The Recurrent Mossy Fiber Pathway of the Epileptic Brain

The dentate gyrus is believed to play a key role in the pathogenesis of temporal lobe epilepsy. In normal brain the dentate granule cells serve as a high-resistance gate or filter, inhibiting the propagation of seizures from the entorhinal cortex to the hippocampus. The filtering function of the dentate gyrus depends in part on the near absence of monosynaptic connections among granule cells. In humans with temporal lobe epilepsy and in animal models of temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network associated with loss of hilar interneurons. This recurrent mossy fiber pathway mediates reverberating excitation that can reduce the threshold for granule cell synchronization. Factors that augment activity in this pathway include modest increases in [K⁺]_o; loss of GABA inhibition; short-term, frequency-dependent facilitation (frequencies of 1–2 Hz); feedback activation of kainate autoreceptors; and release of zinc from recurrent mossy fiber boutons. Factors that diminish activity include short-term, frequency-dependent depression (frequencies <1 Hz); feedback activation of type II metabotropic glutamate receptors; and the potential release of GABA, neuropeptide Y, adenosine, and dynorphin from recurrent mossy fiber boutons. The axon sprouting and reactive synaptogenesis that follow seizure-related brain damage can also create or strengthen recurrent excitation in other brain regions. These changes are expected to facilitate participation of these regions in seizures. Thus, reactive processes that are often considered important for recovery of function after most brain injuries probably contribute to neurological dysfunction in epilepsy.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

A new role for the dynamin GTPase in the regulation of fusion pore expansion

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.     

Opposing interactions of ionophores (valinomycin and monensin) on calcium ion uptake in rat retinal preparations

Valinomycin is a potent inhibitor of taurine-stimulated ATP-dependent calcium ion uptake in rat retinal membrane preparations but had no effect on ATP-dependent calcium ion uptake in the absence of taurine and no effect on ATP-independent calcium ion uptake. The presence of potassium ions in the buffer systems were required for valinomycin to be inhibitory. On the contrary, monensin stimulated calcium ion uptake in the ATP-dependent system but had no effect on ATP-independent calcium ion uptake. The crude retinal homogenate was also fractionated into various subcellular components. The fraction which contained photoreceptor cell synaptosomes (P₁) had a higher, specific activity for taurinestimulated ATP-dependent calcium ion uptake than the crude homogenate or either the fractions which contained synaptosomes derived from the plexiform layer (P₂) or rod outer segments (ROS). No differences in calcium ion uptake were observed in the various subcellular fractions compared to the homogenate when assayed for ATP-dependent calcium ion uptake. Valinomucin inhibited both ATP-dependent and taurine-stimulated ATP-dependent calcium ion uptake in the P₁, P₂, and ROS fractions while monensin stimulated the ATP-dependent calcium ion uptake in the subcellular fractions.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

Effects of 5-hydroxytryptophan on serotonin in nerve endings

—Preparations of synaptosomes (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of 5-hydroxytryptophan (5-HTP) on (a) the levels of serotonin (5-HT) in nerve endings and (b) the release of 5-HT from nerve endings. The levels of 5-HT were significantly higher (3.21 × 0.35 nmol/g original tissue weight) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 50mg/kg of d ,l -5-HTP in comparison to control values (1.42 ± 0.07). A similar twofold increase was observed with the P₂ fraction isolated from the diencephalon plus optic lobes. In addition, the levels of 5-HTP and 5-hydroxyindoleacetic acid also increased significantly in these P₂ fractions isolated from pigeons given d ,l -5-HTP injections in comparison to values obtained for pigeons given saline injections. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5-HT indicated that 0.10 mil l -5-HTP increased the release of [³H]5-HT twofold over control values. A concentration as low as 0.001 mm l -5-HTP was tested on the P₂ fraction from the telencephalon of the pigeon and was found to significantly increase the release of [³H]5-HT over control values. This effect by l -5-HTP was blocked if a decarboxylase inhibitor was added to the medium. l -5-HTP at a concentration of 1.5 mm had no apparent effect on the release of [³H]norepinephrine or [³H]dopamine from synaptosomes prepared from the telencephalon of the rat or pigeon. The results are discussed in terms of the role of serotonin in producing certain types of behavioral depressions exhibited by pigeons and rats given injections of 5-HTP.     

Erythropoietin receptor-mediated inhibition of exocytotic glutamate release confers neuroprotection during chemical ischemia

Erythropoietin (EPO) reduced Ca(2+)-induced glutamate (Glu) release from cultured cerebellar granule neurons. Inhibition was also produced by EPO mimetic peptide 1 (EMP1), a small synthetic peptide agonist of EPO receptor (EPO-R), but not by iEMP1, an inactive analogue of EMP1. EPO and EMP1 induced autophosphorylation of Janus kinase 2 (JAK2), a tyrosine kinase that associates with EPO-R. Furthermore, genistein, but not genistin, antagonized both the phosphorylation of JAK2 and the suppression of Glu release induced by EPO and EMP1. During chemical ischemia, substantial amounts of Glu were released from cultured cerebellar and hippocampal neurons by at least two distinct mechanisms. In the early phase, Glu release occurred by exocytosis of synaptic vesicle contents, because it was abolished by botulinum type B neurotoxin (BoNT/B). In contrast, the later phase of Glu release mainly involved a BoNT/B-insensitive non-exocytotic pathway. EMP1 inhibited Glu release only during the early exocytotic phase. A 20-min exposure of hippocampal slices to chemical ischemia induced neuronal cell death, especially in the CA1 region and the dentate gyrus, which was suppressed by EMP1 but not iEMP1. However, EMP1 did not attenuate neuronal cell death induced by exogenously applied Glu. These results suggest that activation of EPO-R suppresses ischemic cell death by inhibiting the exocytosis of Glu.     

Ouabain induces acetylcholine release from pure cholinergic synaptosomes independently of extracellular calcium concentration

We have studied the correlation between [³H]ouabain binding sites, (Na⁺+K⁺)ATPase (EC 3.6.1.3) activity and acetylcholine (ACh) release in different subcellular fractions ofTorpedo marmorata electric organ (homogenate, synaptosomes, presynaptic plasma membranes). Presynaptic plasma membranes contained the greater number of [³H]ouabain binding sites in good agreement with the high (Na⁺+K⁺)ATPase activity found in this fraction. Blockade of this enzymatic activity by ouabain dose-dependently induced ACh release from pure cholinergic synaptosomes, either in the presence or absence of extracellular calcium ions. We suggest that one of the mechanisms involved in the ouabain-induced ACh release in the absence of Ca²⁺ _o may be an increase in Na⁺ _i that could (a) evoke Ca²⁺ release from internal stores and (b) inhibit ATP-dependent Ca²⁺ uptake by synaptic vesicles.     

Zinc in maturing rat brain: hippocampal concentration and localization

—Alterations in the localization and concentration of zinc in the hippocampus and in other regions of rat brain were studied during postnatal maturation. Histochemical observations indicated increasing levels of zinc in the hippocampal mossy fibre layer at about 20 days of age. Between 18 and 22 days, hippocampal levels of zinc increased by 35 per cent to reach adult levels (121 ng/mg of protein). After subcellular fractionation, more than one-half of the hippocampal zinc was found in the first pellet (P₁; 1085 g– 10 min), where large boutons would be expected. Autoradiographic evidence, revealing the sequential location of positron tracks first in the granular cell layer and later in the mossy fibre layer, raises the possibility of axoplasmic transport of zinc from granule cell perikarya to their terminal boutons. Our data suggest that zinc may be involved in the maturation and function of the mossy fibre pathway.     

The subcellular localization of noradrenaline and dopamine in bovine superior cervical ganglion

The distributions of noradrenaline and dopamine in subcellular fractions of bovine superior cervical ganglia were measured fluorimetrically and were compared with that of acetylcholine. Results indicate that the crude synaptosomal pellet ($\text{P}$₂), which contained the bulk of the bound acetylcholine, was not seriously contaminated with catecholamines. The microsomal fraction showed the highest concentration of noradrenaline relative to protein content, while dopamine was richest in $\text{P}$₂, possibly due to formation of synaptosomes from nerve endings of the dopaminergic interneurones which have been described in this tissue.     

Ca-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP₃) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP₃ mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP₃ mass levels under resting conditions. The IP₃ mass was neither modified by changes in external Ca²⁺ nor by a Ca²⁺-free medium containing EGTA. IP₃ mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K⁺ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca²⁺ emphasizing that Ca²⁺ entry, independently of the influx mechanism involved, leads to an IP₃ increase. The phospholipase C_β inhibitors U-73122 and U-73343 reduced K⁺-stimulated IP₃ levels while K⁺-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP₃ by agents that stimulate ACh release may suggest a possible link between IP₃ metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd     

Levels of glutamate,aspartate, GABA,and taurine in different regions of the cerebellum after X-irradiation-induced neuronal loss

The levels of glutamate (Glu), aspartate (Asp), -amino-n-butyric acid (GABA), and taurine (Tau) were determined in the cortex, molecular layer, and deep nuclei of cerebella of adult rats exposed to X-irradiation at 12–15 days following birth (to prevent the acquisition of late-forming granule cells; 12–15x group) and 8–15 days following birth (to prevent the acquisition of granule and stellate cells; 8–15x group). Also, the levels of the four amino acids were measured in the crude synaptosomal fraction (P₂) isolated from the whole cerebella of the control, 12–15x, and 8–15x groups. The level of Glu was significantly decreased by (1) 6–20% in the cerebellar cortex; (2) 15–20% in the molecular layer; and (3) 25–50% in the P₂ fraction of the X-irradiated groups relative to control values. The content of Glu in the deep nuclei was not changed by X-irradiation treatment. Regional levels of Asp were unchanged by X-irradiation, while its level in P₂ decreased by 15–30% after treatment. The levels of GABA and Tau in the molecular layer, deep nuclei, or P₂ were not changed in the experimental groups. However, there was a 15% increase in the levels of GABA and Tau in the cerebellar cortex of the 8–15x group relative to control values. The data support the proposed role of glutamate as the excitatory transmitter released from the cerebellar granule cells but are inconclusive regarding a transmitter role for either Tau or GABA from cerebellar stellate cells.     

Inhibitory effect of glutamate release from rat cerebrocortical synaptosomes by dextromethorphan and its metabolite 3-hydroxymorphinan

Dextromethorphan (DM), a widely used antitussive, has demonstrated an effective neuroprotective effect. Excessive release of glutamate is considered to be an underlying cause of neuronal damage in several neurological diseases. In the present study, we investigated whether DM or its metabolite 3-hydroxymorphinan (3-HM) could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). DM or 3-HM inhibited the Ca²⁺-dependent release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this presynaptic inhibition was concentration-dependent. Inhibition of glutamate release by DM or 3-HM was resulted from a reduction of vesicular exocytosis, because the vesicular transporter inhibitor bafilomycin A1 completely blocked DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release. DM or 3-HM did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release was blocked by ω-conotoxin MVIIC, an antagonist of N- and P/Q-type Ca²⁺ channel, not by dantrolene, an intracellular Ca²⁺ release inhibitor. DM or 3-HM modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitors GF109203X or Ro318220 all effectively occluded the inhibitory effect of DM or 3-HM. Furthermore, 4-AP-induced phosphorylation of PKC was reduced by DM or 3-HM. These results suggest that DM or 3-HM inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca²⁺ entry and PKC activity. This may explain the neuroprotective effects of DM against neurotoxicity.     

Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain

Conventional subcellular fractionation techniques have been applied to human fetal brain (13–15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P₁), a crude mitochrondrial pellet (P₂) and a supernatant (S₂). Further resolution of the P₂ fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P₂ and subsequently the P₂B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P₂B fraction contains membrane bound structures which resembie synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and -bungarotoxin binding were enriched in fractions P₂ and P₂B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.Special Issue dedicated to Prof. Eduardo De Robertis.     

Effects of taurine on GABA release from synaptosomes of rat olfactory bulb

Summary Superfusion of synaptosomes prepared from rat olfactory bulb revealed constant basal release of endogenous taurine (Tau), aspartate (Asp), glutamate (Glu) and-aminobutyrate (GABA): their release rates were 110.4 ± 13.0, 30.3 ± 6.7, 93.7 ± 13.1, and 53.3 ± 8.8 pmol/min/mg protein, respectively. The depolarizing-stimulation with 30mM KCl evoked 1.17-, 2.18-, 2.55- and 1.53-fold increases, respectively. Tau release was calcium-independent. However, the perfusion of synaptosomes with Tau (10µM) inhibited the evoked increase in GABA release by 63% without changing basal release, although it did not affect release of Asp and Glu. Phaclofen (10µM, a GABA_B receptor antagonist), but not bicuculline (10µM, a GABA_A receptor antagonist), counteracted the Tau-induced reduction in GABA release. These data suggest that Tau may be abundantly released from nerve endings of rat olfactory bulb and that it may regulate GABA release through the activation of presynaptic GABA_B autoreceptors.     

Synthesis and release of prostaglandins by rat brain synaptosomal fractions.

Abstract— Particulate fractions from rat brain homogenate containing the synaptosomes synthesize and release prostaglandins F and E on aerobic incubation. The prostaglandin of the F-typc released could be further identified as proslaglandin F_2α using specific radioimmunoassays for prostaglandins F_1α, and F_2α-. The metabolite 13,14-dihydro-15-keto-prostaglandin F_2α could not be detected. The amount of prostaglandins released is dependent on incubation time and temperature as well as pH and osmolarity of the incubation medium. Total brain homogenate released more prostaglandins than purified synaptosomes per mg protein, indicating that synaptosomes are probably not a main source of prostaglandins when compared with other subcellular brain fractions. While prostaglandin synthesis was only moderately increased by the addition of the precursor fatty acid arachidonic acid, anti-inflammatory drugs like indomethacin, high concentrations of some local anaesthetics and Δ¹-tetrahydrocannabinol inhibited prostaglandin release. The neurotransmitters noradrenaline, dopamine and 5-hydroxytryptamine did not influence prostaglandin release from the synaptosomal rat brain fractions.     

Effects of tryptophan on serotonin in nerve endings.

—Preparations of crude synaptosome fractions (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of l -tryptophan on (a) the levels of serotonin (5-HT), norepinephrinc (NE) and dopamine in nerve endings and (b) the release of radioactive 5-HT, NE and dopamine from nerve endings. The level of 5-HT was significantly higher (P < 0–05) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 300mg/kg of l -tryptophan in comparison to control values (1.11 ± 0.09 vs 0.74 ± 0.13 nmol/g original tissue wt). A smaller but not statistically significant increase in 5-HT was noted in the P₂ fractions isolated from the diencephalon plus optic lobes of pigeons given injections of l -tryptophan. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5–HT demonstrated that 1.0 mm -l -tryptophan caused a 30% increase (P < 0.05) in the release of [³H]5-HT over control values. This effect by l -tryptophan was blocked when a decarboxylase inhibitor was added to the medium. Tryptophan had no effect on the levels of NE or dopamine in these nerve endings nor did it have any effect on the release of these two amines from these preparations of synaptosomes. The results are discussed in terms of the role of serotonin in producing depression in pigeons working on a certain learned behavioural task.     

Effect of Bicuculline-Induced Status Epilepticus on Prostaglandins and Hydroxyeicosatetraenoic Acids in Rat Brain Subcellular Fractions

Abstract: Rat cerebrum, prelabeled in vivo by intraventric-ular injection of [1-¹⁴C]arachidonic acid, was used to assess cyclooxygenase and lipoxygenase reaction products in total homogenates, cytosol, synaptosomes, and microsomes. Effects of bicuculline-induced status epilepticus on arachi-donic acid metabolism in synaptosomes and microsomes were also measured. Lipoxygenase activity, resulting in the synthesis of hydroxyeicosatetraenoic acids (HETEs), and cyclooxygenase activity, resulting in the synthesis of prostaglandins (PGs), were measured by reverse-phase and normal-phase HPLC with flow scintillation detection. Endogenous lipoxygenase products in synaptosomes were identified by capillary gas chromatography-mass spectrometry. PGs and HETEs were detected in all subcellular fractions. The synaptosomal fraction showed the highest lipoxygenase activity, with 5-HETE, 12-HETE, and leukotriene B₄ as the major products. Following bicuculline-induced status epilepticus, endogenous free arachidonic acid and other fatty acids accumulated in synaptosomes, but not in microsomes. Incorporation of [1-^l4C]arachidonic acid into synaptosomal and microsomal phospholipids was decreased after bicuculline treatment. Bicuculline-induced status epilepticus resulted in increased synthesis of HETEs in synaptosomes. PG synthesis increased in the microsomal fraction. When [1-¹⁴C]arachidonic acid-labeled synaptosomes and microsomes were incubated for 1 h at 37°C the synthesis of eicosa-noids, particularly PGD₂, was increased significantly in bi-cuculline-treated rats, as compared with untreated rats. Depolarization (45 mM K⁺) of synaptosomes induced a loss of [1-¹⁴C]arachidonic acid from phosphatidylinositol, and increased the synthesis of PGD₂ and HETEs, an effect that was enhanced in bicuculline-treated rats. This study localizes changes in arachidonic acid metabolism and lipoxygenase activity resulting from bicuculline-induced status epilepticus in the brain subcellular fraction enriched in nerve endings.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.