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Biomedical subjects

A W Cuthbert

Publications and source records attributed to A W Cuthbert.

At least 109 records · Page 6Linked to original sources
PubMed1982

Kinetics of amiloride action in the hen coprodaeum in vitro.

The kinetics of amiloride action on the isolated epithelium of the hen coprodaeum are reported. Tissues were taken from birds fed on low salt diets for 9-10 days, conditions which induce a high resting short circuit current due to sodium and sensitive to amiloride. The relation between the inhibition of amiloride sensitive short circuit current and blocker concentration obeyed simple mass laws with an apparent stoichiometry of 1:1 between amiloride and the sodium entry sites. The concentration of amiloride producing its half maximal effect (Ki) was 1.77 +/- 0.20 microM at a sodium concentration of 130 mM. There was a shallow dependence of Ki on sodium concentration, the value of Ki falling to 0.78 +/- 0.1 microM at 1.3 mM Na. The relation of Ki to Na concentration was linear indicating competitive antagonism. The sodium concentration which half saturates the amiloride site (KNa) was 80 mM. This value is very different from the concentration of sodium which half saturates SCC (Kscc = 5-7 mM) suggesting there are at least two sites at which sodium can modify the transporting characteristics. These data are compared to those for other epithelia where Kscc and KNa are rather similar. The benzyl derivative of amiloride (benzamil) was found to be 11.6 times more potent than amiloride on this tissue. The potency ration is similar to that for other sodium transporting epithelia suggesting that the structure of the ion translocation mechanism is partly conserved between species although the Ki for amiloride may vary by an order of magnitude.

Amiloride↗
PubMed1982

Identification of potential components of the transport mechanism for Na+ in the hen colon and coprodaeum.

The binding of 3H-benzamil to homogenates of epithelium removed from the colon and coprodaeum of hens was studied. A low capacity high affinity binding component was detected. The binding constants for benzamil and amiloride to this component were similar to those obtained when these ligands were used to inhibit transport in intact epithelia. The mean potency ratio of benzamil to amiloride was 12.8 measured by binding compared with 11.6 from functional inhibition. Binding activity was present in tissues taken from animals fed on low sodium diets and those containing a normal sodium content. In the presence of sodium the affinity of benzamil was slightly reduced, but only in tissues taken form animals on a low salt diet. Only a small fraction of the total binding activity was present in the apical surface of low salt tissues indicating that in homogenates a small percentage of the total activity is associated with functional sodium entry sites. It is suggested that the major part of the binding activity detected in homogenates represents components of the sodium ion translocation mechanism which are en route for the apical membrane.

Amiloride↗
PubMed1982

Kinins stimulate net chloride secretion by the rat colon.

1 Short circuit current (SCC), transepithelial conductance and ion fluxes were measured across the isolated descending colon of the rat in response to bradykinin or kallidin. 2 Kinins added to the serosal bath caused immediate increases in SCC but were ineffective when added to the mucosal bath. Increases in SCC were accompanied by significant increases in transepithelial conductance. Threshold kinin concentration was 0.5nM and maximal increases were seen at 50-100 nM. 3 A rat glandular kallikrein (7nM) or mellitin (2 microM) also increased SCC if added to the serosal bath. 4 Responses to kinins were unaffected by mucosal amiloride (100 micron) but attenuated or blocked by serosal frusemide (100 microM), indomethacin (1 microM) or mepacrine (50 microM). 5 Replacement of chloride ion in the serosal bath by gluconate and sulphate ions abolished responses to kinins which reappeared after chloride re-addition. 6 Measurement of 36Cl, 22Na and 86Rb fluxes across the tissue showed that the kinin-induced increase in SCC resulted principally from increased net chloride secretion. Effects upon 22Na or 86Rb flux were minimal and made no contribution to the current responses observed in this tissue. 7 The results prove that kinins stimulate net chloride secretion in the rat colon, most probably via a prostaglandin-dependent pathway.

Animals↗
PubMed1982

Stimulation of sodium and of chloride transport in epithelia by forskolin.

The diterpene, forskolin, is shown to produce a concentration-dependent, increase in short circuit current in two epithelial preparations, amphibian skin and rat colon. In the amphibian tissue the increase is sensitive to amiloride and due to an increase in electrogenic transepithelial sodium transport towards the serosal side. In the rat colon piretanide attenuated the forskolin effect, suggesting the terpene increases electrogenic transepithelial chloride transport towards the mucosal side. Half-maximal activation of both processes was achieved with concentration of 1-3 microM, similar to those required to activate half-maximally the catalytic subunit of adenylate cyclase.

Animals↗
PubMed1982

Irreversible inhibition of epithelial sodium channels by ultraviolet irradiation.

1 The effects of u.v. irradiation at 254 nm and 350 nm on sodium transport across frog skin epithelium have been investigated. 2 Irradiation at 254 nm but not at 350 nm produces a dose-dependent, functionally selective blockade of sodium transport. The effect is apparently due to the irreversible closure of apical sodium channels. 3 The amiloride-sensitive conductance was directly related to sodium transport as measured by short circuit current (SCC) both in normal and irradiated tissues, although both conductance and current were reduced in irradiated tissues. 4 The sensitivity of epithelia to irradiation at 254 nm was defined from the rate constants for the decline in SCC during three 2 min periods of irradiation at 1850 microW cm-2. The rate constant for the initial 2 min irradiation was 0.093 +/- 0.008 min-1. 5 Lowering the sodium concentration to 5.5 mM from 110 mM increased the rate constant to 0.141 +/- 0.014 min-1, consistent with the view that more functional sodium channels exist at lowered sodium concentration. 6 Lowering the temperature to 7 degrees C from 23 degrees C reduced the rate constant to 0.032 +/- 0.007 min-1 suggesting that blockade of channels is not due to a direct interaction with photons. 7 Using a variety of experimental protocols we were unable to demonstrate that bromamiloride or iodoamiloride can act as photoligands for sodium channels in the epithelium of Rana temporaria. This is in contrast to earlier reports with other epithelia.

Amiloride↗
PubMed1981

Mechanisms for the effects of acetylcholine on sodium transport in frog skin.

In frog skin (Rana temporaria) acetylcholine applied to the serosal surface produces either a sustained inhibiton or sustained stimulation of short-circuit current (SCC). The former effect is accompanied by a reduction and the latter by an increase in total tissue conductance. Both effects of acetylcholine can be accounted for, within experimental error, by changes in net sodium flux across the tissue. By use of selective agonists and antagonists it is concluded that acetylcholine interacts with muscarinic receptors in the serosal membrane. The effects of cholinoceptor agents are also seen with isolated epithelium. The stimulatory effect of acetylcholine is potentiated by theophylline and blocked by inhibitors of prostaglandin synthetase and by mepacrine. It is suggested that acetylcholine stimulates transport by liberating prostaglandins which may then activate adenylcyclase. The inhibitory effect of acetylcholine is correlated with a reduction in cyclic AMP content of the epithelium. Calcium appears to be an important determinant of the type of response seen eith acetylcholine, but the mechanism is not known.

Acetylcholine↗
PubMed1981

Adhesive properties of MDCK cell membranes possible role in epithelial histogenesis.

MDCK cells in culture form a functional transporting epithelium. Apical surface of MDCK monolayers are not adhesive to free MDCK cells that fail to grow attached to existing monolayers. In contrast, when plated at high density (106 cm-2) MDCK cells form multilayered structures in which only the outermost layer shows a typical apical surface (microvilli, tight junctions, etc). In the lower layers cell-cell contacts (desmosomes, etc) are made, suggesting that the basolateral surfaces remain adhesive. Using a novel, quantitative, heterotypic cell adhesion reaction with aged human red cells, were have measured the adhesive properties of the apical surfaces of MDCK cells in a variety of conditions. It is found that the adhesion of red cells is dependent on cell density in the monolayer and falls to a low value at confluence. The reaction is considerably stronger at 6 degrees C compared to 22 degrees C (the temperature of most studies), while at 37 degrees C the reaction is expressed less strongly. MDCK cells with adherent red cells fail to divide, suggesting that the cell surface fluence. The expression of the adhesive property is associated with cell division. This was shown by inducing synchrony in cultures either by refeeding starved cultures or by removal of a thymidine " block". Peaks in adhesiveness were related to peaks in thymidine incorporation in synchronized cultures. Because of the precise quantitative way in which adhesion can be measured it is considered that this is an ideal system in which to identify the membrane components responsible for the reaction. Furthermore, it may prove possible to identify signals that arise as an immediate consequence of the adhesion reaction.

Animals↗
PubMed1980

Increase in epithelial cyclic adenosine 3',5'-monophosphate following vanadate.

Vanadate increases the cyclic adenosine 3',5'-monophosphate (cyclic AMP) content of frog skin epithelium and apparently antagonizes the stimulation by isoprenaline. The effect appears to be a direct activation of adenyl cyclase. This new effect of vanadate together with the inhibitory effects on Na-K ATPase may explain the irregular effects on sodium transport.

Animals↗
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↗