive of a blocking model where the cationic amidino group of amiloride, depending on its charge, senses 10 to 13% of the membrane voltage while invading the channel entrance by a single-step process, and rests at an electrical distance corresponding to 24 to 30% of membrane voltage while occupying the blocking position.

Amiloride↗
PubMed1985

Pattern analysis of 5S rRNA.

Some 200 different 5S rRNA sequences from eubacteria, chloroplasts, mitochondria, archaebacteria, and eukaryotes were analyzed for evolutionary kinship relationships and associated sequential features. Group-specific occupation schemes for the 149 positions of an overall alignment were established. Eubacterial, archaebacterial, and intermediate occupation schemes all yield a strongly biased base triplet pattern in one of the three possible reading frames strongest for eubacterial, chloroplastic, and archaebacterial, but still detectable for mitochondrial and eukaryotic cytoplasmic sequences. The frequency of triplets decays in the order RNY greater than RNR greater than YNY greater than YNR; R being a purine (guanine or adenine), Y is a pyrimidine (cytosine or uracil), and N is any base. A strong preference for guanine or cytosine was found in all triplet positions. The effects show no exceptions and are clearly above the level of statistical fluctuations.

Archaea↗
PubMed1984

Leakage-channel conductance of single (Na+ + K+)-ATPase molecules incorporated into planar bilayers by fusion of liposomes.

By fusing liposomes which contain in the mean only one pump unit (one intramembranous particle) to planar bilayers, and provoking the ouabain-blockable leakage conductance by the presence of n-decane, the predominant unit leakage conductance associated with one pump unit was estimated to be 40-50 pS, indicating the channel nature of the leakage pathway.

Electric Conductivity↗
PubMed1984

Feedback inhibition of sodium uptake in K+-depolarized toad urinary bladders.

Ouabain-blocked toad urinary bladders were maintained in Na+-free mucosal solutions, and a depolarizing solution of high K+ activity containing only 5 mM Na+ on the serosal side. Exposure to mucosal sodium (20 mM activity) evoked a transient amiloride-blockable inward current, which decayed to near zero within one hour. The apical sodium conductance increased in the initial phase of the current decay and decreased in the second phase. The conductance decrease required Ca2+ to be present on the serosal side and was more rapid when the mucosal Na+ activity was higher. At 20 mM mucosal Na+ and 3 mM serosal Ca2+ the initial (maximal) rate of inhibition amounted to 20% in 10 min. The conductance decrease could be accelerated by raising the serosal Ca2+ activity to 10 mM. The inhibition reversed on lowering the serosal Ca2+ to 3 microM and, in addition, the mucosal Na+ to zero. Exposure of the mucosal surface to the ionophore nystatin abolished the Ca2+ sensitivity of the transcellular conductance, showing that the Ca2+-sensitive conductance resides in the apical membrane. The data imply that in the K+-depolarized epithelia, cellular Ca2+, taken up from the serosal medium by means of a Na+-Ca2+ antiport, cause feedback inhibition by blockage of apical Na+ channels. However, the rate of inhibition is small, such that this regulatory mechanism will have little effect at 1 mM serosal Ca2+ and less than 20 mM cellular Na+.

Animals↗
PubMed1984

The continuous measurement of tubular volume changes in response to step changes in contraluminal osmolality.

A new method to measure time dependent (t) volume (V) changes in proximal straight tubules (PST) is described. V is calculated from diameter (d) measurements for which a video camera and an integrating circuit are used. A tubular image of high optical contrast is recorded with the TV camera such that the scan lines run crosswise to the tubule. The video signal is analyzed by a special processor which adds 225 tubular diameters of each TV frame and feeds this analog signal to a pen recorder. The fractional error in d measurements is 10(-3). Diameter changes of less than 0.05 micron can be detected, as compared to the usual error of a single measurement of about 0.4 micron. Pcbos, the osmotic water permeability of the contraluminal cell membrane was measured by setting up osmotic steps across it in less than 0.1 s and following the ensuing delta d/delta t. The time delay between solution change and the linear part of the osmotic response was 0.51 +/- 0.05 s. Pcbos was found to be 50.4 (+/- 8.7) X 10(-4) cm3.cm-2 of basement membrane area .s-1.osmolar-1.

Animals↗
PubMed1984

Analysis of additively contaminated Lorentzians by integration.

The two Lorentzian parameters, plateau power and corner frequency, can be retrieved from scattered spectral data points by forming the ratio of two numerical integrals of the data. With a small modification of this strategy, Lorentzians may be stripped from additive background spectra of unknown spectral shape. Recursive curve fitting is not required.

Ion Channels↗
PubMed1984

Fluctuation analysis of sodium channels in epithelia.

When compiling results obtained with various techniques, it appears that the apical Na translocators are channels of high Na, Li selectivity that permit large transport rates at low driving forces. The concentration dependence of the transport rate is linear at least up to 60 mM Nao. The voltage dependence is of the Goldman-Hodgkin-Katz type or closely related to it. Outward facing surface charges have little influence. The flux ratio exponent is close to unity, compatible with single-site channels or multisite channels of low occupancy. Noise analysis has proven particularly suited for following changes in the density of conducting channels in the living preparation, and has shown that cellular and hormonal regulatory mechanisms control Na transport by changing the channel density in a variety of ways.

Aldosterone↗
PubMed1983

Competitive blocking of epithelial sodium channels by organic cations: the relationship between macroscopic and microscopic inhibition constants.

Fluctuation analysis of Na current passing the apical membrane in the skin of Rana ridibunda was used to study the kinetics of Na-channel blocking by several organic cations present in the outer solution together with 60 mM Na. The ratios of the apparent off-rate and on-rate constants (the microscopic inhibition constants) thus obtained for triamterene, triaminopyrimidine (TAP), 5,6-diCl-amiloride, 5H-amiloride and amiloride itself are found to be in the mean about sevenfold smaller than the corresponding inhibition constants obtained from macroscopic dose-response curves. The apparent discrepancy is explicable by competition of the organic blocker with the channel block by Na ions (the self-inhibition effect). The type of interaction between extrinsic blockage and self-inhibition may be purely competitive or mixed. However, in case of mixed inhibition the competitive component must dominate the noncompetitive component by at least seven to one.

Amiloride↗
PubMed1983

Metabolic regulation of apical sodium permeability in toad urinary bladder in the presence and absence of aldosterone.

In the present study, further evidence was adduced for energy-dependent regulation of passive apical transport of Na in toad bladder epithelium. In potassium-depolarized preparations studied by current-voltage analysis, additions of pyruvate or glucose to the media of substrate-depleted bladders evoked proportionate increases in the transepithelial Na current and in apical Na permeability. These responses were large in aldosterone pretreated hemibladders and almost absent in the aldosterone-depleted preparations or when hormonal action was blocked by spironolactone or cycloheximide. The substrate-induced increases in apical Na permeability were fully reversed by appropriate metabolic inhibitors, i.e. 2-deoxyglucose and oxythiamine. Moreover, the inhibitory effect of 2-deoxyglucose was bypassed by the addition of pyruvate to the serosal medium. Thus apical Na permeability is clearly sensitive to the supply of cellular energy. The possibility that changes in intracellular free Na activity may mediate metabolic regulation of apical Na permeability was evaluated by prolonged exposure to Na-free mucosal and serosal media, with and without inhibition of the Na/K-pump by ouabain. The stimulatory and inhibitory effects of pyruvate, 2-deoxyglucose and oxythiamine on Na currents and Na conductances were preserved under these circumstances. Furthermore, reduction of serosal Ca to a minimal level of 3 microM, was without effect on the response to metabolic inhibition. These experiments demonstrate the existence of Na-independent metabolic regulation of apical Na transport and imply that neither basal-lateral nor mitochondrial Na/Ca exchange is required for this regulatory process under the imposed conditions. The possibility that a Na-independent, Ca transport mechanism in mitochondria or endoplasmic reticulum may be involved in metabolic regulation of apical Na transport, however, remains to be evaluated.

Aldosterone↗