A. Favit

Ubiquinone protects cultured neurons against spontaneous and excitotoxin-induced degeneration.

Ubiquinone is an endogenous quinone with pharmacological actions mainly related to its antioxidant properties. Here we report that ubiquinone protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. In control cultures at 9 days of maturation in vitro (DIV), a 30-min exposure to 100 μM glutamate induced neuronal degeneration, as reflected by the great percentage (>90%) of cells labeled with propidium iodide 24 h after the exposure. Glutamate-induced neuronal death was dramatically reduced in cultures treated daily with ubiquinone since the second DIV. In these cultures, glutamate failed to induce a “delayed” increase in the influx of 45Ca2+, an established parameter of excitotoxicity. Similarly, repeated addition of ubiquinone attenuated in a concentration-dependent manner the age-dependent degeneration of granule cells that is due to the toxic action of the endogenous glutamate progressively released into the medium. These results suggest that ubiquinone may be a useful drug in the therapy of acute and chronic neurodegenerative diseases related to hyperactivity of excitatory amino acid neurotransmission.

Estrogen modulates stimulation of inositol phospholipid hydrolysis by norepinephrine in rat brain slices

The influence of estrogen on stimulation of inositol phospholipid hydrolysis by norepinephrine and carbamylcholine has been studied by measuring the accumulation of [3H]inositol-monophosphate ([3H]InsP) in cortical, hippocampal and striatal slices from ovariectomized rats. Repeated (but not a single) subcutaneous injections of estradiol benzoate (EB) (2 micrograms/animal once every 2 days for 10 days) markedly reduced stimulation of inositol phospholipid hydrolysis by norepinephrine in hippocampus and corpus striatum. Conversely, the efficacy of norepinephrine was increased in cortical slices. Estrogen treatment did not affect basal or carbamylcholine-stimulated [3H]InsP formation. In vitro addition of 17 beta-estradiol (1-100 nM) failed to modify norepinephrine- or carbamylcholine-induced [3H]InsP production in all regions examined. An increased density of alpha 1-adrenergic binding sites in cortical membranes paralleled the enhanced responsiveness of inositol phospholipid hydrolysis to norepinephrine induced by EB treatment in this area, whereas no significant changes in [3H]prazosin binding were found in membranes from hippocampus and corpus striatum. These results indicate that estrogen may affect inositol phospholipid hydrolysis in discrete brain areas, suggesting a complex role for estradiol in modulating noradrenergic receptor activity in the central nervous system.

Pituitary Adenylate Cyclase-Activating Polypeptide Activates Different Signal Transducing Mechanisms in Cultured Cerebellar Granule Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel 38-residue neuropeptide which stimulates adenylate cyclase activity in rat pituitary cells as well as in other neuronal and non-neuronal tissues. In this study we have investigated whether PACAP27 and PACAP38 may stimulate either cyclic AMP accumulation or phosphoinositide formation in cultured cerebellar granule cells. In cultures at 8 days of maturation in vitro (DIV), a 15-min exposure to PACAP27 or PACAP38 equally promoted a concentration-dependent increase in intracellular cAMP content: the effect was significant at 1-5 nM and maximal between 10 and 100 nM, while VIP was 1,000-fold less potent in elevating cAMP levels. In the presence of 3-isobutyl-1-methylxanthine (200 microM), stimulation by PACAP was present already at 0.1 nM and was maximal (6-fold increase) at 1 nM. A rapid elevation in intracellular cAMP (about 80%) was observed within a 30-second exposure to 10 microM PACAP38 or PACAP27; the maximal activity of PACAP was present between 15 and 30 min and progressively declined at 60 min without reaching basal values. PACAP27 and PACAP38, but not VIP, were also able to stimulate inositol phospholipid hydrolysis: PACAP38 (EC50: 0.16 nM) was 10-fold more potent than PACAP27 (EC50: 2.1 nM) in stimulating [3H]inositol phosphate formation. The effect of PACAP was rapid: fractionation of [3H]inositol phosphates revealed that inositol trisphosphate and inositol bisphosphate increased earlier (within 20 s) than inositol monophosphate (within 60 s).(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol Esters Attenuate Glutamate‐Stimulated Inositol Phospholipid Hydrolysis in Neuronal Cultures

Abstract: The phorbol diesters 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and phorbol‐12,13‐dibutyrate, but not 4–α‐phorbol‐didecanoate, inhibited the stimulation of inositol phospholipid hydrolysis by excitatory amino acids and carbamylcholine in primary cultures of cerebellar neurons. This inhibition was mimicked by the synthetic diacylglycerol 1,2‐dioleoyl‐rac‐glycerol (DOG) and was selective for a specific glutamate‐phosphoinositide receptor subtype (GP2 receptor) activated by glutamate and quis‐qualate. TPA was nearly inactive in inhibiting the stimulation of inositol phospholipid hydrolysis by N‐methyl‐d‐aspartate, a selective agonist of the GP1 receptor. Phorbol diesters and DOG attenuated the stimulation of inositol phospholipid hydrolysis by glutamate and quisqualate also in cerebellar slices from 9–15‐day‐old rats; however, using this preparation, their action was weak and required high concentrations (> 1 μM). The inhibition of signal transduc‐tion by phorbol diesters was not consequent to a reduced binding of glutamate to its membrane recognition sites. In fact, TPA induced only a small increase in the KD but no change in the Bmax of [3H]glutamate binding in cerebellar membranes. Phorbol diesters may act to inhibit specific GTP‐binding proteins or particular molecular forms of phosphoinositidase C associated with GP2 or muscarinic cholinergic receptors.

Receptor-mediated stimulation of inositol phospholipid hydrolysis in human brain

Stimulation of inositol phospholipid hydrolysis by transmitter receptor agonists was studied in slices from the occipital, temporal and cerebellar cortex that were surgically removed from three patients during ablation of brain tumors. Norepinephrine and the muscarinic acetylcholine agonist carbamylcholine increased the content of [3H]inositol monophosphate (InsP) in all the regions examined, whereas the glutamate receptor agonist quisqualate was effective only in the cerebellar slices. These results provide evidence that neurotransmitter receptors are coupled to inositol phospholipid hydrolysis in human brain tissue.

Repeated calcitonin treatment reduces the stimulation of inositol phospholipid by norepinephrine and serotonin in rat hippocampus and cerebral cortex

Stimulation of inositol phospholipid hydrolysis by norepinephrine or 5-hydroxytryptamine was reduced in hippocampal or cortical slices from rats repeatedly injected with (Asu1.7)eel-calcitonin (2.5 IU/kg i.p.). This effect was specific, as the basal or carbamylcholine-stimulated inositol phospholipid hydrolysis was unchanged in slices from calcitonin-injected animals. The reduced responsiveness to norepinephrine did not reflect a decreased number or affinity of alpha 1-adrenergic recognition sites, suggesting that calcitonin treatment leads to a reduced coupling between alpha 1-adrenoceptors and phospholipase C.