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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 86 (1985), S. 255-265 
    ISSN: 1432-1424
    Keywords: amiloride ; relaxation ; admittance ; current-voltage curves ; epithelial Na channels ; toad urinary bladder
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary 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.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1424
    Keywords: amiloride ; blocking kinetics ; structure-activity relationship ; Na channels ; frog skin ; fluctuation analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary 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〈Br〈I〈F〈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. pK a 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.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 105 (1988), S. 233-243 
    ISSN: 1432-1424
    Keywords: chemoreception ; olfaction ; periodic discharge ; cyclic AMP ; amiloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Olfactory receptor cells were isolated from the nasal mucosa ofRana esculenta and patch clamped. Best results were obtained with free-floating cells showing ciliary movement. 1)On-cell mode: Current records were obtained for up to 50 min. Under control conditions they showed only occasional action potentials. The odorants cineole, amyl acetate and isobutyl methoxypyrazine were applied in saline by prolonged superfusion. At 500 nanomolar they elicited periodic bursts of current transients arising from cellular action potentials. The response was rapidly, fully and reversibly blocked by 50 μm amiloride added to the odorant solution. With 10 μm amiloride, the response to odorants was only partially abolished. 2)Whole-cell mode: Following breakage of the patch, the odorant response was lost within 5 to 15 min. Prior to this, odorants evoked a series of slow transient depolarizations (0.1/sec, 45 mV peak to peak) which reached threshold and thus elicited the periodic discharge of action potentials. These slow depolarizing waves were reversibly blocked by amiloride, which stabilized the membrane voltage between −80 and −90 mV. We conclude that amiloride inhibits chemosensory transduction of olfactory receptor cells, probably by blocking inward current pathways which open in response to odorants.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 105 (1988), S. 245-255 
    ISSN: 1432-1424
    Keywords: sensory cells ; taste-and chemoreception ; gustatory senses ; salt-taste ; patch-clamp whole-cell recording ; Na channels ; amiloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Isolated taste receptor cells from the frog tongue were investigated under whole-cell patch-clamp conditions. With the cytosolic potential head at −80 mV, more than 50% of the cells had a stationary inward Na current of 10 to 700 pA in Ringer's solution. This current was in some cells partially, in others completely, blockable by low concentrations of amiloride. With 110mm Na in the external and 10mm Na in the internal solution, the inhibition constant of amiloride was (at −80 mV) near 0.3 μm. In some cells the amiloride-sensitive conductance was Na specific; in others it passed both Na and K. The Na/K selectivity (estimated from reversal potentials) varied between 1 and 100. The blockability bysmall concentrations of amiloride resembled that of channels found in some Na-absorbing epithelia, but the channels of taste cells showed a surprisingly large range of ionic specificities. Receptor cells, whichin situ express these channels in their apical membrane, may be competent to detect the taste quality “salty.” The same cells also express TTX-blockable voltage-gated Na channels.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 95 (1987), S. 171-185 
    ISSN: 1432-1424
    Keywords: amiloride ; analogs ; blocking kinetics ; structure-activity relationship ; Na channels ; frog skin ; fluctuation analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary 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. (1) 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. (2) 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. (3) 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. (4) 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.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 95 (1987), S. 91-103 
    ISSN: 1432-1424
    Keywords: nystatin ; amiloride ; basolateral membrane ; toad urinary bladder ; Na+ transport ; epithelial impedance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 Ω cm2. The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm 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 SO 4 2− Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K+. This pathway can be blocked by serosal amiloride (K i=13.1 μm) or serosal Na+ ions (K i∼ 10 to 20mm). It also conducts Li+ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec−1 at 0 mV.
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  • 7
    ISSN: 1432-1424
    Keywords: sensory cells ; chemoreception ; taste ; gustatory senses ; taste receptor cells ; epithelial action potentials ; amiloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Apical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-μm opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10–100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50–900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250mm, the maximal collective spike rate of a bud was in the range of 6–10 sec−1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5–2 sec−1. Buds of pure phasic response were also observed. Amiloride (30 μm) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 μm). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud. This is understandable since the stationary current of a bud arises from a multitude of taste cells, while each current spike is presumably generated by just one taste cell. We suggest that, in a Na-sensitive receptor cell, (i) the apical amiloride-blockable Na inward current serves as a generator current causing cell depolarization and firing of action potentials, and (ii) each current spike recorded from the taste pore arises mainly from a modulation of the apical Na inward current of this cell, because the action potential generated by the taste cell will transiently decrease or abolish the driving force for the apical Na inward current. The transients are indicators of receptor cell action potentials, which appear to be physiological responses of taste cellsin situ.
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