Robert V. Dorman

Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.

ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells.

The accumulation of radiolabeled arachidonic acid (AA), immunoblot analysis of subcellular fractions, and immunofluorescence tagging of proteins in intact cells were used to examine the coupling of ANG II receptors with the activity and location of a cytosolic phospholipase A2(cPLA2) in vascular smooth muscle cells (VSMC). ANG II induced the accumulation of AA, which peaked by 10 min and was downregulated by 20 min. A large proportion of the AA released in response to ANG II was due to the activation of a Ca2+-dependent lipase coupled to an AT1 receptor. However, regulation of Ca2+ availability failed to completely block AA release, and a small but significant reduction in ANG II-mediated AA release was observed in the presence of an AT2 antagonist. These findings, coupled with a 25% reduction in the ANG II-induced AA release by an inhibitor specific for a Ca2+-independent PLA2, are consistent with the presence and activation of a Ca2+-independent PLA2. In contrast, immunoblot analysis and immunofluorescence detection showed that the ANG II-mediated translocation of cPLA2to a membrane fraction was exclusively AT1 dependent and regulated by Ca2+ availability. Furthermore, the nucleus was the membrane target. We conclude that ANG II regulates the Ca2+-dependent activation and translocation of cPLA2 through an AT1 receptor and that this event is targeted at the nucleus in VSMC.

12‐Lipoxygenase Products Attenuate the Glutamate Release and Ca2+ Accumulation Evoked by Depolarization of Hippocampal Mossy Fiber Nerve Endings

Abstract: Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium‐dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12‐lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5‐ or 15‐lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1–10 μM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12‐lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.

Prostaglandin involvement in the evoked release of D-aspartate from cerebellar Mossy fiber terminals

Isolated cerebellar glomeruli, containing mossy fiber terminals, were used to investigate the mechanisms involved in the evoked release of acid amino acids. The glomeruli contain a high affinity uptake system for D-aspartate, with a KT of 384 pmol/mg protein/min and the incorporated D-[3H]aspartate was released in response to various depolarizing agents, as well as exogenous arachidonic acid and prostaglandins. There was a marked inhibition of the release evoked by high K+ and exogenous arachidonate when the cyclooxygenase inhibitor ibuprofen was present. Also, exposure of the glomeruli to depolarizing conditions resulted in an increase in the amount of unesterified [3H]arachidonate. It appears that accumulation of unesterified arachidonate and subsequent production of prostaglandins are involved in the evoked release of the acidic amino acids from cerebellar mossy fiber terminals.

Effects of calcium antagonists on the evoked release of dynorphin A(1–8) and availability of intraterminal calcium in rat hippocampal mossy fiber synaptosomes

The pharmacological properties of presynaptic calcium (Ca) channels on rat hippocampal mossy fiber synaptosomes were characterized by determining the inhibitory potencies for various classes of Ca antagonists on depolarization-induced Ca mobilization and the release of dynorphin A(1-8)-like immunoreactivity (Dyn-LI). Flunarizine and cinnarizine were the most potent inhibitors of both parameters (IC50 values less than 10(-5) M). Gadolinium and omega-conotoxin (omega-CgTx) were also effective inhibitors of Dyn-LI release (IC50 values less than 3 x 10(-5) M), but omega-CgTx only partially reduced the level of cytosolic free Ca. The release of Dyn-LI was relatively insensitive to both the L-type (dihydropyridines, verapamil and diltiazem) and T-type (amiloride and phenytoin) channel blockers. It appears that presynaptic N-type Ca channels make the most substantial contribution to the Ca influx required for the exocytosis of Dyn-LI from hippocampal mossy fiber nerve endings.

Modulation of Glutamate Release From Hippocampal Mossy Fiber Nerve Endings By Arachidonic Acid And Eicosanoids

Arachidonic acid has been implicated in normal synaptic transmission processes, including those related to the development of hippocampal long-term synaptic potentiation. Hippocampal mossy fiber (MF) synaptosomes were used to investigate the role of arachidonate in the evoked accumulation of presynaptic Ca2+ and the release of endogenous glutamate, since these nerve terminals express long-term potentiation and selectively release glutamate as the excitatory transmitter. It was demonstrated that membrane depolarization evoked the accumulation of Ca2+, the release of glutamate, and the production of unesterified arachidonic acid. These events may be functionally related, since exogenous arachidonate and phospholipase A2 activation mimicked the effects of depolarization on Ca2+ availability and glutamate release, while secretion processes were attenuated in the presence of phospholipase A2 inhibitors. In addition, pretreatment of the nerve terminals with arachidonate or melittin allowed for the facilitated release of glutamate in response to a subsequent depolarizing stimulus. Inhibition of cyclooxygenase or lipoxygenase activities also potentiated presynaptic responses to membrane depolarization. In contrast, 12-lipoxygenase products attenuated the depolarization-evoked accumulation of intraterminal free Ca2+ and glutamate release. It is suggested that arachidonic acid acts as a positive modulator of mossy fiber secretion processes, including those involved in the increased glutamate release required for the induction of long-term potentiation, while 12-lipoxygenase metabolites provide negative feedback signals designed to limit neurotransmitter secretion.

DISPLACEMENT OF ENDOGENOUS GLUTAMATE WITH D-ASPARTATE : AN EFFECTIVE STRATEGY FOR REDUCING THE CALCIUM-INDEPENDENT COMPONENT OF GLUTAMATE RELEASE FROM SYNAPTOSOMES

Abstractd-aspartate was used in the present study to partially deplete the cytosolic pool of glutamate, which is released independent of extracellular Ca2+, prior to measuring the K+-evoked release of this endogenous acidic amino acid from rat hippocampal mossy fiber synaptosomes. This pretreatment is known to be an effective method for substantially reducing the Ca2+-independent component of glutamate release. The rate of glutamate efflux is dependent on the concentration of sodium ions in the external medium and can be stimulated by exposure of hippocampal mossy fiber synaptosomes to externald-aspartate (50 μM). Following the partial displacement of this cytosolic pool of glutamate withd-aspartate, the K+-evoked release of the residual, presumably vesicular, pool of endogenous glutamate has a strict requirement for external calcium and is highly dependent on the extent to which depolarization elevates the level of free cytosolic calcium. It is concluded that the protocol described in this study for the displacement of cytosolic glutamate withd-aspartate provides a useful alternative method of controlling for the Ca2+-independent component of glutamate release in synaptosomal preparations.

Characterization of the presynaptic calcium channels involved in glutamate exocytosis from rat hippocampal mossy fiber synaptosomes

Calcium antagonists inhibit both the Ca2(+)-dependent and -independent release of endogenous glutamate from intact synaptosomes. In the present study, the inhibitory potency of several different classes of calcium antagonists were determined under conditions that control for an effect of these compounds on the Ca2(+)-independent component of glutamate release. The following order of inhibitory potency was derived: flunarizine and cinnarizine greater than diltiazem greater than verapamil, nifedipine and nimodipine greater than omega-conotoxin much greater than amiloride, phenytoin, gadolinium and nickel. Only the diphenylpiperazine derivatives inhibited Ca2(+)-dependent glutamate release with an IC50 value of less than 10(-5) M. This finding indicates that no one type of presynaptic calcium channel predominantly mediates Ca2(+)-dependent glutamate release from hippocampal mossy fiber terminals. It is suggested that the exocytosis of glutamate from rat hippocampal mossy fiber synaptosomes may be mediated by multiple types of calcium channels.

Arachidonic Acid and Oleoylacetylglycerol Induce a Synergistic Facilitation of Ca2+-Dependent Glutamate Release from Hippocampal Mossy Fiber Nerve Endings

Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization‐evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca2+‐dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca2+ ionophore ionomycin. The Ca2+ dependence of this effect was established, as removal of Ca2+ eliminated evoked release and the lipid‐dependent potentiation. Also, Ca2+ channel blockers attenuated ionomycin‐ and KCI‐evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca2+, it may not involve an enhancement of evoked Ca2+ accumulation, because ionomycin‐dependent glutamate release was potentiated under conditions that did not increase ionomycin‐induced Ca2+ accumulation. Also, the facilitation may not depend on inhibition of K+ efflux, because enhanced release was observed in the presence of increasing concentrations of 4‐aminopyridine and diazoxide did not reduce the lipid‐dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid‐dependent facilitations of both KCI‐ and ionomycin‐evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid‐dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca2+ and involves multiple presynaptic effects, some of which depend on protein kinase C.

Effects of Cerebral Ischemia and Reperfusion on Prostanoid Accumulation in Unanesthetized and Pentobarbital-Treated Gerbils

Cerebral ischemia was induced in unanesthetized gerbils using bilateral carotid artery ligations. The effects of 20 min of global ischemia on the concentrations of prostaglandin F2α (PGF2α), PGE2, 6-keto-prostaglandin F1α (6-keto-PGF1α), and thromboxane B2 were determined after 0–24 h of reperfusion. Ischemia had little effect on eicosanoid production, but significant increases were observed by 5 min of reperfusion, with maximal levels reached by 15 min of reperfusion. PGF2α was the most concentrated prostaglandin in postischemic brain, whereas PGE2 was most concentrated in control cerebra. Pretreatment with anesthetic doses of pentobarbital supported increased accumulation of PGF2α in postischemic cerebra, increased accumulation of 6-keto-PGF1α during the ischemic episode, and decreased accumulation of PGE2 at 120 min of reperfusion. It appears that the protective effects of barbiturate anesthesia are not expressed by the reduced accumulation of the above eicosanoids.