Eric Grange

Lithium decreases turnover of arachidonate in several brain phospholipids

In vivo rates of incorporation and turnover of palmitate and arachidonate in brain phospholipids were measured in awake rats treated chronically with lithium, following intravenous infusion of radiolabeled palmitate and arachidonate, respectively. Chronic lithium, at a brain level considered to be therapeutic in humans, decreased turnover of arachidonate within brain phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine by up to 80% (P < 0.001). In contrast, lithium had a minimal effect on turnover of palmitate, causing only a 26% reduction in turnover in phosphatidylcholine (P < 0.01). These results suggest that a major therapeutic effect of lithium is to reduce turnover specifically of arachidonate, possibly by inhibiting phospholipase A2 involved in signal transduction. The effect may be secondary to the known action of lithium on the phosphoinositide cycle, by inhibiting the activity of inositol monophosphatase.

Dynamics of Docosahexaenoic Acid Metabolism in the Central Nervous System: Lack of Effect of Chronic Lithium Treatment

Using a method and model developed in our laboratory to quantitatively study brain phospholipid metabolism, in vivo rates of incorporation and turnover of docosahexaenoic acid in brain phospholipids were measured in awake rats. The results suggest that docosahexaenoate incorporation and turnover in brain phospholipids are more rapid than previously assumed and that this rapid turnover dilutes tracer specific activity in brain docoshexaenoyl-CoA pool due to release and recycling of unlabeled fatty acid from phospholipid metabolism. Fractional turnover rates for docosahexaenoate within phosphatidylinositol, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserine were 17.7, 3.1, 1.2, and 0.2 %.h−1, respectively. Chronic lithium treatment, at a brain level considered to be therapeutic in humans (0.6 μmol.g−1), had no effect on turnover of docosahexaenoic acid in individual brain phospholipids. Consistent with previous studies from our laboratory that chronic lithium decreased the turnover of arachidonic acid within brain phospholipids by up to 80% and attenuated brain phospholipase A2 activity, the lack of effect of lithium on docosahexaenoate recycling and turnover suggests that a target for lithiums action is an arachidonic acid-selective phospholipase A2.

Specific Activity of Brain Palmitoyl‐CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10‐3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl‐CoA, pools. [9,10‐3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline‐containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl‐CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60‐fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl‐CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.

In Vivo Imaging of Fatty Acid Incorporation into Brain to Examine Signal Transduction and Neuroplasticity Involving Phospholipids

An in vivo method is presented that allows quantification and imaging of fatty acid incorporation into different brain phospholipids in relation to membrane synthesis, neuroplasticity, and signal transduction. The method can be used with positron emission tomography, and may help to evaluate brain phospholipid metabolism in humans with brain tumors, neurodegenerative disease, cerebral ischemia or trauma, or neurotoxic effects of drugs or other agents.

Changes in Cerebral Acyl‐CoA Concentrations Following Ischemia‐Reperfusion in Awake Gerbils

Abstract: Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl‐CoAs via acyl‐CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia‐reperfusion on brain fatty acid and acyl‐CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia‐reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4‐fold in the ischemic‐reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl‐CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl‐ and arachidonoyl‐CoA and a marked decrease in palmitoyl‐ and docosahexaenoyl‐CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl‐CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.

Selective Acceleration of Arachidonic Acid Reincorporation into Brain Membrane Phospholipid Following Transient Ischemia in Awake Gerbil

Abstract: Awake gerbils were subjected to 5 min of forebrain ischemia by clamping the carotid arteries for 5 min and then allowing recirculation. Radiolabeled arachidonic or palmitic acid was infused intravenously for 5 min at the start of recirculation, after which the brains were prepared for quantitative autoradiography or chemical analysis. Dilution of specific activity of the acyl‐CoA pool was independently determined for these fatty acids in control gerbils and following 5 min of ischemia and 5 min of reperfusion. Using a quantitative method for measuring regional in vivo fatty acid incorporation into and turnover within brain phospholipids and determining unlabeled concentrations of acyl‐CoAs following recirculation, it was shown that reperfusion after 5 min of ischemia was accompanied by a threefold increase compared with the control in the rate of reincorporation of unlabeled arachidonate that had been released during ischemia, whereas reincorporation of released palmitate was not different from the control. Selective and accelerated reincorporation of arachidonate into brain phospholipids shortly after ischemia may ameliorate specific deleterious effects of arachidonate and its metabolites on brain membranes.

Manoalide, a Phospholipase A2 Inhibitor, Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A2. Awake rats received an i.v. injection of a phospholipase A2 inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-14C]arachidonate or [3H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A2 activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A2 in vivo, and that phospholipase A2 plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.

Effects ofEGb 761 on fatty acid reincorporation during reperfusion following ischemia in the brain of the awake gerbil

Transient cerebral ischemia (5 min) releases unesterified fatty acids from membrane phospholipids, increasing brain concentrations of fatty acids for up to 1 h following reperfusion. To understand the reported anti-ischemic effect of Ginkgo biloba extract (EGb 761), we monitored its effect on brain fatty acid reincorporation in a gerbil-stroke model. Both common carotid arteries in awake gerbils were occluded for 5 min, followed by 5 min of reperfusion. Animals were infused intravenously with labeled arachidonic (AA) or palmitic acid (Pam), and rates of incorporation of unlabeled fatty acid from the brian acyl-CoA pool were calculated by the model of Robinson et al. (1992), using quantitative autoradiography and biochemical analysis of brain acyl-CoA. Animals were treated for 14 d with 50 or 150 mg/kg/d EGb 761 or vehicle. Ischemia-reperfusion had no effect on the rate of unlabeled Pam incorporation into brain phospholipids from palmitoyl-CoA; this rate also was unaffected by EGb 761. In contrast, ischemia-reperfusion increased the rate of incorporation of unlabeled AA from brain arachidonoyl-CoA by a factor of 2.3-3.3 compared with the control rate; this factor was further augmented to 3.6-5.0 by pretreatment with EGb 761. There is selective reincorporation of AA compared with Pam into brain phospholipids following ischemia. EGb 761 further accelerates AA reincorporation, potentially reducing neurotoxic effects of prolonged exposure of brain to high concentrations of AA and its metabolites.

Arecoline stimulation of radiolabeled arachidonate incorporation from plasma into brain microvessels of awake rat

The cholinergic agonist, arecoline, was used to examine the effects of cholinergic stimulation upon incorporation of radiolabeled arachidonic acid from blood into cerebral microvessels of awake rats. Animals received a single I.P. injection of arecoline (1 mg/kg) followed 3 to 5 minutes later by a 5 minute intravenous infusion of [1-14C]arachidonic acid (AA) (170 μCi/kg) via the femoral vein. Timed arterial blood samples were collected over 20 minutes following the start of infusion, after which the animal was killed, and the brain was removed. The incorporation coefficient k* for [1-14C] AA was approximately 2-fold higher in microvessels isolated from arecoline-injected than from sham-injected animals. The data demonstrate in an in vivo paradigm, that activation of cholinergic pathways within the rat CNS stimulates arachidonic acid turnover in cerebral microvessels. This suggests a direct involvement of this fatty acid in second messenger function within microvessel endothelial cells and possibly attached pericytes.