Biochemical Pharmacology. Vol. 28. pp. 2799-2805. FJ Pergamon Press Ltd. 1979. Printed in Great Britain. 0006.2952/79lO91?-2799 $02.0010 IN k%‘-7tO EXPERIMENTS ON THE ACCUMULATION AND RELEASE OF 14C-HISTAMINE BY SNAIL (HELIX POMA TIA) NERVOUS TISSUE N. N. OSBORNE,* K.-D. WOLTER and V. NEUHOFF Max-Planck-Institut fir experimentelle Medizin, Forschungsstelle Neurochemie. Hermann-Rein-Str. 3, 3400 GGttingen, West Germany zyxwvutsrqponmlkjihgfedcbaZY (Received 3 1 January 1979; accepted 10 April 1979) Abstract-When isolated snail ganglia were incubated at 25’ in a medium containing 14C-histamine, tissue:medium ratios of about 4 were obtained after only 5 min. Metabolism of the accumulated amine is rapid, for even within this short incubation period, 20 per cent of the substance was metabolised. The process responsible for this accumulation showed properties of an active transport system: it was temperature- sensitive, sodium dependent, and particularly inhibited by ouabain, phenoxybenzamine, chlorpromazine and desimipramine. The accumulation of “‘C-histamine showed saturation kinetics typical of a carrier-mediated process and can be divided into sodium-sensitive and -insensitive components. Kinetic analysis of the data revealed similar K, values for both sodium-sensitive and sodium-insensitive plus -sensitive components, i.e. 1O-5 M. A rapid efRux of radioactivity from tissue loaded with ‘Y-histamine was observed when exposed to 70 mM KCl. This release was inhibited when the calcium content in the medium was reolaced bv sucrose. Moreover. cobalt ions added to the incubation medium counteracted the effect of KC< ’ The discussion of the present results is based on the hypothesis that histamine has a transmitter function in the snail CNS. Despite the existence of a vast amount of biochemical and physiological data on histamine in the vertebrate central nervous system, the actual function of this substance is not understood, although a transmitter role is suspected (see ref. [ 11). Recent studies on certain invertebrate preparations (viz. gastropod molluscs) containing large neurones have now fortified the suppo- sition that histamine can function as a transmitter. For example, it has been shown that certain neurones in the gastropods contain this specific amine i2-41 and the enzyme which is required to synthesise histamine from histidine, histidine-decarboxylase, exists only in these neurones [ 5,61. Furthermore, it has been demonstrated that neurones receiving monosynaptic connections from a defined histamine-containing neurone respond to exogenously applied histamine in a manner qualita- tively similar to the effect produced by histamine stimu- lation [7]. These data thus provide convincing argu- ments in favour of histamine functioning as a transmitter in the gastropods (e.g. the snail Heltipoma- tia) and therefore probably in the vertebrate CNS as well. One requisite of a transmitter substance is that it is rapidly inactivated after delivery of its message. Acetyl- choline is inactivated by the enzyme acetylcholinester- ase at cholinergic synapses [S, 91 whereas dopamine and S-hydroxytryptamine are inactivated by specific high affinity re-uptake mechanisms in both vertebrate and invertebrate (i.e. Helix) nervous tissue [ 10-121. The aim of the present work was to see whether an active high affinity uptake system for histamine exists in the snail CNS. Turner and Cottrell’s report [ 3 I suggests - * Present address: Nuffield Laboratory of Ophthalmol- ogy, The University of Oxford, Walton Street, Oxford, England. that such a mechanism may exist. By means of autora- diography they demonstrated that a certain neurone in the pond snail Ly mnea specifically accumulated hista- mine. Experiments have also been undertaken on the KCl-induced release of radioactivity from snail tissue loaded with 14C-histamine in order to gain more infor- mation on the functional role of the amine. METHODS Helix pomatia were obtained from Alfred Koch, 3450 Holzminden, G.F.R., and kept at room tempera- ture in a moist atmosphere for 24 hr before use. Suboe- sophageal ‘ganglia from a number of animals were rapidly dissected and placed in a beaker containing cold snail saline. The snail saline [ 131 consisted of NaCl (3.45 g/l), KC1 (0.43 g/l), CaCl, (1.17 g/l), NaHCO, (1.0 g/l) and MgCl, (1.55 g/l). Each ganglion was subsequently blotted dry on filter paper, weighed (4- 6 mg) and placed in avial containing 2 ml ice-cold snail saline. After a preincubation of 5 min at 25’ in a shaking water bath, various amounts of [2- 14C Ihistamine (Radiochemical, Amersham, 5 9.7 mC i/ mmole) were added to the incubation medium and the incubation was continued for varying periods of time. At the end of the incubation the ganglia were recovered with forceps and rapidly rinsed in 20 ml ice-cold saline. It had been previously established that no significant release of radioactivity from the tissues occurred during the washing process. Individual ganglia were then placed in vials containing 0.5 ml tissue solubilizer (So- luene-350 Packard) for at least 2 hr at room tempera- ture before adding 10 ml Dimilume (Packard). Radio- activity was measured in a Packard liquid scintillation spectrometer. A small amount (50- 100 ~1) of radioac- tive incubation mixture dissolved in 10 ml Dimilume 2799