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It was previously shown that in a radiochemical assay using anion exchange resin the capacity to synthesize acetylcholine in salivary glands was markedly overestimated unless suitable controls were introduced. By the use of paper electrophoresis of the incubation mixture it is shown that the labelled acetyl group is not only transferred from acetyl-Coenzyme A to choline but also to carnitine in both salivary glands and urinary bladders. The two enzymes responsible for these processes, choline acetyltransferase and carnitine acetyltransferase, are shown to behave differently following denervation of the glands and bladders. Further, the adequacy of the introduced controls is demonstrated.

The biosynthesis of acetylcholine and the fate of intravenously administered choline [methyl- 3-H] were studied in guinea pigs anesthetized with pentobarbital. Choline and acetylcholine were isolated by paper electrophoresis and estimated by use of a specific enzymatic (choline kinase) - radioisotopic assay. The concentration of acetylcholine ranged from 25.5 to 1.1 nmol/g in the following tissues (in order of decreasing concentration): duodenum, corpus striatum, stomach, cerebral cortex, spinal cord, abdominal fat, submaxillary gland, kidney, adrenal gland, spleen, liver, lung, heart and diaphragm. Choline [methyl- 3-H] was converted in the tissues to acetylcholine within 3 minutes after intravenous administration of the precursor. Virtually all the radioactivity in plasma at that time was present as free choline, suggesting that free choline from plasma is the immediate precursor for acetylcholine synthesized in the tissues cited. The concentration of free choline in tissues ranged from 344 nmol/g in adrenals to 40 nmol/g in heart, while that in plasma was 15 nmol/g. The initial half-life of choline in plasma, estimated from the rate of disappearance of choline after intravenous administration of either a tracer dose of choline [methyl- 3-H] (0.031 mumol/kg) or a high dose of choline chloride (200 mumol/kg), was less than 1 minute. This rapid removal of choline from plasma resulted from uptake (or binding) by tissues, with kidney and liver removing about 50% of the administered dose of choline [methyl- 3-H] within 3 minutes after its administration. Uptake of choline occurred in all tissues cited above, but there was a 20-fold difference in the uptake by the most active tissues (kidney and adrenals), as compared to that of the least active (central nervous system). Within 60 minutes after administration of choline [methyl- 3-H], most of the radioactive choline taken up by tissues had been converted to organic-soluble metabolites and to water-soluble metabolites that behaved like either phosphorylcholine or betaine during paper electrophoresis and chromatography. Betaine was the principal metabolite of choline in plasma. Radioactivity was excreted slowly into urine, which contained primarily free choline, betaine and a large amount of an unidentified metabolite. These findings indicate that the principal mechanism for the rapid removal of choline from plasma is uptake into tissues followed by metabolism.

Innervated strips of longitudinal muscle of guinea pig ileum were incubated with 3H-choline in the presence of physostigmine, after which the efflux of tritiated metabolites was studied. Under resting conditions 3H-acetylcholine, 3H-choline and other tritiated metabolites were released into the incubation medium. Analysis of the kinetics of the release of the metabolites indicated that the release occurred from at least 2 different compartments. Under the unstimulated conditions used, between 72 and 78% of the radio-activity remained in a third, non-releasable compartment. The release of 3H-acetylcholine showed spontaneous fluctuations which were positively correlated to spontaneous variations of the isometric tension of the preparation. Electrical field stimulation at low frequency (0.5 Hz) consistently increased the release of 3H-acetylcholine. The release of 3H-choline was increased only slightly or not at all by the electrical stimulation. For studying stimulation-evoked release of 3H-acetylcholine, it is suggested that the measurement of the total quaternary amine release is of similar value to measurement of 3H-acetylcholine itself. This approach considerably simplified the method. During 5 consecutive stimulation periods of 150 pulses each, the increase in the release of total quaternary amines diminished with time. By taking into account the reduction of the stores of radioactivity in the tissue during the experiment, correction for the negative trend could be made. When the preparation was stimulated with between 30 and 3000 pulses a close correlation was found between the increase in the release of total quaternary amines and the number of pulses delivered. The volley output of the quaternary amines was not constant, however, but diminished by increasing stimulation frequency.

Human retinas have the capacity to synthesize and release [3H]acetylcholine ([3H]ACh) after an incubation in [3H]choline ([3H]Ch). Synthesis of [3H]Ch by retinal homogenates was determined using either high-voltage paper electrophoresis (HVPE) or a two-step enzymatic/extraction assay for separating [3H]ACh from [3H]Ch. The enzymatic/extraction assay is shown to be accurate over a wide range of concentrations (10(-6)-10(-12) M). Homogenates of human retina synthesize [3H]ACh from [3H]Ch. We find an approximate Km of 50 microM and a Vmax of about 20 nmol/mg protein/h (at 37 degrees C) for the synthesis of labeled ACh by retinal homogenates. Human retinas also release [3H]ACh after a pulse of [3H]Ch. Release of labeled transmitter is stimulated by potassium depolarization. The potassium-stimulated release is partially blocked by magnesium or cobalt ions. Release data were analyzed by both the enzymatic/extraction assay and HVPE; the results are qualitatively identical in both cases. The data reported here provide additional evidence for cholinergic neurotransmission in the human retina.

For determination of possible neurotransmitters synthesized by photoreceptor cells, turtle retinas were dissociated into single cells with proteolytic enzymes. These cells were partially separated by velocity sedimentation to yield a fraction rich in photoreceptors. Individual photoreceptor cells were then sucked into a micropipette and incubated with labeled precursors of known or suspected neurotransmitters. After incubation, the radioactive products were analyzed by high-voltage electrophoresis. Of all the chemicals tested, turtle photoreceptor cells synthesized only acetylcholine, suggesting that these cells may be cholinergic.