Gianfrancesco Goracci

Botulinum-A Toxin Injections Into the Detrusor Muscle Decrease Nerve Growth Factor Bladder Tissue Levels in Patients With Neurogenic Detrusor Overactivity

PURPOSE We investigated the effects of BTX-A on visceral afferent nerve transmission by measuring bladder tissue NGF levels in patients with neurogenic detrusor overactivity before and after intravesical treatment with BTX-A. We also compared the bladder tissue NGF content with clinical and urodynamic data. MATERIALS AND METHODS A total of 23 patients underwent clinical evaluation and urodynamics with detection of the UDC threshold, maximum pressure and maximum cystometric capacity before, and at the 1 and 3-month followups. Endoscopic bladder wall biopsies were also obtained at the same time points. NGF levels were measured in tissue homogenate by enzyme-linked immunosorbent assay (Promega, Madison, Wisconsin). RESULTS At 1 and 3 months mean catheterization and incontinent episodes were significantly decreased (p <0.05 and <0.001, respectively). On urodynamics we detected a significant increase in the UDC threshold and maximum cystometric capacity, and a significant decrease in UDC maximum pressure at the 1 and 3-month follow-ups compared to baseline (each p <0.001). At the same time points we detected a significant decrease in NGF bladder tissue content (each p <0.02). CONCLUSIONS BTX-A intravesical treatment induces a state of NGF deprivation in bladder tissue that persists at least up to 3 months. As caused by BTX-A, the decrease in acetylcholine release at the presynaptic level may induce a decrease in detrusor contractility and in NGF production by the detrusor muscle. Alternatively BTX-A can decrease the bladder level of neurotransmitters that normally modulate NGF production and release.

The reverse reaction of cholinephosphotransferase in rat brain microsomes a new pathway for degradation of phosphatidylcholine

The synthesis of phosphatidylcholine is catalyzed by cholinephosphotransferase (EC 2.7.8.2) which is known to be reversible in liver. The reversibility of cholinephosphotransferase in rat brain in demonstrated in this paper. Labeled microsomes were prepared from young rats which had been given an intracerebral injection of labeled choline or oleate 2 h before killing. During incubation of choline-labeled microsomes with CMP, label was lost from ;choline glycerophospholipids and labeled CDPcholine was produced. The Km for CMP was 0.35 mM and V was 3.3 nmol/min per mg protein. Neither AMP nor UMP could substitute for CMP. Oleate-labeled microsomes were pretreated with e mM diisopropylfluorophosphate (lipase inhibitor). During incubation with CMP, label was lost from choline, and ethanolamine glycerophospholipid and labeled diacylglycerols were produced. When the lipase was not inhibited, labeled oleate was produced. We propose that a principal pathway for degradation of phosphatidylcholine, particularly during brain ischemia, is by reversal of cholinephosphotransferase, followed by hydrolysis of diacylglycerols by the lipase.

BASE-EXCHANGE ENZYMIC SYSTEM FOR THE SYNTHESIS OF PHOSPHOLIPIDS IN NEURONAL AND GLIAL CELLS AND THEIR SUBFRACTIONS: A POSSIBLE MARKER FOR NEURONAL MEMBRANES

Abstract— The calcium‐dependent incorporation of L‐[3‐14C]serine and [1,2−14C]ethanolamine into the phospholipid of isolated neuronal and glial cells from rabbit brain was studied, and the distribution of the enzymic system among the correspondent subfractions was examined. The neuronal cell‐enriched fraction was found to possess a much higher rate of exchange of both bases than the glial cell‐enriched fraction. Among the sub‐fractions isolated from the neuronal and glial cells, those corresponding to neuronal plasma membranes and microsomes showed a noticeably higher exchange of serine and ethanolamine compared to the corresponding subfractions from glia. Neuronal/glial ratios of about 6–8 were found for the exchange activity in both plasma membrane‐enriched fraction and in microsomes. Synaptosomes and synaptosomal subfractions contained low activities. It is concluded that the calcium‐dependent enzymic system for the exchange of serine, ethanolamine and other nitrogenous bases with endogenous phospholipid is concentrated mostly in the neuronal perikaryal membranes, and could be used as a neuronal marker.

Metabolism and functions of phosphatidylserine in mammalian brain.

Phosphatidylserine (PtdSer) is involved in cell signaling and apoptosis. The mechanisms regulating its synthesis and degradation are still not defined. Thus, its role in these processes cannot be clearly established at molecular level. In higher eukaryotes, PtdSer is synthesized from phosphatidylethanolamine or phosphatidylcholine through the exchange of the nitrogen base with free serine. PtdSer concentration in the nervous tissue membranes varies with age, brain areas, cells, and subcellular components. At least two serine base exchange enzymes isoforms are present in brain, and their biochemical properties and regulation are still largely unknown because their activities vary with cell type and/or subcellular fraction, developmental stage, and differentiation. These peculiarities may explain the apparent contrasting reports. PtdSer cellular levels also depend on its decarboxylation to phosphatidylethanolamine and conversion to lysoPtdSer by phospholipases. Several aspects of brain PtdSer metabolism and functions seem related to the high polyunsaturated fatty acids content, particularly docosahexaenoic acid (DHA).

THE SYNTHESIS OF CHOLINE AND ETHANOLAMINE PHOSPHOGLYCERIDES IN NEURONAL AND GLIAL CELLS OF RABBIT IN VITRO

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine‐5′‐phosphate choline (cytidine‐5′‐phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell‐enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine‐5′‐phosphate choline and cytidine‐5′‐phosphate ethanolamine, respectively. Several kinetic properties of the choline‐phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. Km of cytidine‐5′‐phosphate ethanolamine, Km of diacyl glycerol, pH optimum, need for divalent cations), but the Km value for cytidine‐5′‐phosphate choline in glial cells was much lower (2.3 × 10−4m) than in neurons (1 × 10−3m). The Kmfor cytidine‐5′‐phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine‐dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.

Properties of PAF-synthesizing phosphocholinetransferase and evidence for lysoPAF acetyltransferase activity in rat brain

Several reports have indicated that platelet-activating factor (PAF) may play a role in the physiopathology of nervous tissue. We previously have demonstrated the presence, in the microsomal fractions of rat brain, of a phosphocholinetransferase which is able to synthesize PAF by thede novo pathway. The presence of dithiothreitol in the medium increases the rate of PAF biosynthesis, whereas it inhibits the synthesis of long-chain alkylacyl- and diacyl-glycerophosphocholines (GPC), including dioctanoyl-GPC. This and other properties, such as pH dependence and thermal stability, indicate that rat brain may have two distinct enzymes for the synthesis of PAF and other choline phospholipids. The affinity of these enzymes for CDPcholine is similar to that reported for other tissues, the Km being 42 μm and 55 μm with alkylacetylglycerol and dioctanoylglycerol as lipid substrates, respectively. The Vmax values were 3.0 and 2.2 nmol/mg prot/min for PAF and dioctanoyl-GPC, respectively. In addition, it was shown that the microsomal fraction of rat brain contains an acetyltransferase which can convert lysoPAF to PAF. Since it has been reported previously that brain tissue possesses phospholipase A2 activity that can hydrolyze alkylacyl-GPC to lysoPAF, we conclude that brain tissue has all enzymic activities for the synthesis of PAF by the “remodeling pathway”. The role of the two routes of PAF biosynthesis in nervous tissue remains to be established.

Rat Brain Cortex Mitochondria Release Group II Secretory Phospholipase A2 under Reduced Membrane Potential

Activation of brain mitochondrial phospholipase(s) A2 (PLA2) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca2+-dependent PLA2 having biochemical properties characteristic to secreted PLA2 (sPLA2) and immunoreacting with the antibody raised against recombinant type IIA sPLA2 (sPLA2-IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA2 from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC6(3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA2 release. The increase of sPLA2 specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA2 has mainly nuclear localization. In addition to sPLA2, mitochondria contain another phospholipase A2 that is Ca2+-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA2-IIA that might contribute to cell damage.

Enzymic synthesis of ethanolamine plasmalogens through ethanolaminephosphotransferase activity in neurons and glial cells of rabbit in vitro

The de novo synthesis of ethanolamine plasmalogen in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labeled cytidine-5′-diphosphate ethanolamine as lipid precursor. The neuronal cell enriched fraction was found to possess a twofold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction. The neuronal/glial ratio was similar both in the absence and in the presence of saturating alkenylacyl glycerol. Under the most favorable conditions, rates of 31 nmoles and 16 nmoles ethanolamine plasmalogen/mg protein/30 min were obtained for neurons and glia, respectively. Several kinetic properties of the phosphotransferase were found to be similar both in neurons and glia, e.g., Km of cytidine-5′-diphosphate ethanolamine, pH optimum, need for divalent cations; the Km value for alkenylacyl glycerol was twofold higher in glia (4 mM) than in neurons (2 mM). The neuronal/glial ratio for the phosphatidylethanolamine synthesizing activity was 2, 4.5, and 6 on using diacyl glycerols prepared from ox heart, ox brain, and soybean, respectively. It is concluded that the cytidine-dependent system for ethanolamine plasmalogen and phosphatidylethanolamine synthesis is concentrated prevalently in the neuronal cells, as compared to glia.

Enzymic synthesis of plasmalogen and O-alkyl glycerolipid by base-exchange reaction in the rat brain

It has been demonstrated recently that a Ca2+-dependent bassexchange system occurs in subcellular fractions of brain which can convert in vitro, at the expenses of endogenous phospholipid, labelled ethanolamine, serine, choline or other nitrogenous bases into EPG, SPG, CPG or other lipids [l-3]. Membranes from the endoplasmic reticulum of chick, rat and rabbit brains were found to possess the highest rate of incorporation, and the requirement for Ca2+ appeared to be absolute. The reaction, which takes place also in non-nervous tissue (see [2]), is regarded as an exchange process, not requiring energy, between the bound-base of the exchanging membrane phospholipid and the free base [2,3], and cannot be compared for several reasons with any of the known pathways of lipid synthesis. On studying this system we obtained evidence that the greater part of the incorporated ethanolamine or serine (about 85%) was confined to labelled diacylGPE or diacyl-GPS, respectively; the remainder of the radioactivity was not accounted for. The experiments described here were aimed at examining whether alkenylacyl-GPE and alkenylacyl-GPS could also be produced by similar mechanisms. It is shown that the microsomal fraction from rat brain converts by base-exchange labelled L-serine or ethanolamine into the plasmalogen derivatives [l-3], and the reaction restem are similar to those described for the synthesis of the diacyl derivative [l-3], and the reaction requires Ca2+ as the only factor to obtain the exchange.

Relative contribution of the de novo and remodelling pathways to the synthesis of platelet-activating factor in brain areas and during ischemia

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 than that 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.