In an attempt to study the properties of acetylcholine receptors in intestinal smooth muscle, measurements have been made of the uptake of tritium-labelled atropine and methylatropinium, and of
14
C-labelled methylfurmethide by the longitudinal muscle of guinea-pig small intestine
in vitro
. Substantial amounts of atropine were taken up from very dilute solutions, a clearance of 160 ml. per g tissue (wet weight) being achieved at the lowest concentration tested (1.5 × 10
-10
M). Analysis of the curve relating atropine uptake at equilibrium to the bath concentration, which was explored over a concentration range 1.5 × 10
-10
M to 2.5 × 10
-3
M, enabled three components to be distinguished: (1) A binding site with a capacity of 180 pmoles/g, and equilibrium constant 1.1 × 10
-9
M. (2) A binding site of capacity about 1000 pmoles/g and equilibrium constant about 5 × 10
-7
M. (3) A compartment with a clearance of 4.7 ml./g (nonsaturable). The equilibrium constant of the first binding site agreed exactly with that measured for acetylcholine antagonism in the same tissue. Methylatropinium was taken up in rather smaller amounts than atropine, and analysis of the uptake curve showed a binding site of capacity about 90 pmoles/g with an equilibrium constant 6.5 × 10
-10
M, an ill-defined series of binding sites with much higher equilibrium constants, and a constant clearance of about 0.4 ml. /g. Analysis of this curve was much less clear cut than that of atropine. The equilibrium constant for blockade of acetylcholine receptors by methylatropinium was 4.7 × 10
-10
M. Atropine was not taken up appreciably by striated muscle, nerve or tendon of the guineapig; hydrolysed atropine was not taken up by smooth muscle (and lacks atropinic activity); cocaine and
d
-tubocurarine in high concentrations did not affect atropine uptake; lachesine and benzhexol blocked atropine uptake competitively at low concentrations, and with lachesine the equilibrium constant for this interaction agreed with that measured for acetylcholine antagonism (1.4 × 10
-9
M). These findings suggested that the atropine taken up could be related to receptor-bound drug. The kinetics of atropine uptake and washout were studied over the concentration range 0.5-5 × 10
-9
M. Uptake and washout took place approximately exponentially between 2½ and 50 min, and the rate constant was 4.5-5 × 10
-4
s
-1
for both uptake and washout. The uptake rate constant did not increase with concentration. This contrasted with the kinetics of receptor blockade, which took place much faster, with a rate constant which increased linearly with concentration, in accordance with the theoretical kinetic behaviour of a single binding site. This finding precluded a simple identification of atropine taken up with receptor-bound drug. Studies with various metabolic inhibitors suggested that no metabolic energy was required for the accumulation of atropine, and by dialysis experiments, the atropine taken up was shown to be bound in homogenized tissue. A theoretical study, using an analogue computer, was made of the kinetic properties of three passive binding systems, in order to see whether the observed kinetic behaviour could be simulated. It was found that a system of four binding sites in series, with only one communicating directly with the surrounding medium, could show these kinetic properties, and the outermost binding site could still show the kinetic behaviour of receptors. Experimental testing of this model demands more accurate kinetic measurements than can be made by the method used in this study. The acetylcholine-like stimulant, methylfurmethide, was taken up very slowly (taking more than 24 h to reach equilibrium), reaching a clearance of about 5 ml. /g after 6 h. This uptake was unaffected by atropine in a concentration sufficient to block 80% of acetylcholine receptors, but was blocked by depolarization in high potassium solution, suggesting that it was behaving passively as a slowly permeant cation. No uptake referable to acetylcholine receptors was detected. These findings are discussed in relation to the abundance and chemical behaviour of acetylcholine receptors in smooth muscle, and in relation to current theories of drug action.
Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change