Journal of lipid mediators and cell signalling最新文献

Neuroblastoma × glioma hybrid NG108-15 and neuroblastoma × fibroblast hybrid NL308 cells possess endogenous bradykinin B₂ receptors and m4 muscarinic acetylcholine receptors (mAChRs), which couple to phospholipase C and adenylate cyclase, respectively. Four genetic subtypes of mAChRs differed in their effects when stimulated in NG108-15 and NL308 cells overexpressing mAChRs. Broadly speaking, the principal effects fell into two categories: the odd-numbered receptors (ml and m3) activated phospholipase C and increased inositol trisphosphate/Ca²⁺, as bradykinin did, whereas the even-numbered receptors (m2 and m4) inhibited adenylate cyclase via a pertussis toxin (PTx)-sensitive G-protein in NG108-15 cells. But all four types of NL308 cells overexpressing each m1, m2, m3 and m4 receptor activated phospholipase C, while keeping the PTx-sensitivity in m2/m4, but not in m1/m3 receptors. Coupling to ion channel effectors showed a comparable dichotomy in NG108-15 cells, while cross-activation occurred in NL308 cells.

It is apparent that the phosphoinositide signalling pathway is subject to a variety of regulatory features which will ultimately dictate the magnitude and profile of cellular responses to agonist occupation of PIC-linked receptors. Our understanding of these mechanisms is far from complete but will be crucial in revealing both the specificity of receptor signalling and the integration of signals arising from the potentially wide variety of metabotropic and ionotropic receptors on individual cells.

Anandamide is an endogenous ligand for cannabinoid receptors. We tried to isolate and purify ‘anandamide amidohydrolase’ which hydrolyzes anandamide to arachidonic acid and ethanolamine. The enzyme activity was found in the microsomal fraction of procine brain homogenate. The enzyme was solubilized in 1% Triton X-100, and partially purified by hydrophobic chromatography to a specific activity of about 0.3 μmol/min per mg protein (37°C). Apparent K_m for anandamide was about 60 μM. The enzyme reacted also with also converted arachidonic acid to anandamide in the presence of 250 mM concentration of ethanolamine. Several lines of evidence including experiments using various inhibitors suggested that the anandamide synthase and amidohydrolase activities were derived from a single enzyme protein.

Two distinct pathways for the synthesis of platelet-activating factor (PAF) have been demonstrated in the nervous tissue. This potent lipid mediator is involved in physiological and pathological processes. The relative contribution of the two pathways to its synthesis during various conditions needs to be defined, thus the activities of the enzymes directly responsible for PAF synthesis, PAF-synthesizing phosphocholinetransferase (PAF-PCT) and lyso-PAF acetlytransferase (lyso-PAF AcT), have been assayed in rat brain areas. The former catalyses the last reaction of the de novo pathway and the latter that of the remodelling one. PAF-PCT activity was always more elevated thant hat of lyso PAF AcT. No differences were observed among different brain areas when enzyme activities were assayed in their homogenates. In microsomes, the highest PAF-PCT activity was found in cerebellum whereas lyso-PAF AcT activity was greater in cerebellum and in hippocampus than in the other brain areas. The activity of PAF-synthesizing enzymes was also studied in the gerbil during ischemia and reperfusion. After 6 min from bilateral occlusion of the carotid arteries, a significant increase of lyso-PAF AcT activity was observed in the hippocampus. This enzyme activity remained relatively high up to 3 days after reperfusion whereas, in other brain areas it reached basal levels much earlier. Since it has been shown that the PAF levels increase in the brain of animals during ischemia, these results suggest that the remodelling pathway may provide an important contribution to its synthesis particularly in the hippocampus, where a selective neuronal death is observed. In this area during reperfusion, a further contribution to PAF synthesis might be also provided by the de novo pathway.

Platelet-activating factor (PAF) receptor and somatostatin receptor (SSTR4) were cloned, and their primary structures were identified. They are both highly expressed in the rat hippocampus. When expressed in Chinese hamster ovary cells, these receptors activated mitogen-activated protein (MAP) kinase cascade and phosphilipase A₂. Arachidonic acid or its derivatives, thus produced by the activation of these receptors may play some roles in synaptic transmission and synaptic plasticity.

Phospholipase A₂ (PLA₂) activity is an important contributor to destructive cellular processes in the central nervous system. Two cytosolic forms of calcium independent PLA₂ have been characterized in the gerbil brain and the neuronal cultures from rat brain. PLA₂ enzymatic activity in cell free extracts from cortical neuronal cultures is upregulated after cells are exposed to glutamate. Brief exposure to a calcium ionophore or phorbol 12-myristate 13-acetate (PMA) stably enhanced PLA₂ activity. Stable activation of the two cytosolic forms of PLA₂ occur prior to evidence of cell death and this activation is reversible. The larger molecular mass form was characterized as cPLA₂. The smaller form (∼ 14 kDa) was distinct from Group I and II PLA₂. Exposure to glutamate shifted the calcium activation curve of the smaller form to the left suggesting a novel mechanism of regulation of PLA₂. Glutamate-induced stable enhancement of PLA₂ activity, by processes involving calcium and protein kinase C activation, is a potential molecular switch likely mediating changes in synaptic function and contribution to excitotoxicity.

The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [³H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C₂-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation.

There is evidence available suggesting that membrane alterations occur in Alzheimer's disease including the metabolism of membrane phospholipids. We have quantitated in vitro the phospholipase D activity of homogenates from Alzheimer's disease brain tissue. There was a significant increase of this enzyme activity as compared to controls. Amyloid β protein is the predominant protein of the characteristics senile plaques found in Alzheimer's disease. Treatment of LA-N-2 cells, a human cholinergic neuroblastoma clone, with amyloid β protein results in an activation of phospholipases A, C and D.

SK-N-BE cells were differentiated (D) to neurons with retinoic acid. Total n - 6 polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA), were increased in D versus ND cells. AA synthesis from linoleic acid (LA) and phospholipid (PL) synthesis from glycerol were initially elevated, whereas both processes were reduced approaching differentiation. At this stage, the incorporation of glycerol in triglycerides was enhanced. Formation of long chain PUFA and synthesis of acceptors (PL) for esterification, during RA-induced SK-N-BE differentiation, appear to be associated.