Ether lipid content of phosphoglycerides from the retina and brain of chicken and calf.

Phospholipid contents and compositions were determined for chicken and calf retinas, chicken brain and calf gray matter. Retinal phospholipid compositions differ from brain phospholipid compositions by including a higher percentage of choline phosphoglycerides and lower percentages of ethanolamine and serine phosphoglycerides. The proportion of sphingomyelin is lower in calf retina than in calf brain. Among the ethanolamine phosphoglycerides, the proportions of the alk-1-enylacyl (plasmalogen) type are lower and the proportions of alkylacyl and diacyl types are higher in retina than in brain. The alkyacyl glycerophosphoethanolamines accounted for 7.6% and 8.9% of the ethanolamine phosphoglycerides from chicken and calf retinas respectively. Lower proportions of plasmalogens in the choline phosphoglycerides were found in retina as compared with brain. The alkylacyl glycerylphosphocholines comprised 4.0% of the retinal choline phosphoglycerides. Overall, a smaller proportion of retinal phosphilipids than of brain phospholipids contained alkyl or alk-1-enyl ether groups and the ratio of alkyl groups to alk-1-enyl groups was greater in retina than in brain.

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 F2 alpha (PGF2 alpha), PGE2, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), 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. PGF2 alpha 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 PGF2 alpha in postischemic cerebra, increased accumulation of 6-keto-PGF1 alpha 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.

PGF2 alpha synthesis in isolated cerebellar glomeruli: effects of membrane depolarization, calcium availability and phospholipase activity.

The controlling factors for PGF2 alpha production were assessed in isolated cerebellar glomeruli, since this prostaglandin has been shown to stimulate the release of neurotransmitters from the mossy fiber terminals associated with this synaptic preparation. The metabolism of PGE2 was also examined, in order to determine the specificity of any treatment effects. It was observed that K(+)-dependent membrane depolarization or the activation of voltage-sensitive Na+ channels with veratradine stimulated the production of PGF2 alpha. The syntheses of both prostanoids were dependent on available calcium and were blocked by cyclooxygenase inhibitors. The lipoxygenase inhibitor NDGA also reduced the accumulation of PGE2 and PGF2 alpha. In addition, PGF2 alpha synthesis was stimulated by the phospholipase A2 activator melittin and was reduced due to phospholipase inhibition with dibucaine. These results are consistent with a role for PGF2 alpha in the evoked release of neurotransmitter from cerebellar mossy fiber terminals.

Prostaglandins F2 alpha synthesis in the hippocampal mossy fiber synaptosomal preparation: II. Effects of receptor activation.

Mossy fiber nerve endings were isolated from rat hippocampi and used to determine the effects of receptor activation on the production of prostaglandin F2 alpha (PGF2 alpha). Glutamate and its agonists had no effect on PGF2 alpha synthesis. Similarly, acetylcholine, gamma-aminobutyric acid, histamine and purinergic receptor agonists did not affect PGF2 alpha accumulation in this preparation. However, norepinephrine, serotonin and dopamine exerted receptor-mediated stimulations of PGF2 alpha production. The agonist-evoked increases in PGF2 alpha production were attenuated by phospholipase A2 inhibitors, L-type voltage-sensitive Ca2+ blockers and a K+ channel activator, but they were insensitive to tetrodotoxin. In addition, a kappa opioid agonist decreased PGF2 alpha synthesis in unstimulated and depolarized synaptosomes. It appeared, therefore, that certain receptor agonists were able to modulate PGF2 alpha synthesis in the hippocampal mossy fiber synaptosomal preparation.

Prostaglandin F2 alpha synthesis in the hippocampal mossy fiber synaptosomal preparation: I. Dependence in arachidonic acid, phospholipase A2, calcium availability and membrane depolarization.

Isolated hippocampal mossy fiber synaptosomes were used to characterize control mechanisms of prostaglandin F2 alpha (PGF2 alpha) synthesis at a central mammalian synapse. Exogenous arachidonic acid stimulated the dose-dependent synthesis of PGF2 alpha, as did the addition of phospholipase A2 or the activation of endogenous phospholipase A2. Phospholipase A2 inhibitors attenuated prostaglandin synthesis, but phospholipase C inhibitors had no effect. However, a diglyceride kinase inhibitor reduced PGF2 alpha accumulation. The cyclooxygenase inhibitor ibuprofen eliminated PGF2 alpha production, while the lipoxygenase inhibitors baicalein and NDGA reduced PGF2 alpha accumulation. The CA(2+)-ionophore-dependent stimulation of PGF2 alpha synthesis was abolished by Cd2+ or Ni2+. Further more, PGF2 alpha production appeared to be dependent on Ca2+ influx via L-type, but not N- or T-type, voltage-sensitive Ca2+ channels. Membrane depolarization with KC1, veratridine or 4-aminopyridine stimulated the synthesis of PGF2 alpha. This depolarization-dependent stimulation of PGF2 alpha synthesis was attenuated by L-type voltage-sensitive Ca2+ channel blockers, phospholipase A2 inhibitors, a K+ channel activator and a Na+ channel blocker. The activation of protein kinase C also led to a reduction of PGF2 alpha accumulation in depolarized nerve endings. These results may be used to suggest that PGF2 alpha production by hippocampal mossy fiber synaptosomes was controlled by the Ca(2+)- and phospholipase A2-dependent accumulation of unesterified arachidonic acid and was modulated by membrane depolarization and the activity of protein kinase C.

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.

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.

Synergistic potentiation of glutamate release by arachidonic acid and oleoyl-acetyl-glycerol.

Both the activation of protein kinase C (PKC) and the facilitation of depolarization-evoked glutamate release have been implicated in the induction of hippocampal long-term synaptic potentiation. These observations may be functionally related, since stimulation of PKC activity enhances evoked glutamate release. Recently, it was shown that arachidonic acid and the diacylglycerol analog oleoyl-acetyl-glycerol activate brain PKC in a synergistic fashion. We report the facilitation of depolarization-induced glutamate efflux from hippocampal mossy fiber synaptosomes due to a combination of arachidonic acid and oleoyl-acetyl-glycerol. The potentiating effects appeared to depend on the activation of PKC since they were attenuated by staurosporine. In addition, the effects of arachidonic acid and oleoyl-acetyl-glycerol appeared to be limited to calcium-dependent processes.

Depolarization-induced [3H]arachidonic acid accumulation: effects of external Ca2+ and phospholipase inhibitors.

Isolated cerebellar glomeruli were prelabeled with [3H]arachidonate prior to assessment of the roles of external Ca2+ and phospholipases in the depolarization-induced accumulation of unesterified arachidonate. The glomerular particles have previously been shown to release neurotransmitters upon exposure to depolarizing conditions, calcium influx, exogenous arachidonate and added prostaglandins. It was observed that membrane depolarization caused an increased accumulation of [3H]arachidonate, which was inhibited by EGTA, verapamil or the lipase inhibitors mepacrine and dibucaine. The major effect of EGTA was expressed on the catabolism of [3H]triglycerides, while verapamil prevented the loss of radioactivity from inositol glycerophospholipids and the lipase inhibitors reduced by triglyceride and inositol glycerophospholipid catabolism.

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.

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.

Relationship between prostaglandin synthesis and release of acidic amino acid neurotransmitters.

The importance of glutamate as an excitatory neurotransmitter in the central nervous system has become increasingly clear. However, the presynaptic mechanism of amino acid neurotransmitter release does not appear to be consistent with existing models. A major source of controversy has been the finding that a significant amount of the amino acid release evoked by membrane depolarization is calcium-independent. It is often implied that this component of release is of cytosolic origin and subserves no neurotransmitter function. In this report, an alternative model is presented which suggests that the depolarization-induced movement of calcium ions into the nerve terminal acts, simultaneously, to stimulate the release of acidic amino acid neurotransmitters from both a vesicular and cytosolic compartment. According to this model, the influx of calcium may indirectly stimulate the biosynthesis of prostaglandins which lower the plasma membrane potential and reverse the electrogenic transport of amino acids to cause a net efflux. Such a mechanism could explain how the vesicular and cytosolic neurotransmitter pools of release are functionally related.

[3H]arachidonic acid metabolism in rat brain minces: effects of nucleotide triphosphates, CDPcholine and CMP.

Rat brain minces were used to investigate the effects of nucleotides on the metabolism of arachidonic acid in nerve tissue. Brain free fatty acids, neutral lipids and phospholipids, were radiolabeled in vivo following intracerebral injection of [3H]arachidonic acid. Minces were prepared from the radiolabeled cerebra and were incubated in a modified Krebs-Ringer buffer with and without various nucleotides. The incubation-induced accumulation of unesterified [3H]arachidonate was reduced in the presence of CDPcholine, ATP, CTP, GTP, and UTP. These nucleotides inhibited choline and inositol glycerophospholipid hydrolysis. They also reduced the amount of labeled diglycerides. However, CDPethanolamine had no effect on arachidonic acid metabolism in the mince preparation and CMP appeared to stimulate further hydrolysis of choline glycerophopholipids, resulting in increased accumulation of [3H]arachidonic acid and labeled diglycerides. We suggest that the production of unesterified [3H]arachidonate and labeled diglycerides is due to the involvement of more than one catabolic reaction, since the high energy nucleotides had similar effects on fatty acid accumulation, but different effects on phospholipid labeling.

Ischemia-induced development of cerebral edema in awake and anesthetized gerbils.

General anesthesia is often used to immobilize experimental animals prior to the induction of cerebral ischemia. However, anesthetics are known to alter many of the biochemical and physiological parameters used for the assessment of stroke-induced brain damage. We examined the effects of bilateral carotid artery ligations on mortality and the development of cerebral edema in unanesthetized gerbils. We found that increasing the length of the ischemic episode resulted in increased mortality, both during the ischemic period and during cerebral reperfusion. The duration of the ischemic episode was also correlated with the rate and degree of the development of cerebral edema. Both of these estimates of ischemia-induced brain damage were significantly reduced by the pretreatment of the animals with pentobarbital. Based on the variable effects of different anesthetics on CNS activities, and the observed effects of barbiturate anesthesia on ischemia-induced mortality and edema development in the present model, we suggest that it may be inappropriate to anesthetize experimental animals when investigating certain aspects of stroke-induced brain damage.

Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings.

Depolarization-evoked increases in intraterminal free Ca2+ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca2+ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca2+ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca2+ channel blockers. In addition, the K(+)-dependent effects on Ca2+ concentrations were attenuated in the presence of phospholipase A2 inhibitors, but were mimicked by the phospholipase A2 activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca2+ were reduced by voltage-sensitive Ca2+ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca2+. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A2 and the subsequent accumulation of unesterified arachidonic acid.

Arachidonic acid metabolism in gerbil cerebra: effects of ischemia and pentobarbital.

Pentobarbital pretreatment may be used to predict biochemical events involved in ischemic brain damage following bilateral carotid artery ligations in the gerbil, since it reduces the subsequent edema and mortality. The effects of this anesthetic on the ischemia-induced modifications of cerebral arachidonic acid metabolism were investigated, in order to correlate observed alterations with tissue damage. Cerebral lipids were radiolabeled in vivo with [3H]arachidonic acid prior to 10 min of cerebral ischemia and 0-120 min of perfusion. Ischemia stimulated a 97.3% increase in unesterified [3H]arachidonate, which was due to the loss of label from choline, inositol, and ethanolamine glycerophospholipids. Tissue reperfusion stimulated further reductions in [3H]choline and [3H]inositol glycerophospholipids, while ethanolamine glycerophospholipid and triglyceride labeling increased. Inositol glycerophospholipid, but not choline glycerophospholipid, labeling returned to control level by 60 min of reperfusion. Pentobarbital pretreatment reduced the accumulation of [3H]arachidonate by 56.2% during ischemia. It increased the recovery of [3H]ethanolamine glycerophospholipids during the ischemic period and [3H]choline glycerophospholipids during the first 5 min of reperfusion. These effects accounted for the reduction of unesterified [3H]arachidonate observed during ischemia and reperfusion.

Calcium-dependent phospholipid catabolism and arachidonic acid mobilization in cerebral minces.

Cerebral minces were used to investigate the role of calcium influx on trauma-induced alterations of brain lipid metabolism. Cerebral phospholipids, nonpolar lipids, and free fatty acids were radiolabeled in vivo with [3H]arachidonic acid. Tissue incubation stimulated the time-dependent catabolism of choline and inositol glycerophospholipids, and resulted in the accumulation of [3H]free fatty acids. These effects were attenuated in Ca2(+)-free incubations, and when EGTA or verapamil were present. The inhibition of calcium influx also reduced the labeling of diglycerides, whereas ethanolamine and serine glycerophospholipids were not affected by incubation or treatments. Replacing Ca2+ with other cations also attenuated the incubation-dependent alterations in lipid metabolism. However, only cadmium was able to compete with calcium and reduce the accumulation of [3H]free fatty acids. It appeared that about half of the observed phospholipid catabolism was dependent on Ca2+ influx and that at least 80% of the [3H]free fatty acid accumulation required calcium.

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.