P. Benda

The presence of 2′, 3′‐cyclic AMP 3′‐phosphohydrolase in glial cells in tissue culture

Abstract— The Enzyme 2′, 3′‐cyclic AMP 3′‐phosphohydrolase (CNP) is regarded as a marker for myelin (KURI‐ HARA and MANDEL, 1970) on the basis of its regional and subcellular distribution (Kurihara and Tsukada, 1967), its ontogenetic characteristics (KURIHARA and TSUKADA, 1968), and its behaviour in two strains of myelin‐deficient mutant mice (Kurihara, Nussbaum and Mandel, 1969). However we have isolated highly‐purified preparations of neuronal plasma membrane from rat brain synaptosomes which contain this enzyme activity (Morgan, Wolfe, Mandel and Gombos, 1971). Two explanations of this finding are possible. The activity could be due to the presence of myelin, but this explanation is ruled out by electron microscopy and by the low level of cerebrosides in the synaptosomal plasma membrane preparations. Myelin is extremely rich in cerebrosides (norton and Autilio, 1966). The second possibility is that the enzyme, 2′, 3′‐cyclic AMP 3′‐phosphohyrolase, may also be found in the glial cells from which myelin is derived (Bunge, Bunge and Pappas, 1962). To test our hypothesis that 2′, 3′‐cyclic AMP 3′‐phosphohydrolase is not a specific marker for myelin, but is also found, in glial cells, we have examined a tumoral glial cell line maintained in myelin‐free tissue culture.

MOLECULAR FORMS OF ACETYLCHOLINESTERASE: THEIR DE NOVO SYNTHESIS IN MOUSE NEUROBLASTOMA CELLS

Rat mouse AChE molecular forms are indistinguishable with respect to their sedimentation coefficients and their evolutive proportions during brain maturation. Among rat or mouse erythrocytes, rat C6 glial cells, and mouse 2A and NS 20 neuroblastoma cells, only neuroblastoma cells showed both the ES and HS molecular forms with a 1:1 proportion for NS 20 cells. All these cells lack a third molecular form (16S), which is present in rat and mouse superior cervical ganglia. After irreversible inhibition of pre‐existing NS 20 neuroblastoma AchE, the ES form is first synthesized (de novo synthesis). The HS form begins to appear after a lag time of several hours and represents, 24 h after inhibition, only 15% of the total recovered activity, which is near the initial level. The initial relative proportions return by 2 to 3 days after inhibition. The recovery of the HS form is, for the most part, blocked by actinomycin D, which does not block the recovery of activity itself, which remains as an ES form.

Probenecide sensitive 3′-5′-cyclic AMP secretion by isoproterenol stimulated glial cells in culture

Very little is known at the present time about the biological functions of glial cells in the central nervous system (CNS). The close anatomical relationships between glial cells and neurones has been taken as evidence for a functional or metabolic coupling between these two types of cells [ 1,2]. The small volume of extracellular fluid between glial cells and neurones raises the possibility that glial cells control the chemical composition of this extracellular compartment. On the other hand, it has recently been shown that glial cells in culture are able to accumulate large quantities of 3’-5’-cyclic AMP in response to catecholamines stimulation [3,4]. Thus it seems likely that the glial cells are at least partly responsible for the increase in the 3’-5’-cyclic AMP content of CNS slices observed after incubation in presence of catecholamines or other neurotransmitters [5-71. Besides its role as an intracellular messenger for the action of neurotransmitters on glial cells per se, 3’-5’-cyclic AMP could exert a regulatory action on the neurone if the glial cell is able to secrete 3’-5’cyclic AMP and to modulate the nucleotide concentration in the interspace between neurones and glial cells. The existence of 3’-5’~cyclic AMP secretion has been clearly demonstrated for at least one cell type. Davoren and Sutherland [ 81 reported that pigeon erythrocytes respond to epinephrlne stimulation by increasdd cyclic AMP production and secretion into the extracellular fluid. The nucleotide secretion occurred against a concentration gradient and was partially blocked by probenecide. In this paper we show that actively growing glial cells in culture are also able to secrete 3’-5’-c3cllc AMP into the external medium and that this secretion is sensitive to probenecide.

THE EFFECT OF Cl− ON CHOLINE ACETYLTRANSFERASE KINETIC PARAMETERS AND A PROPOSED ROLE FOR Cl− IN THE REGULATION OF ACETYLCHOLINE SYNTHESIS

Abstract— Crude or purified rat brain choline acetyltransferase (ChAc) is activated by anions. Among anions, Cl− is the most effective and may promote an up to 60 fold increase in Vmax. In the absence of Cl−, at low ionic strength, acetylcholine (ACh) is a good ChAc inhibitor (Ki= 0.310 mm). The ACh inhibition becomes negligible when Cl− is increased to 145 mm (ACh Ki= 45 mm). These results are discussed in terms of regulation of ACh synthesis by nerve terminals. It is proposed that ChAc is part of a presynaptic membrane bound multienzymatic complex under direct control of the ion fluxes promoted by nerve impulses.

Antibodies to rat brain choline acetyltransferase: species and organ specificity.

The production of antibodies against enzymes in- volved in neurotransmission has been developed in the last few years. In the case of catecholamines, the use of antibodies against enzymes involved in their syn- thesis has led to the fine localization of catecholamin- ergic neurons by using immunohistochemical tech- niques [

Restricted lateral diffusion of concanavalin A receptors of different malignant cells of the nervous system.

It is well known [l] that the properties of many surface components of malignant cells are different from their non-malignant counterparts (composition, enzymes, cellular transport etc.). One of the most characteristic properties of malignant cells is the absence of contact inhibition. This should be dependent on the state of the surface receptors, including their mobility [l] . A number of measurements of this mobility, as well as a number of theories, were based on data concerning the formation of caps and patches [2]. This type of lateral mobility, however, could not reflect the lateral diffusion of the receptors. For example, the concanavalin A (Con A) receptors of malignant C 1 1 D fibroblasts which collected into caps did not demonstrate any detectable (< lo-r2 cm2 s-‘)lateral diffusion [3]. In the present study we used a direct method to measure lateral diffusion. This technique allows the measurement of the return of fluorescence to a spot bleached on an otherwise uniformly labelled cell surface [4-71. Previous results furnished by this method showed that the lateral diffusion of Con A receptors of malignant fibroblasts is essentially slower that that of the same receptors of their non-malignant counterparts [3]. In the present investigation we utilized malignant cells originating from various types of neural cells to measure the lateral diffusion of Con A receptors. We found that this diffusion in the malignant cells was much slower that in their nonmalignant counterparts described elsewhere [8]. This fact could perhaps reflect an essential difference between all malignant and primary cells.