Bernd Hamprecht

ADENOSINE REGULATES VIA TWO DIFFERENT TYPES OF RECEPTORS, THE ACCUMULATION OF CYCLIC AMP IN CULTURED BRAIN CELLS

Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β‐adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon–glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon–glia metabolic coupling serves a physiological function.

Influence of opiates on the levels of adenosine 3':5'-cyclic monophosphate in neuroblastoma X glioma hybrid cells.

Abstract In neuroblastoma x glioma hybrid cells prostaglanddin E 1 (PGE 1 ) increases the level of adenosine 3′: 5′-cyclic monophosphate. This response to PGE 1 is strongly enhanced in cells that were incubated with morphine, methadon, noradrenaline or carbamylcholine for several hours. All these compounds increase the level of cyclic GMP in the cells. As in untreated cells, the effect of PGE 1 can be inhibited by morphine or noradrenaline. The development of the increased response to PGE 1 is dependent on protein synthesis. The increased response to PGE 1 is discussed in connection with morphine tolerance and withdrawal.

Rhythmic changes of hydroxymethylglutaryl coenzyme a reductase activity in livers of fed and fasted rats.

During studies on the cholesterol biosynthetic activity in liver slices of rats kept under different nutritional conditions, we found that the rate of acetate incorporation into cholesterol depends on the time of day when the rats were killed. The activity exhibits a diurnal rhythm with the highest values at midnight and the lowest at noon [ 1,2] . Using a new sensitive radiogaschromatographic assay procedure for the microsomal enzyme HMG-CoA reductase @-hydroxyf3-methylglutaryl_CoA reductase, E.C. 1 .1.1.34) [3-51 we could demonstrate similar rhythmic changes for this enzyme [3]. This was expected since in other studies HMG-CoA reductase was demonstrated to be the rate limiting enzyme of cholesterol biosynthesis from acetate [ 1,6,7] . Therefore the regulation of cholesterol synthesis is most effectively exerted at this step. As rats feed mainly at night, investigations were made whether the rhythm existed independently of food intake. Livers from fasted rats have extremely low activities of HMGCoA reductase. The difficulty in detecting significant differences in these low levels did not arise, when the new test procedure was used. A recent publication [S] described the increase, during the time between 8.30 and 20.30 hr, in the incorporation of acetate into liver 3@terols of mice fed ad libiturn. This increase agrees with the discovery in this laboratory of a 24 hr cycle of cholesterol synthetic activity in rat liver. These authors also confirm our findings of the time dependence of HMGCoA reductase [3], when they show the activity of this enzyme at 16.30 hr to be 5.5 times higher than that at 8 hr.

Prostaglandin production by neuroblastoma, glioma and fibroblast cell lines; Stimulation by N 6, O2′-dibutyryl adenosine 3′:5′-cyclic monophosphate

Prostaglandin E, (PGE1) elevates the levels of intracellular adenosine 3’: 5’-cyclic monophosphate (cyclic AMP) in clonal lines of mouse neuroblastoma cells [ 1, 21 , of fibroblasts [3, 41 and of hybrid cells involving cell lines derived from nervous tissues or fibroblasts [4]. If the cell lines preserve properties of the nervous tissue cells from which they were derived, their sensitivity to PGE, should reflect a physiological function. PGE compounds injected into an animal disap.pear very rapidly from the circulation [5], as they are oxidized to inactive derivatives [6]. Consequently, the PGE to which the cells would respond in situ would most probably have to be produced in the close neighbourhood of those cells. Therefore, it was of interest to investigate the capability of a neuroblastoma, a glioma and a fibroblast cell line to synthesize PGE. It has been shown that many actions of cyclic AMP can be mimicked by N6 ,02’-dibutyryl adenosine 3’:s’~cyclic monophosphate (dibutyryl cyclic AMP) [7-l 1 ] . The influence of this cyclic AMP analogue on the production of prostaglandins (PG) was measured in order to obtain information on the significance of cyclic AMP (and of neurohormones acting via cyclic AMP) in the regulation of PG synthesis. All 3 cell lines essentially produce PGE; only minor amounts of PGF and PGA were detected.

Measurements of adenosine 3′:5′‐cyclic monophosphate and membrane potential in neuroblastoma X glioma hybrid cells: Opiates and adrenergic agonists cause effects opposite to those of postaglandin E1

Hybrid cells obtained by fusion of rat glioma and mouse neuroblastoma cells display many properties characteristic of differentiated neurons [ 1,2] . On treatment with N6 -2’0dibutyryl adenosine -3’: 5’cyclic monophosphate (dibutyryl cyclic AMP) they extend long processes [l-4] . In containing choline acetyltransferase of high specific activity [ 1,2,5,6] and dopamine-/3-hydroxylase [3] they have marker enzymes of both cholinergic and adrenergic nerves. They fire action potentials on electrical stimulation or when acetylcholine is applied to their plasma membrane. The response to acetylcholine can be blocked by the cholinergic antagonists d-tubocurarine, atropin or cr-bungarotoxin [ 1,2] . When exposed to prostaglandin El, neuroblastoma [7,8] and hybrid cells [9] increase their intracellular concentration of cyclic AMP. In both cell types this effect can be antagonized by morphine [lo] . The basal level of cyclic AMP in unstimulated cells or the elevated level in cells exposed to a phosphodiesterase inhibitor is lowered in the presence of noradrenaline (NA) [9]. In extension of this work we now report that in the hybrid cells NA at low concentrations also prevents the increase of intracellular levels of cyclic AMP evoked by PGEi . In doing so it is a more potent agent than dopamine, phenylephrine and isoproterenol (order of decreasing potency). The adrenergic a-blocker phentolamine antagonizes the action of NA more effectively than the I(-blocker propranolol. The data suggest the adrenergic receptor to be classified as a-receptor [ 111. Also opiates, in a highly stereospecific

THE INFLUENCE OF SECRETIN, GLUCAGON AND OTHER PEPTIDES, OF AMINO ACIDS, PROSTAGLANDIN ENDOPEROXIDE ANALOGUES AND DIAZEPAM ON THE LEVEL OF ADENOSINE 3′,5′-CYCLIC MONOPHOSPHATE IN NEUROBLASTOMA GLIOMA HYBRID CELLS

Abstract— Neuroblastoma glioma hybrid cells display many properties of neurons. A series of compounds, among them a number of amino acids, peptides and peptide hormones were tested for their ability to influence the level of adenosine 3′,5′‐cyclic monophosphate (cyclic AMP) in the hybrid cells. Two prostaglandin endoperoxide analogues exhibit a weak stimulatory action, if applied in at least micromolar concentrations. At nanomolar concentrations, only the gastrointestinal hormones secretin and glucagon stimulate the formation of cyclic AMP, as detected in the presence of a phosphodiesterase inhibitor. The effect of secretin but not that of glucagon is antagonized by secretin‐(5–27), suggesting that secretin and glucagon act on the cells via different receptors. These results appear to be noteworthy since (a) an effect of secretin or glucagon on a cell with neuronal characteristics has not yet been described, and (b) many peptide hormones have been detected both in the gastrointestinal tract and the nervous system.

Na+‐Dependent Uptake and Release of Taurine by Neuroblastoma × Glioma Hybrid Cells

Abstract In neuroblastoma × glioma hybrid cells, a cell line of neuronal character, a saturable uptake system for taurine is found which displays high affinity and high capacity (Km= 38 μm, V= 1.25 nmol mg−1 min−1)‐ Only the closely related structural analogues hypotaurine and β‐alanine are able to inhibit the transport of radioactively labeled taurine. Imipramine or haloperidol at 100 μmeffectively blocks taurine uptake. High‐affinity taurine uptake shows an absolute and highly specific requirement for Na+. The hybrid cells internalize taurine very slowly and, with 1 mm extracellular taurine, attain a plateau only after more than 20 h, at which time approximately 34 mm labeled taurine has accumulated in the cytosol. Generally there is hardly any spontaneous release of accumulated taurine. Efflux can, however, be induced by increasing the intracellular Na+ content and is then accelerated by lowering the extracellular Na+ concentration. The hypothesis is forwarded that taurine may exert its function by driving the extrusion of Na+ in emergency situations.

Parathyrin and calcitonin stimulate cyclic AMP accumulation in cultured murine brain cells.

Despite the key role Ca2+ plays in the nervous system, biochemical actions on neural tissue of the Ca2+‐regulating peptide hormones parathyrin and calcitonin were unknown. Until a few years ago only neurons, but not glial cells, were considered as targets for peptide hormones. Our recent observation that peptide hormones do indeed act on glial cells is extended by the present report that these cells respond to the calcaemic peptide hormones parathyrin and calcitonin. In cultured murine brain cells mainly consisting of glioblasts, parathyrin stimulates the accumulation of cyclic AMP. The half‐maximal effect is elicited at 30 nM parathyrin. With rat brain cells the effects are three times those observed with mouse brain cells. Calcitonin, which is less potent than parathyrin, elevates the concentration of cyclic AMP only in rat brain cells. If properly occupied, the inhibitory receptors present on the cells lower the increase in the level of cyclic AMP evoked by parathyrin and, to some extent, that elicited by calcitonin. The results suggest that: (i) these or closely related hormones might exert regulatory functions in brain; and (ii) glial cells must be considered in discussions of the targets of the calcaemic and other peptide hormones.

Influence of cations on the electrical activity of neuroblastoma × glioma hybrid cells

Electrical excitability is one of the various neuronal properties of neuroblastoma X glioma hybrid cells. At a Ca2+ concentration of 1.8 mM the action potential is inhibited by tetrodotoxin, suggesting that the inward current is carried by Na+ ions. In contrast, at a Ca2+ concentration of 20-36 mM and even in the absence of Na+, spikes (sometimes repetitive) with strong hyperpolarizing afterpotential occur, which are no longer affected by tetrodotoxin. They are, however, blocked by antagonists of Ca2+ like La3+, Co2+, Mn2+, and the synthetic compounds D-600 and BAY a-1040. This seems to indicate that at high concentrations of Ca2+, the inward current of the action potential is essentially carried by Ca2+. Sr2+, but not Mg2+ can effectively substitute for Ca2+. It slows down the time course of the action potential. Ba2+ depolarizes the membrane gradually. If Ca2+ is also present, Ba2+ causes a reduced depolarization and spontaneous action potentials with no hyperpolarizing after-potential are observed.