Mn2+-stimulated ATPase in rat brain.

Divalent cation ATPases were prepared from rat brain synaptic vesicles, synaptosomal plasma membranes, and plasma membranes from the brain stem and sciatic nerve and tested for optimal stimulation by Mn2+, Mg2+, or Ca2+. ATPase in the synaptic vesicle subfraction was optimally stimulated by Mn2+. All plasma membrane preparations were optimally stimulated by Mg2+. Separate Mn2+ and Mg2+ ATPases could not be distinguished by either chemical inactivation or substrate preference criteria. Mn2+ stimulated ATPase in the micromolar range and it is suggested that Mn2+ interaction with ATPase may be of physiological and/or toxicological importance by being related to the cellular metabolism of this element.

Ecto-5'-nucleotidase regeneration after chemical modification of the plasma membrane.

Treatment of the C6 glioblastoma cell with trinitrobenzenesulfonic acid (TNBS) resulted in the selective inactivation of ecto-5'-nucleotidase under conditions which maintained cell viability. Cells respond to ecto-enzyme inactivation by replacing 80% of lost activity within 24 hrs. A lag time of 4-6 hrs before ecto-5'-nucleotidase replacement began and its complete blockage by cycloheximide indicated that the source of replaced enzyme was de novo synthesis and not an intracellular pool. Release of 5'-nucleotidase activity into culture medium in the form of membraneous vesicles slowed during the active recovery period and then steadily increased with time as the plasma membrane enzyme level approached normal. TNBS did not exert a direct inhibitory action upon the exfoliative process as release of vesicular GM1 and protein were little affected. Decrease in exfoliated 5'-nucleotidase activity may be due to a selective conservation of the enzyme in the exfoliative process.

Exfoliation of membrane ecto-enzymes in the form of micro-vesicles.

Cultures from various normal and neoplastic cell lines exfoliated vesicles with 5'-nucleotidase activity which reflected the ecto-enzyme activity of the parent monolayer culture. The ratio of 5'-nucleotidase to ATPase activity in the microvesicles indicated that cellular ecto-ATPase was conserved in the exfoliative process. Phospholipids of the microvesicles contained significantly increased amounts of sphingomyelin and total polyunsaturated fatty acids. It was concluded that the shedded vesicles constituted a select portion of the plasma membrane. Examination by electron microscopy showed the vesicles had an average diameter of 500 to 1000 nm and often contained a second population of vesicles about 40 nm in diameter. As much as 70% of the plasma membrane ecto-5'-nucleotidase activity of a culture was released into the medium over a 24-h period. Phosphoesterhydrolases from C-6 glioma or N-18 neuroblastoma microvesicles dephosphorylated cell surface constituents when in contact with monolayer cultures. Exfoliated membrane vesicles may serve a physiologic function; it is proposed that they be referred to as exosomes.

Selective chemical modification of plasma membrane ectoenzymes.

As part of an investigation of the organization of cell surface macromolecular assemblies, we have treated intact central nervous system cells with chemical probes which react convalently with proteins and aminophospholipids. Selective alterations of the enzymatic activities of ecto-ATPases, ecto-5'-nucleotidases and cholinesterases were obtained under appropriate reaction conditions. The cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene, was a potent inactivator of ecto-ATPase of C6 glioblastoma, IMR-32 neuroblastoma and of a primary rat astroblast cell line (RB). Ecto-5'-nucleotidase and acetylcholinesterase were less sensitive to difluorodinitrobenzene. 1-Fluoro-2,4-dinitrobenzene at concentrations which inactivated ecto-ATPase had little effect on ecto-5'-nucleotidase. Conversely, 2,4,6-trinitrobenzenesulfonic acid was a potent inactivator of ecto-5'-nucleotidase but had no effect on ecto-ATPase. The difluorodinitrobenzene inactivation of ecto-ATPase and of ecto-5'-nucleotidase as well as the fluorodinitrobenzene inactivation of ecto-ATPase could be prevented by the presence of the appropriate substrates in the reaction medium. In the presence of protecting nucleotide substrates, a decrease in reactivity with proteins and lipids was observed when the isotopic probe fluorodinitro[3H]-benzene was used.

On the evolution of neurochemical transmission.

A discussion of the evolution of neurochemical transmission is divided into three main topics: evolution of biochemical signalling devices, evolution of neurotransmitter substances, and evolution of signal meaning. Models of signalling devices are developed from a primitive chemoceptive process through open and closed loop communications to a neuronal communications network and to its development into a symbolic logic exchange. The evolution of neurotransmitter substances is extrapolated from experimental evidence which has been obtained under primitive earth atmosphere conditions. Examples from comparative biology suggest that the evolution of transmitter use was not unidirectional and that purine derivatives may well have been the primordial transmitter substances. The classical neurotransmitters, such as acetylcholine and norepinephrine have a limited information content in their molecular structure, whereas inherent message content of peptidergic transmitters is potentially significant. If there are mnemotypic genes, they may be expressed as informational macromolecules which specify behavioral patterns. Such information transfer would represent a second order of neurochemical transmission and its evolution would be closely coupled to that of molecules which contain a universal meaning.

Antiserum to the milk fat globule membrane. Preparation and capacity to suppress milk secretion.

A procedure is described for preparing rabbit antiserum to goat milk fat globule membrane. This membrane is derived from the secretory surface of the lactating cell. Immunoelectrophoresis and enzyme-linked immunosorbent assay showed that antibody development reached maximal levels in about 6--8 weeks. Infusion of 5--10 ml of this antiserum into the lactating mammary gland of goats via the teat canal depressed milk yields temporarily on the infused side to 60--80% of normal. Ordinary serum from rabbit, goat or human did not evoke such a response and rabbit complement was not essential for the effect. Fractionation showed that the globulin fraction of the antiserum contained the milk-suppressing principle. Milk from the antiserum-infused side of the udder showed extensive and tenacious clumping of fat globules on standing 12--24 h. The inhibition of milk flow by antibodies to the secretory membrane resembles a previously observed inhibition following infusion of concanavalin A or its succinyl derivative. Binding of antibodies or lectins which recognize specific surface protein components of the lactating cell appears to be involved in the suppression mechanism. The possible relevance of our findings to autoimmune suppression of exocytosis is noted.

A comparative study of brain Ca2+-ATPases.

1. Particulate brain ATPases from various vertebrates were optimally activated by Ca2+, Mg2+ or Mn2+. 2. Specific enzyme activity with AT32P as substrate was low in lower vertebrates and increased on the evolutionary scale. 3. The properties of the brain ATPases suggested that most of the activity was associated with plasma membrane ecto-ATPase.

On the activation of plasma membrane ecto-enzymes by treatment with neuraminidase.

It had been proposed that sialyl-residues on the surface of the cell control the activity of certain plasma membrane ecto-enzymes. We have tested the effects of several established (or presumptive) ecto-enzymes in tissue cultures of CNS-derived cells. Application of neuraminidases to cultured mouse neuroblastoma (N-18), neonatal Syrian hamster astrocytes (NN), human astrocytoma (Cox clone) and two lines of primary mouse astroblasts failed to change the activity of ecto-ATPase and 5'-nucleotidase. Only two of the seven neuraminidase preparations produced marked or moderate increases in inorganic pyrophosphatase, p-nitrophenylphosphatase and cholinesterase. We have concluded that the stimulation of these enzymes was not due to removal of sialyl-residues. We suggest that contaminants (haemolysins?) in neuraminidase preparations of Clostridium perfringens increased membrane permeability and facilitated substrate-product translocation.

Adenosine deaminase of cultured brain cells.

Two types of adenosine deaminase (EC 3.5.4.4) were found in cultured cells of central-nervous-system origin. The predominant and more active enzyme was obtained in soluble form from the cytosol of mouse neuroblastoma (N-18), neonatal hamster astrocytes (NN), human oligodendroglioma (HOL) and human astrocytoma (Cox Clone). Particulate adenosine deaminase was probably associated with the plasma membrane. When radioactive adenosine was added to superfusates of monolayer cultures it was rapidly converted into inosine and hypoxanthine. The metabolic conversion required adenosine uptake by the cells, a probable transition through the intracellular ATP pool(s) and a rapid excretion into the superfusate of the catabolic products. We discuss the evidence that points to adenosine and its derivatives as neurohumoral modulators of central-nervous-system function.