Donald F. Bogdanski

MICROANALYTICAL PROCEDURES FOR FLUORO-METRIC ASSAY OF BRAIN DOPA-5HTP DECARBOXYLASE, NOREPINEPHRINE AND SEROTONIN, AND A DETAILED MAPPING OF DECARBOXYLASE ACTIVITY IN BRAIN

THE presence of the amines, norepinephrine (NE)? and serotonin (5HT), and of the enzymes, 3 :4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5HTP) decarboxylases, in brain has aroused considerable conjecture regarding the role of the arnines in the central nervous system. A detailed mapping of the decarboxylases and the amines in brain might outline the regions where the amines act and throw some light on their normal function. Previous papers have described the fluorometric estimation of tissue NE (SHORE and OLIN, 1958) and 5HT (BOGDANSKI eta/ . , 1956) and the Auorometric (BERTLER and ROSENGREN, 19594, or manometric (HOLTZ and WESTERMANN, 1956), estimation of SHTP and DOPA decarboxylases. Although these methods are suitable for the estimation of these substances in relatively gross areas of the brain, they are not sensitive enough for their estimation in more precisely defined brain regions. This paper describes modifications of the fluorometric procedures for NE and 5HT estimation whereby they can be assayed in amounts of brain containing as little as 40 pmg of NE or 200 pmg of 5HT. In addition, methods are described for measuring the activities of SHTP and DOPA decarboxylases in 20 to 100 mg of brain tissue. With these methods the activity of DOPA decarboxylase in the cat brain has been mapped in considerable detail and has been related to the activity of 5HTP decarboxylase and to the levels of 5HT and NE.

Role of sodium, potassium, quabain and reserpine in uptake, storage and metabolism of biogenic amines in synaptosomes

Abstract PREVIOUS reports have shown that after homogenization of rat brain in isotonic sucrose the apparently intact nerve endings are pinched off and can be separated by centrifugation in a discontinuous sucrose gradient (1,2). These so-called synaptosomes contain synaptic vesicles which store acetylcholine, norepinephrine (NE) and serotonin (5HT). However, the synaptosomes appeared to be functionally different from nerve endings in the intact brain in that at 37°C they rapidly lose their content of NE and 5HT (3). This laboratory has previously reported that the storage of NE in slices of rat heart is greatly enhanced by Na+ and impaired by K+ (4). In subsequent studies it was shown that Na+ is essential for the system that stores NE in sympathetic nerve endings and that this process is competitively inhibited by K+ (5,6). Other workers have also postulated that Na+ is required for the uptake of NE by sympathetic nerve endings (7,8). The present paper shows that Na+ is essential for the process that stores and accumulates NE and 5HT and that in synaptosomes the action of Na+ is inhibited by K+. Moreover, evidence is presented which suggests that biogenic amines in synaptosomes are synthesized from endogenous precursors.

Occurence of serotonin and catecholamines in brain and peripheral organs of various vertebrate classes

Abstract A number of studies suggest that norepinephrine (NE) and serotonin (5HT) in mammalian brain modulate the activities of the functionally opposing systems, postulated by W. Hess (1). This concept has proved useful in merging physiological, anatomical and chemical data into a working hypothesis (2). We have now undertaken studies on the distribution, storage and release of the amines to determine whether these processes in the nerve endings of lower vertebrates are basically similar to those of mammals. Reports have appeared on the distribution of 5HT nervous system of various vertebrates (3,4,5), and others on the distribution of the catecholamines (6,7,8,9). As far as we know, however, a phylogenetic comparison of the two brain amines has not been reported.

Mechanisms of biogenic amine transport and storage IV. Relationship between K+ and the Na+ requirement for transport and storage of 5-hydroxytryptamine and norepinephrine in synaptosomes

The transport of the biogenic amines, 5-hydroxytryptamine and norepinephrine, was studied using isolated nerve endings (synaptosomes) prepared from rat and rabbit brains. The transport was absolutely dependent upon Na+, but maximum transport required K+. In the absence of K+, transport increased with increasing [Na+] up to about 75 mM, then decreased. High [K+] decreased transport in the presence of Na+. In the absence of K+, a lapse of time preceded the blockade of transport. Moreover, preliminary incubation of synaptosomes in K+-free media containing 150 mM Na+ blocked initial transport more than media containing 50 mM Na+. It is concluded that K+-lack blocks transport as a result of changes in transmembrane ion gradients secondary to the inhibition of (Na+ + K+)-ATPase. K+-lack slightly increased the efflux of stored norepinephrine.

Mechanism of transport and storage of biogenic amines. III. Effects of sodium and potassium on kinetics of 5-hydroxytryptamine and norepinephrine transport by rabbit synaptosomes

The kinetics of biogenic amine transport by a process located in the membrane of synaptosomes were studied in order to determine the mechanism of the transport process and the effects of inorganic ions thereon. The following points were shown: 1. 1. Exogenous 5-hydroxy[14C]tryptamine is taken up by the sum of two processes; a saturable process predominating at low concentrations. At concentrations sufficient to saturate the first, a linear relationship between the velocity of uptake and substrate concentration suggests uptake occurs by passive diffusion. 2. 2. The saturable processes for 5-hydroxy[14C]tryptamine and [3H]norepinephrine could be described by the Lineweaver-Burk representation of Michaelis-Menten kinetics, suggesting that uptake was mediated by a carrier mechanism. 3. 3. The affinities of the carriers for 5-hydroxy[14C]tryptamine and [3H]norepinephrine were increased by Na+. 4. 4. The transport of 5-hydroxy[4C]tryptamine in the presence of Na+ was decreased by a high K+ concentration. 5. 5. These findings are presented in support of an ion gradient model for the transport mechanism located in the membrane of nerve endings.

Binding of serotonin and other amines by blood platelets.

Abstract 1. 1. Platelets have been found to be able to bind a variety of amines both in vitro and in vivo . 2. 2. Factors influencing the in vitro binding of serotonin by dog platelets have been studied. 3. 3. It is suggested that platelets may have the function of rapidly binding circulating amines, so as to render them physiologically inactive.

Gastric Mucosal Erosion in the Rat Following Administration of the Serotonin Precursor, 5-Hydroxytryptophan:

Summary 5-Hydroxytryptophan administered to rats in a dose of 300 mg/kg produced hemorrhagic mucosal erosions in the glandular portion of the stomach. When atropine sulfate was injected prior to 5-hydroxytryptophan administration, the lesions were decreased in number or their occurrence prevented. Administration of reserpine also produced similar lesions which were delayed in appearance.

Biogenic Amine Transport

Comparisons were made of the inhibitory effects of ouabain and potassium-deficient media on the transport of sero tonin and norepinephrine by synaptosomes. The following parallel effects were observed

Biogenic Amines and Drug Action in the Nervous System of Various Vertebrate Classes

Publisher Summary The problem of the functional significance of catecholamines and serotonin in the brain has stimulated considerable research in recent years, and has led to the view that norepinephrine and serotonin might be neurohormoces of the functionally opposing systems postulated by Hess. Considerable research is also being carried out on the nature of the biophysical units at nerve endings, which synthesize, store, and release the biogenic amines. The understanding of these processes might be enhanced by comparative studies of the amines in the nervous system of the various vertebrate classes. Reports have appeared on the distribution of serotonin in the nervous system of various vertebrates, as well as on the distribution of catecholamines. As far as it is known, however, a phylogenetic comparison of the two brain amines has not been published. Recently, on a study of the formatioil, storage and release of the amines has been embarked to see whether these processes in the nerve endings of lower vertebrates are basically similar to those in mammals. The present chapter reports the findings to date.

Inhibition of Gastric Acid Secretion in the Dog by the Precursor of Serotonin, 5-Hydroxytryptophan

Summary The effect of the serotonin precursor, 5-hydroxytryptophan (5HTP), on gastric secretion in the dog has been studied. Administration of 5HTP increases both blood and tissue serotonin with a maximum effect evident in about one hour. 5HTP inhibits gastric secretion when it is spontaneous or when stimulated by Urecholine. The onset of inhibition of gastric secretion by 5HTP is coincident with the degradation of this substance to serotonin. Gastric secretion stimulated by large doses of histamine is not inhibited by 5HTP.