Catecholamine secretion from the adrenal gland treated with neuraminidase, protamine or alkylating agent.

It has 1-ccn shown that brain gangliosides are concentrated in microsomal fractions and in synaptic membranes (1-3) and that the increased respiratory responses of cere bral cortex slices to stimulation are inhibited by basic proteins such as histones and protannines and all:ylating agents which form covalent linkages in aqueous solutions with carboxylic and other acidic groupings (4, 5). inhibition of the responses can be restored by polyacidic substances such as ga:iglioside and phosphatidylserine (4). On the other hand a possible role of ganglioside in release of acetyl~eholine from synaptic vesicles was also shown (6). It may be of interest to determine the intracellular distribution of gangliosides in the adrenal medulla as well as the possible effect of protamines, neuraminidase and nitro gen mustard of the adrenal medullary secretion. Soon after death bovine adrenal glands obtained from the local slaughterhouse were placed on ice, then prepared for medullary cell-fractionation and perfusion. The medullae were honsoge=?izcd in two volumes of 0.25 M sucrose. Each 0.8 iii] of the homogenate was centrifuged in a 0,?-2.3 iM sucrose gradient in the 5-ml Spinco preparative ultracentri fuge tubes at 125,000 x g for 30 min according to the method of Potter and Axerlod (7). The subceliular fr tctions were dialized for 18 hr in running tap water, dried in vacuum, and suspended in a homogenizer in [00 ml of cl-rloroform-methanol (2:1, v/v). The ganglio side fraction was extracted and estimated as described by Burton (8). Succinic dehydro genase activity was estimated by the method of Slater and Bonner (9). Catecholainines (CA) in the subcellular fractions and the perfusa`e were assayed by the ethylenediamine condensation method (10). Method of perfusion of bovine adrenal glands was similar to that described by Trifaro et al. (11). CA secretion was evoked by switching perfusion from Locke's solution to Locke's containing 5 x l0 M of acetylcholine (ACh), or from Ca-free Locke's solution to Locke's, using perfusion pump. Flor rate was 15 ml/min. For the determination of the effect of neuraminidase, protamine or nitrogen mustard on the induced CA secretions, the following calculation was used.