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A W Cuthbert

Publications and source records attributed to A W Cuthbert.

18 recordsLinked to original sources
PubMed1979

Synthesis, properties and biological activity of tritiated N-benzylamidino-3,5-diamino-6-chloro-pyrazine carboxamide -- a new ligand for epithelial sodium channels.

A method is described for the synthesis and purification of tritiated N-benzylamidino-3,5-diamino-6-chloro-pyrazine carboxamide (benzamil). The tritium was inserted at the meta position of the benzyl ring, from which it apparently does not exchange with solvent hydrogen. When stored in ethanol at -4 degrees C the radioligand remains stable for at least 15 months. The pharmacology of benzamil is very similar to that of amiloride in terms of its effects on sodium transporting epithelia except that it has a higher affinity. The affinity of benzamil for sodium channels in amphibian epithelia in the absence of sodium is approximately 10(9) M-1. The new ligand can be used to label sodium channels in epithelia, and may be useful in channel isolation procedures.

Amiloride↗
PubMed1979

Estimation of the density of sodium entry sites in frog skin epithelium from the uptake of [3H]benzamil.

1. The inhibition of short circuit current in frog skin by benzamil (N-benzyl-amidino-3,5-diamino-6-chloropyrazine carboxamide) was investigated. When skins were bathed on both sides by Ringer solution (pH 7.6) the affinity was 5 x 10(7) M-1. When the sodium concentration was reduced to 1.1 mM and the pH adjusted to 6.5 the affinity increased to 8.5 x 10(8) M-1. 2. A method is described for measuring uptake of [3H]benzamil into the mucosal (outer) surface of pieces of isolated epithelium, 0.95 cm2 in area, under open circuit conditions. 3. The relation of [3H]benzamil uptake at the mucosal surface to its concentration was measured in solutions containing 1.1 mM-sodium and adjusted to pH 6.5. Uptake could be resolved into a linear component (10.2 f-mole nM-1) and a saturable component (21.5 f-mole cm-2) with a half saturating concentration of 1 nM. 4. In the presence of amiloride (1 microM) or unlabelled benzamil (1 microM) uptake was linear with concentration, and was, respectively, 9.2 f-mole nM-1 and 8.8 f-mole nM-1. When the pH was reduced to 3.5 uptake was again linear but reduced to 3.3 f-mole nM-1. 5. The identity of the saturable component of [3H]benzamil uptake to sodium entry sites is discussed. The results suggest a sodium entry site density of around 130 micron-2 of mucosal surface.

Amiloride↗
PubMed1979

Uptake of [3H]benzamil at different sodium concentrations. Inferences regarding the regulation of sodium permeability.

1. The effect of benzamil on short-circuit current in frog skin was measured at different external sodium concentrations. A linear relationship exists between the concentration of benzamil reducing short-circuit current by 50% and the external sodium concentration, indicative of some form of competitive antagonism between sodium and benzamil. 2. Uptake of [3H]benzamil into isolated frog skin epithelium and whole skin (0.95 cm2 pieces) was measured at different external sodium concentrations. With a sodium concentration of 111 mM in the external medium the uptake of [3H]benzamil is linear with concentration. Uptake amounted to 8.8 f-mole nM-1, a value similar to the linear component of the uptake measured at low (1.1 mM) sodium concentration. 3. Using a variety of other conditions the maximal number of specific binding sites for [3H]benzamil was calculated from displaceable binding and the fractional occupancy, the latter being derived from the inhibition of short-circuit current. This approach gave similar binding site densities to those reported previously at low sodium concentrations. 4. The reduction in specific [3H]benzamil uptake at high sodium may result from two mechanisms, competition of sodium with the ligand for an external binding site and a reduction in the site density as the intracellular sodium concentration increases. 5. It is concluded that the saturation of sodium transport which occurs at high sodium concentration is likely a consequence of the reduced availability of entry sites, rather than saturation of the uptake process.

Amiloride↗
PubMed1978

Interdependence of the two borders in a sodium transporting epithelium. Possible regulation by the transport pool.

Specific binding of 14C-amiloride to the mucosal surface of frog skin epithelium (Rana temporaria) has been used as a measure of the number of sodium entry sites. All binding measurements were made with the mucosal surface bathed in a solution containing 1.1 mM sodium. When manipulations were used which increased the intracellular concentration of sodium the amount of amiloride bound was reduced. The manipulations included flushing the mucosal surface with solutions containing 111 mM sodium after serosal efflux was inhibited with ouabain or potassium removal. Similar results were obtained when cells were loaded with lithium. These effects on amiloride binding did not appear to depend on changes in membrane potential or upon changes in affinity of amiloride for its binding site. It appears that inhibition of serosal sodium efflux from the epithelium causes a reduction of mucosal sodium influx by making entry sites unavailable. This latter may be a result, directly or indirectly, of the sodium concentration in the sodium transport pool.

Amiloride↗
PubMed1978

Effects of some pyrazinecarboxamides on sodium transport in frog skin.

1 The inhibitory effect of amiloride (N-amidino-3,5-diamino-6-chloropyrazinecarboxamide) on sodium transport in isolated skin of frog has been compared with 17 of its analogues. The dissociation constant of amiloride for passive sodium channels was 181.9 +/- 8.9 nM, and the maximal percentage inhibition of sodium transport was 101.3 +/- 0.4% (means of 123 measurements) when measured at a sodium concentration of 111 mM. 2 The N-benzylamidino and N-o-chlorobenzylamidino compounds had affinities approximately 20 times larger than those for amiloride, and produced maximal inhibition of transport. 3 Substitution of chlorine in the 6-position by other halogens showed that the bromo-compound was equally active to amiloride, whereas the iodo derivative had an affinity equal to 15% of that for amiloride. 4 Substitution in the 5-amino group in 10 compounds reduced the affinities to less than 1% of that of amiloride, without affecting their ability to produce complete inhibition of transport. 5 N-Amidino-3,5-diaminopyrazinecarboxamide was unique in that it produced an unusual concentration-response relationship.

Amiloride↗
PubMed1976

Similarities between sodium channels in excitable membranes and in epithelia.

The inhibitory effects of the pyrazine derivative, amiloride, on sodium transport in an amphibian epithelium has been studied as a function of pH. It is concluded that the charged (guanidinium) group interacts with a negatively charged acid grouping in the membrane. Similarities between sodium channels in excitable membranes and epithelia are highlighted.

Amiloride↗
PubMed1976

Estimation of the lifespan of amiloride binding sites in the membranes of toad bladder epithelial cells.

1. Sodium entry sites in the membranes of isolated epithelial cells prepared from bladders of toads (Bufo marinus) have been labelled with amiloride. The number of binding sites remained constant in suspensions for up to 100 hr. 2. In the presence of a protein synthesis inhibitor (cycloheximide, 0-5 mug/ml.) there was a decline in the density of binding sites was approximately exponential. Regression analysis gave a half-life of approximately 60 hr. 3. Aldosterone (5 X 10(-8) M) caused a significant (P less than 0-001) increase (50%) in the density of amiloride binding sites. Cells which had been treated with aldosterone had populations of binding sites which declined, in the presence of cycloheximide, at rates indistinguishable from those of untreated cells.

Aldosterone↗