Mitochondrial-synaptosomal Fraction Research Articles

Preparation of purified perikaryal and synaptosomal mitochondrial fractions from relatively small hypothalamic brain samples

In order to measure the activity of neuronal mitochondria, a representative proof of neuronal processes, physiologically relevant mitochondrial samples need to be gained as simply as possible. Existing methods are, however, either for tissue samples of large size and/or homogenous microstructures only, or are not tested for mitochondrial function measurements. In the present article we describe a gradient fractionation method to isolate viable and well-coupled mitochondria from relatively heterogeneous histological microstructures such as the hypothalamus. With this new method, we are able to isolate a sufficient amount of functional mitochondria for determination of respiratory activity, in a short period of time, using affordable equipment.•Verified by electron microscopy, our method separates highly enriched and well-preserved perikaryal and synaptosomal mitochondria. Both fractions contain minimal cell debris and no myelin. Respiratory measurements (carried out by Clark-type electrode) confirmed undisturbed mitochondrial function providing well-evaluable records. The demonstrated protocol yields highly viable mitochondrial subfractions within 3h from small brain areas for high-precision examinations. Using this procedure, brain regions with relatively heterogeneous histological microstructure (hypothalamus) can also be efficiently sampled.•Up to our present knowledge, our method is the shortest available procedure with the lowest sample size to gain debris-free, fully-viable mitochondria.

Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4- (adult), 28-, and 33-mon-old (aged) rats. PI-4,5-bisphosphonate (PIP2)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP2-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP2-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.

Pro- or anti-oxidant manganese: a suggested mechanism for reconciliation

The neurodegeneration induced by manganese has been attributed to its ability to undergo redox cycling, and catalysis of reactive oxygen species (ROS) formation, as with other transition metals. However, the characterization of manganese as a pro-oxidant is confounded by increasing evidence that the metal may scavenge superoxide anions and protect cells from oxidative damage. The current study was designed to address conflicting reports pertaining to the oxidative capacity of manganese. We found that the metal has distinctive redox dynamics in which the divalent reduced form, unlike iron, possessed no intrinsic oxidative capacity. The apparent ability of Mn 2+ to promote the formation of ROS within a cortical mitochondrial-synaptosomal fraction was quenched by the depletion of contaminating nanomolar concentrations of trivalent metals. The addition of manganic ions at trace concentrations dose-dependently restored the oxidative capacity attributed to divalent manganese, whereas the presence of the ferric ion retarded the rate of ROS generation. This result was paralleled by the spectrophotometric demonstration that the kinetics of iron oxidation is accelerated by trivalent but not divalent manganese. The markedly different capacities of the lower and higher valence states of manganese to promote free-radical formation in cortical fractions and to modulate the process of iron oxidation may account for earlier contradictory reports of anti- and pro-oxidant properties of manganese.

Dopamine Oxidation Products Inhibit Na+, K+-ATPase Activity in Crude Synaptosomal–mitochondrial Fraction from Rat Brain

The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50–200 μM) causes dose-dependent inhibition of Na+, K+-ATPase activity of rat brain crude synaptosomal–mitochondrial fraction during in vitro incubation up to 2 h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na+, K+-ATPase. Under similar conditions of incubation, DA (200 μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal–mitochondrial proteins and the phenomenon is also prevented by glutathione (5 mM) or cysteine (5 mM).The available data imply that the inactivation of Na+, K+-ATPase in this system involves both H2O2 and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na+, K+-ATPase from DA-mediated damage. The inactivation of neuronal Na+, K+-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease.

Dopamine induced protein damage in mitochondrial-synaptosomal fraction of rat brain

Dopamine during in vitro oxidation induced covalent cross-linking of membrane proteins in rat brain crude mitochondrial-synaptosomal fraction. The process is not inhibited by hydroxyl radical scavengers, lipid soluble anti-oxidants, metal-chelator or catalase, but reduced glutathione produced a dramatic inhibition of cross-linking. The protein cross-linking mediated by dopamine is not associated with any detectable membrane lipid peroxidation but significant formation of protein bound quinone takes place during incubation. Our results indicate that reactive quinones rather than oxygen free radicals are involved in dopamine induced protein cross-linking in rat brain membrane fraction.

Interactions of Dynorphin A-(1–13) and Nociceptin with Cardiac D2 Binding Sites: Inhibition of Ischemia-evoked Release of Noradrenaline from Synaptosomal–mitochondrial Fractions

The effect of dynorphin A (Dyn A)-related peptides and nociceptin on the binding of the D2 receptor antagonist, [3H]raclopride, was examined in membrane preparations of rat heart. Non-linear regression saturation binding analysis of [3H]raclopride binding revealed the presence of a single high-affinity binding site with a Kdof 4.1 n M and a Bmaxof 220 fmol/mg protein. The D2 stereospecificity of [3H]raclopride binding was demonstrated by competition experiments using two enantiomer pairs of antagonists. (+)-Butaclamol (IC50: 8.0 n M) and (−)-sulpiride (IC50: 112.3 n M) were 27 000 and 24 times more potent than (−)-butaclamol (IC50: >100 μ m) and (+)–sulpiride (IC50: 2666 n M), respectively. Nociceptin and Dyn A-(1–13) were also potent inhibitors of the binding of [3H]raclopride with shallow inhibition curves that fitted best with two sites model. Their order of potency on the low affinity site [α -Neo-endorphin>nociceptin>Dyn A-(2–13)>Dyn A-(1–13)>Dyn B>Dyn A-(6–10)] correlated well with their ability to inhibit the binding of [3H]nociceptin (r=0.82). The indirect nature of the inhibitory effects of the peptides on the D2 receptor was demonstrated by their inability to inhibit [3H]raclopride binding to a membrane preparation (Sf9 cells transfected with the human D2longreceptor) that does not contain the ORL1receptor and the lack of effect of raclopride (0.1 nM –10 μ m) on both [3H]nociceptin and [3H]Dyn A-(1–13) binding. Isolated cardiac mitochondrial–synaptosomal fractions submitted to ischemic conditions (1 m M iodoacetate +2 m M NaCN, 5 min at 37°C) released 10.9% of their content in preloaded [3H]noradrenaline ([3H]NA). Dyn A-(1–13) (10 μ m), nociceptin (10μ m) and the selective D2 receptor agonist, quinpirole (10 μ m) were potent blockers of the release of [3H]NA evoked by the ischemic conditions. The inhibitory effect of Dyn A-(1–13), nociceptin and quinpirole were antagonized by the selective D2 receptor antagonist, raclopride (10 μ m); whereas naloxone, at a concentration (1 μ m) known to affect the ORL1receptor, blocked the effects of the peptides but not those of quinpirole. The results demonstrate the presence of D2 receptors in rat heart and suggest that Dyn A-(1–13) and nociceptin modulate ischemia-induced NA release by a mechanism that involves the participation of both ORL1and D2 receptors.

Glucocorticoids regulate protein synthesis in hippocampal slices under mild heat shock conditions.

Glucocorticoid hormones potentiate the toxic effects of neuronal stressors. Alteration of gene expression by glucocorticoids could contribute to neuronal susceptibility by downregulating the synthesis of proteins necessary to adapt to challenge. Using heat shock of hippocampal slices as a model for cellular insult, protein synthesis has been examined in response to acute glucocorticoid administration to rats. Incubation of hippocampal slices at 39 degrees C produces a heat-shock pattern of protein synthesis in that total incorporation of labeled amino acid is diminished, whereas synthesis of the major heat-shock proteins, HSP90 and HSP70, is increased. Prior administration of corticosterone to rats does not affect subsequent synthesis of HSP90 or HSP70 in slices. However, at 4 or 24 h following a single corticosterone injection, the synthesis of two acidic proteins is found to be altered: a 25-kDa protein is downregulated in the nuclear and synaptosomal-mitochondrial fraction of the hippocampus, and a 47-kDa protein is downregulated in all three fractions of the hippocampus, cortex, and cerebellum. These effects are mimicked by administration of RU-28362, a specific glucocorticoid (GR or Type II) receptor agonist. Since decreased synthesis of p25 and p47 is the only glucocorticoid-mediated response observed in slices under heat-shock conditions, these proteins may be related to the adaptation to heat shock.

Nonexocytotic noradrenaline release from rat cardiac synaptosomal-mitochondrial fractions.

Nonexocytotic noradrenaline (NA) release was examined in rat cardiac synaptosomal-mitochondrial fractions prelabeled with [3H]NA (300 nM; 1 h at 37 degrees C). Ischemic conditions (1 mM iodoacetate + 2 mM NaCN; 15 min at 37 degrees C) evoked a Ca(2+)-independent release of [3H]NA from isolated synaptosomes, which represented 33.4% of total content, whereas the release evoked by 56 mM K+ was Ca2+ dependent and represented 5.8% of total content. Tyramine, phencyclidine (PCP), and rimcazole also caused important Ca(2+)-independent releases of [3H]NA (from 12 to 45% of total content) with median effective concentrations (EC50s) of 6.8, 182, and 41.8 microM, respectively. The release responses evoked by ischemic conditions, tyramine, PCP, and rimcazole were mimicked by the delta-receptor ligand, 1,3-ditolyl guanidine (DTG), and blocked by the uptake 1 inhibitor, desipramine (100 microM). The delta 1-receptors ligands, (+)-3-hydroxyphenyl-N-(1-propyl)piperidine ((+)-3-PPP) and (+)N-allylnormetazocine [(+)SKF-10047], were potent blockers of the release of [3H]NA evoked by ischemic conditions but not by PCP or rimcazole. These data indicate that ischemic conditions and PCP/delta 2-receptor ligands induce carrier-mediated NA efflux from cardiac sympathetic nerve terminals, whereas delta 1-receptor ligands produce marked inhibition of the ischemic response.

Isolation of 1-Methyl-1,2,3,4-Tetrahydroisoquinoline-Synthesizing Enzyme from Rat Brain: A Possible Parkinson's Disease-Preventing Enzyme

An endogenous parkinsonism-preventing substance, 1-methyl-1,2,3,4-tetrahydroisoquinoline, is enzymatically formed from 2-phenethylamine and pyruvate in rat brain. The enzyme involved was localized in the mitochondrial-synaptosomal fraction of rat brain, solubilized by treatment with sodium deoxycholate. In order to purify the enzyme, gel filtration was carried out. This enzyme may be important in the pathogenesis of Parkinson's disease.

Inhibitory Effects of Dynorphin-A on Norepinephrine Uptake by Cardiac Synaptosomal-Mitochondrial Fractions

The effect of dynorphin A-(1-13) (Dyn A-(1-13)) on [3H]norepinephrine ([3H]NE) uptake was examined in cardiac synaptosomal-mitochondrial fractions of control rats (Wistar, WR; Wistar-Kyoto, WKY) and spontaneously hypertensive rats (SHR). In adult WR, Dyn A-(1-13) caused naloxone-insensitive dose-dependent inhibition of [3H]NE uptake with an IC50 of 4.0 microM. The nonopioid Dyn A fragments Dyn A-(2-13) and Dyn A-(6-10) displayed 89 and 11% of the potency of Dyn A-(1-13), respectively, whereas Dyn A-(1-8), Leu-enkephalin, and the selective opioid agonists [D-Ala2, N-MePhe4, Glyol5]enkephalin (DAGO, mu), [D-Ser2, Thr6]Leu-enkephalin (DSLET, delta), and U-50488H (kappa) were inactive. The relative potency of various analogues and fragments of Dyn A in inhibiting [3H]NE uptake correlated well (r = 0.96) with their potency in inhibiting binding of [3H]Dyn A-(1-13) to nonopioid sites on cardiac membrane preparations. Dyn A-(1-13) showed the same potency in inhibiting [3H]NE uptake in prehypertensive (4-week-old) SHR as in age-matched WR and WKY. However, at ages 8 and 16 weeks Dyn A-(1-13) was twice as potent in SHR as in WR and WKY. The increased inhibitory potency of Dyn A-(1-13) in 8-week-old SHR was accompanied by a 1.3-fold increase in number of cardiac nonopioid [3H]Dyn A-(1-13) binding sites. Dyn A and related peptides inhibit [3H]NE uptake by cardiac synaptosomes by a nonopioid mechanism. The possible involvement of such a mechanism in development of hypertension in SHR is discussed.

Possible role of the non-opioid dynorphin A-(1–13) receptor in the inhibition of [ 3H]noradrenaline uptake in rat heart

Recently, our laboratory has demonstrated the presence of low affinity and hiBh capacity non-opioid binding sites for [ 3 H]Dynorphin A-(1-13) ([ 3 H]Dyn A-(1-13)) in membrane preparations of rat heart (1). In order to determine if these sites could be linked to some cardiovascular function, we measured the effects of Dyn A and related peptides as well as selective κ, μ and δ opioid receptor ligands on the uptake of [ 3 H]noradrenaline ([ 3 H]NA) in a heart synaptosomal-mitochondrial fraction. The uptake of [ 3 H]NA was temperature, Na + and nisoxetine sensitive and blocked by Dyn A and related peptides. A good correlation (r=0.96) was established between the ability of the peptides to inhibit [ 3 H]NA uptake and to compete with the binding of [ 3 H]Dyn A-(1-13) to non-opioid sites

Central mu-opioid receptors are down-regulated in a rat model of cholestasis

Ameliorations of the pruritus of cholestasis by opioid antagonists are consistent with this form of pruritus being centrally mediated by the opioid system. To determine whether the central opioid system is altered in cholestasis, the specific binding of a selective mu-opioid receptor ligand, 3H-DAMGO, to mu-opioid receptors was studied in rats with acute cholestasis due to bile duct resection. Using whole brain membranes and subcellular mitochondrial-synaptosomal fractions the density of mu-receptor sites was 30% ( p < 0.01) and 22% ( p = 0.03) less in bile-duct-resected rats than in sham-resected rats. Using membranes from individual brain regions specific binding of 3H-DAMGO was reduced by 43–53% in the cerebral cortex, hippocampus and caudate nucleus of bile-duct-resected rats. Thus mu-opioid receptors in the brain are down-regulated in a classical model of cholestasis. This alteration of the central opioid system could be a consequence of increased exposure of opioid receptors to endogenous opioids in cholestasis and may reflect an important mechanism in the pathogenesis of the pruritus of cholestasis.

Characterization of norepinephrine accumulation by a crude synaptosomal-mitochondrial fraction isolated from rat heart

Norepinephrine (NE) uptake into a heart synaptosomal-mitochondrial fraction was assessed under conditions where neuronal uptake (type 1) was linear with respect to both time and protein concentration. The NE accumulation process was sensitive to incubation temperature, sodium ion concentration and medium osmolality. Furthermore, NE uptake was attenuated by the neuronal uptake inhibitor desmethylimipramine (DMI) in a concentration dependent manner; the IC 50 value was approximately 10 nM and maximum inhibition was obtained at 100 nM. In contrast, the extraneuronal uptake inhibitor, metanephrine did not significantly attenuate NE uptake. Kinetic analysis demonstrated that the DMI sensitive NE accumulation is saturable with a K M of approximately 400 nM and that NE uptake occurs via a single uptake process. This demonstration of neuronal type NE uptake by a synaptosomal-mitochondrial fraction constitutes a successful demonstration of the preparation of a rat heart subcellular fraction containing functional synaptosomes.

A re-examination of the Na+-independent binding of [3H]beta-alanine to rat brain stem-spinal cord.

The Na+-independent binding of [3H]beta-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described beta-alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na+-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na+-independent binding, using frozen/thawed/washed synaptosomal-mitochondrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200-415 micrograms/ml incubation medium. Binding was completely inhibited by glycine, alanine, alpha-aminobutyric acid, beta-aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl-beta-alanine, brucine and gelsemine. It was insensitive to taurine, gamma-aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (KD 20 microM), and heat sensitive.

An endogenous regulator of diacylglycerol kinase

During the initial steps of the subcellular fractionation of rat brain homogenate, we recovered more than 100% of diacylglycerol kinase activity. The unusually high yields prompted us to examine the possibility that we had removed an endogenous inhibitor from diacylglycerol kinase during those steps. Our study revealed the existence of a potent inhibitor of diacylglycerol kinase in the crude synaptosomal-mitochondrial fraction (P2 pellet). The inhibitory substance was water soluble upon organic solvent extraction. The inhibitory activity of the substance was retained after extensive dialysis, suggesting the macromolecular nature of this compound. This substance may represent an important physiological regulator of diacylglycerol kinase.

In vitro transformation elicited by Creutzfeldt-Jakob-infected brain material.

Previous studies indicated that tissue culture cells derived from Creutzfeldt-Jakob disease (CJD) brains possess neoplastic properties. In order to see if CJD brain material could itself transform cells in vitro, BALB/C-3T3 and normal hamster brain monolayer cultures were exposed to whole brain homogenate or synaptosomal-mitochondrial fractions derived from CJD or control brains. Cells were exposed for seven days to CJD or control brain material, washed, and then passaged at weekly intervals. Transformation was evaluated by loss of contact inhibition, replication of cells with absent or low serum content of the medium and significant colony formation in soft agar. Seven parallel experiments were done, and six were viable. All cultures exposed to CJD material containing greater than or equal to 10(4) LD50 infectious units were positive for transformation (five of six studies); the single negative experiment contained only 2 X 10(3) infectious units. Definitive transformation could be observed at 12-16 weeks after application of both mouse and hamster CJD material. BALB/C-3T3 cells exposed to control hamster or mouse brain material were studied for greater than 120 passages in vitro and showed no comparable changes or evidence of spontaneous transformation, i.e. they required serum for replication and produced essentially no colonies in soft agar. Similarly, a normal adult hamster brain culture exposed to control brain material showed no evidence of transformation, whereas the identically passaged normal brain culture exposed to CJD material not only became a permanent line, able to grow in soft agar, but also was capable of producing tumors in nude mice. Thus CJD transformation was not confined to special potentials of "permanent" BALB/C-3T3 cells. These experiments suggest that a genomic alteration can be effected by CJD material.

Detection of Scrapie-associated Fibril (SAF) Proteins Using Anti-SAF Antibody in Non-purified Tissue Preparations

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.

Does 6-hydroxydopamine treatment change the rat's behavioural sensitivity to noradrenergic drugs?

Intraperitoneal (i.p.) injection of 250 μgkg of oxotremorine (OT) caused a 50% decrease in the glycine content of the synaptosomal-mitochondrial fraction of spinal cord homogenates prepared from rats killed 15 min after treatment. The glycine content of the supernatant fraction was correspondingly raised. In synaptosomes isolated from the spinal cord of OT-treated rats, the decrease in glycine content was 30%. Prior administration of atropine, but not of methylatropine, abolished this effect of OT on synaptosomal glycine content. Eserine exerted a potentiating effect on the action of OT in lowering the glycine content of spinal synaptosomes. Prior administration of l-dopa, apomorphine, haloperidol, muscarine or mecamylamine had no significant effect on the action of OT on synaptosomal glycine content. OT alone or in combination with eserine, and acetylcholine (ACh) in combination with eserine, did not alter the rate of release of glycine from spinal synaptosomes of untreated rats incubated under appropriate conditions. OT was also without effect on the rate of release of glycine from normal spinal synaptosomes subjected to electrical stimulation, as well as on the eventual glycine content of the synaptosomes. On the basis of the present findings it has been suggested that (i) glycine may be released from Renshaw cells at their synapses with motoneurones in response to the muscarinic action of OT; (ii) dopaminergic modulation may not be involved in the OT-induced glycine release from Renshaw cells; and (iii) excessive release of glycine onto motoneurones may be the causative factor of the akinesia observed in OT-induced experimental Parkinsonism.

Phosphatidate phosphatase activity in isolated rod outer segment from bovine retina

Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2- 3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated K m value was 6.7 · 10 −4 M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using as substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70–75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg 2+, further additions of the ion (0.2–2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F − (50 mM), Ca 2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent activity present in rod outer segments was stimulated by Triton X-100 and taurocholate. Similar values were observed in the enzyme activity of entire rod outer segments and in that of disks obtained from them, showing the enzyme to be associated with disk membrane. The [ 3H]diacyIglycerol] production from [ 3H]phosphatidic acid was analyzed in synaptosomal-mitochondrial and microsomal fractions and in a crude pigment epithelium preparation. The degree of dephosphorylation of phosphatidic acid in these subcellular fractions was as follows: microsomes > synaptosomal-mitochondrial fraction > rod outer segment > crude pigment epithelium. The present report is the first evidence of phosphatidic acid phosphohydrolase activity associated with rod outer segment membranes.

3H]Kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain

Binding of [ 3H]kainic acid to the synaptosomal-mitochondrial fraction (P2) of the goldfish brain was studied. Specific binding to this fraction represents about half of the total binding capability of the homogenate particulate material and is enriched in synaptic membranes; it is greater by about two orders of magnitude than those given for rat brain and pigeon optic tectum membranes. Association of the ligand-site complex has a time constant lower than 1 min and the same is true for the main component of the dissociation process. The binding equilibrium is apparently not affected by substance contained in the fraction material. The analysis of the dose- response data showed a main receptor population (B max= 139pmol/mg protein) which displayed posperativity (n H = 1.29). The same behaviour was shown by washed membranes from the same fraction but, in this case, the affinity for the ligand was lower (apparent affinity constants: K′ D = 0.28nM for the intact fraction and K′ D = 0.38nM for membranes). A smaller population of sites with higher affinity was also detected both in the intact fraction and in membranes. Among the substances tested as displacers of kainic acid from the synaptosomal sites, the most effective were quisqualate and l-glutamate. Folic acid and its dihydro and tetrahydro derivatives were half as potent as glutamate whereas methyltetrahydrofolic acid and folinic acid had a very weak action. The difference between these sites and those detected on rat brain membrane preparations is discussed.