Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum

Abstract: The effects of lithium on muscarinic cholinoceptor-stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [³H]inositol or [³H]cytidine prelabelling and inositol 1,4,5-trisphosphate [lns(1,4,5)P₃] and inositol 1,3,4,5-tetrakisphosphate [lns(1,3,4,5)P₄] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P₃ and lns(1,3,4,5)P₄ mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30-min incubation period. Carbachol-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations were inhibited by lithium in all brain regions studied in a time-and concentration-dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC₅₀ values for inhibition of agonist-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [³H]CMP-phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo-inositol to reverse these effects, as well as lithium-suppressed lns(1,4,5)P₃ accumulation, implicates myo-inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [³H]inositol monophosphate and [³H]CMP-phosphatidate levels and inhibitions of lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations. However, the inability of striatal slices to sustain carbachol-stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo-inositol may indicate differences in phosphoinositide signalling in this brain region.     

Chronically Administered Lithium Alters Neither myo-Inositol Monophosphatase Activity nor Phosphoinositide Levels in Rat Brain

Rats were exposed to either 29 consecutive days of LiCl injections or 27 and 39 days of dietary Li2CO3, followed by injected LiCl at the end of the diet to insure a constant level of exposure to the drug. At the end of the period of chronic exposure to lithium, the rats were sacrificed and brain myo-inositol-1-phosphate phosphohydrolase (myo-inositol monophosphatase) activity was measured. In none of the experiments was there any difference in the lithium-sensitive activity toward myo-inositol monophosphatase when comparing the control and chronic groups. These brains and those from another group of rats that had been given Li2CO3 in their diet for 41 days, followed by 7 additional days of LiCl injections, were also examined for changes in the levels of the phosphoinositides. No reproducible differences in the absolute tissue levels of those lipids were found when control and chronic lithium groups were compared. These results are contrary to published reports which suggest that myo-inositol monophosphatase activity increases and that the phosphatidylinositol level decreases in rat brain as a result of chronic administration of lithium.     

In Vivo Determination of 5-Hydroxytryptamine Receptor-Stimulated Phosphoinositide Turnover in Rat Brain

Phosphoinositide turnover stimulated by 5-hydroxytryptamine (5-HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H-inositol phosphates, [3H]inositol-1-monophosphate [( 3H]IP1), [3H]inositol-1,4-bisphosphate [( 3H]IP2), and a mixture of [3H]inositol-1,4,5-trisphosphate and [3H]inositol-1,3,4-trisphosphate [( 3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of [3H]IP1 and [3H]IP2. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and quipazine, 5-HT agonists, significantly increased the amount of 3H-inositol phosphates under lithium pretreatment. The response to 5-MeODMT was inhibited by ritanserin, a 5-HT2 antagonist, but not by (-)-propranolol, a 5-HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5-HT2 receptor activation. It is considered that this method will be useful for measurement of 5-HT2 receptor-stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.     

Effect of Electrical Stimulation on Phosphoinositide Metabolism in Rat Sciatic Nerve In Vivo

The metabolism of phosphoinositides in rat sciatic nerves in vivo during electrical stimulation was studied. Nerves were prelabeled by injection of [2-3H]-myo-inositol alone for periods of 2 and 20 h or together with [32P]orthophosphate for 2 h and then electrically stimulated (100 Hz) for 5 or 20 min. Contralateral unstimulated nerve served as the control. When tritiated myo-inositol was used alone for prelabeling the nerves, approximately 6% and 14% of the label was incorporated into lipids after 2 h and 20 h, respectively. Both 5 and 20 min of electrical stimulation caused an insignificant change in the percentage of radioactivity recovered in lipids from the nerves prelabeled with either myo-inositol or with a mixture of myo-inositol and phosphate. The proportion of label associated with phosphoinositides of nerves prelabeled with myo-inositol for both 2 h and 20 h showed an increase in phosphatidyl-inositol-4-phosphate at the expense of phosphatidylinositol in stimulated nerves. Similar results were obtained with nerves prelabeled for 2 h with a mixture of [32P]orthophosphate and [2-3H]myo-inositol. No significant changes in the radioactivity associated with water-soluble inositol phosphates were found in stimulated versus control nerves.     

Enhanced Coupling of Neonatal Muscarinic Receptors in Rat Brain to Phosphoinositide Turnover

The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo-[2-3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day-18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1-10 mM) was at the adult level (i.e., three-fold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose-response curve was shifted to the left, giving a half-maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine-2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of "spare" muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.     

Effects of Oxygen Depletion on Norepinephrine- and Carbachol-Stimulated Phosphoinositide Turnover in Rat Brain Slices

We examined the effects of in vitro anoxia and in vivo hypoxia (8% O2/92% N2) on norepinephrine (NE)- and carbachol-stimulated phosphoinositide (PI) turnover in rat brain slices. The formation of 3H-labeled polyPI in cortical slices was impaired by in vitro anoxia and fully restored by reoxygenation. Accumulation of 3H-labeled myo-inositol phosphates (3H-IPs) stimulated by 10(-5) M NE was significantly reduced by anoxia (control at 60 min, 1,217 +/- 86 cpm/mg of protein; anoxia for 60 min, 651 +/- 82 cpm/mg; mean +/- SEM; n = 5; p less than 0.01), and reoxygenation following anoxia resulted in overshooting of the accumulation (control at 120 min, 1,302 +/- 70 cpm/mg; anoxia for 50 min plus oxygenation for 70 min, 1,790 +/- 126 cpm/mg; n = 5; p less than 0.01). The underlying mechanisms for the two phenomena--the decrease caused by anoxia and the overshooting caused by reoxygenation following anoxia--seemed to be completely different because of the following observations. (a) Although the suppression of NE-stimulated accumulation at low O2 tensions was also observed in Ca2+-free medium, the overshooting in response to reoxygenation was not. (b) Carbachol-stimulated accumulation was significantly reduced by anoxia and was restored by reoxygenation only to control levels. Thus, the postanoxic overshooting in accumulation of 3H-IPs seems to be a specific response to NE. (c) The decrease observed at low O2 tensions was due to a decrease in Emax value, whereas the postanoxic overshooting was due to a decrease in EC50 value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that Phosphoinositide Metabolism in Rat Cerebral Cortex Stimulated by Pilocarpine, Physostigmine, and Pargyline In Vivo Is Not Changed by Chronic Lithium Treatment

The effect of chronic versus acute administration of lithium on receptor-linked phosphoinositide metabolism was assessed by comparing the change in the cerebral cortex levels of myo-inositol 1-phosphate in response to pilocarpine, physostigmine, or pargyline in rats. Rats were exposed to either 29 consecutive days of LiCl injections or 27 and 39 days of dietary Li2CO3, followed by injected LiCl at the end of the diet to insure a constant level of exposure to the drug. In each experiment, an acute group received a single injection of LiCl 20-24 h before they were killed. One hour before being killed, some of the animals acutely exposed to lithium and some of the animals chronically exposed to lithium each received pilocarpine, physostigmine, or pargyline. At the conclusion of the experiment, the rats were killed and brain levels of myo-inositol 1-phosphate and lithium were determined. A differential production of myo-inositol 1-phosphate in groups receiving acute versus chronic lithium would provide evidence of a change in receptor-linked phosphoinositide metabolism due to the chronic administration of lithium. Brain levels of myo-inositol 1-phosphate are dependent on tissue lithium concentrations; consequently, significant differences observed in brain lithium levels between the groups receiving acute versus chronic lithium prevented a meaningful assessment of the effect of the mode of lithium administration on the production of myo-inositol 1-phosphate in those groups. Stepwise multiple regression analysis and the measured brain lithium levels were used to assess the response of myo-inositol 1-phosphate levels to stimulation in animals receiving acute or chronic lithium treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Uptake of Lithium Isotopes by Rat Cerebral Cortex and Its Effect on Inositol Phosphate Metabolism

Twenty hours following the subcutaneous administration of 5 mEq/kg doses of 6LiCl and 7LiCl to two groups of rats, the cerebral cortex molar ratio of 6Li+/7Li+ is 1.5. The effects of the lithium isotopes on cortex myo-inositol and myo-inositol-l-phosphate levels are the same as we have reported earlier: a Li+ concentration-dependent lowering of myo-inositol and increase in myo-inositol-1-phosphate. Thus 6LiCl, when administered at the same dose as 7LiCl, produces the larger effect on inositol metabolism. When the 6LiCl and 7LiCl doses were adjusted to 5 mEq/kg and 7 mEq/kg, respectively, the cortical lithium myo-inositol and myo-inositol-1-phosphate levels of each group of animals became approximately equal, suggesting that the isotope effect occurs at the level of tissue uptake, but not on inositol phosphate metabolism. The inhibition of myo-inositol-1-phosphatase by the two lithium isotopes in vitro showed no differential effect. The isotope effect on cerebral cortex uptake of lithium is in the same direction as that reported by others for erythrocytes and for the CSF/plasma ratio, but of larger magnitude.     

Interaction of σ-Compounds with Receptor-Stimulated Phosphoinositide Metabolism in the Rat Brain

Sigma-receptors are nonopioid, nondopaminergic receptors that bind with high affinity several antipsychotic drugs and appear to be involved in regulation of posture and movement. The second messenger system coupled to these receptors is still unknown. Recently, an inhibitory effect of various sigma-compounds on carbachol-stimulated phosphoinositide metabolism has been reported. We have investigated the effect of six sigma-compounds on carbachol- and norepinephrine-stimulated 3H-inositol phosphate accumulation in rat cerebral cortex slices. All compounds tested had a dose-dependent inhibitory effect on both systems, although their order of potency differed between neurotransmitters. Pentazocine and 1,3-di-o-tolylguanidine were the most potent inhibitors of carbachol-stimulated phosphoinositide turnover (IC50 = 31.5 and 45.7 microM, respectively), while haloperidol showed the greatest potency on the norepinephrine-coupled system (IC50 = 3.5 microM). In the presence of IC50 concentrations of these inhibitors, the dose-response curves for the agonists were shifted to the right and the EC50 values were significantly increased. Sigma-compounds also inhibited the binding of [3H]quinuclidinyl benzilate to muscarinic receptors and of [3H]prazosin to alpha 1-adrenoceptors in cortical membranes. In the presence of IC50 concentration (11 microM) of 1,3-di-o-tolylguanidine, no differences were found in the maximal number of muscarinic binding sites, whereas the dissociation constant increased approximately fivefold, indicating a decrease of the radioligand's affinity for the receptor. These results indicate that sigma-compounds, at micromolar concentrations, inhibit muscarinic and alpha 1-adrenergic receptor-coupled phosphoinositide metabolism, probably through an interaction with the neurotransmitter recognition sites.     

Measurement of Receptor-Activated Phosphoinositide Turnover in Rat Brain: Nonequivalence of Inositol Phosphate and CDP-Diacylglycerol Formation

Abstract: Two methods for the measurement of receptor-activated phosphoinositide turnover were evaluated for their degree of correspondence in slices of rat brain; they involved the Li⁺-dependent accumulations of either [³H]-inositol-labeled inositol phosphates or [³H]cytidine-labeled CDP-diacylglycerol. In contrast to the expectation that the ratio of these two responses would remain approximately constant, varying degrees of correspondence were obtained. The two extremes are exemplified by carbachol, which elicited large increases in both inositol phosphate and CDP-diacylglycerol labeling, and endothelin, which gave a robust inositol phosphate response with little or no accumulation of ³H-CDP-diacylglycerol. No instance of the presence of the latter response in the absence of ³H-inositol phosphate accumulation was observed. Measurement of ³H-CDP-diacylglycerol accumulation thus may add additional insight into the regulation of phosphoinositide turnover and the complex actions of Li⁺.     

Regulation of Endogenous ADP-Ribosylation by Acute and Chronic Lithium in Rat Brain

Abstract: In the present study, we investigated the effects of lithium on endogenous ADP-ribosylation in rat brain. It was found that addition of lithium in vitro inhibits endogenous ADP-ribosylation activity in extracts of frontal cortex at therapeutically relevant concentrations. Inhibition is observed at concentrations as low as 0.3 m M and is maximal at 1 m M when 50% inhibition is obtained. A similar degree of inhibition of endogenous ADP-ribosylation was observed for all substrate proteins identified, including G_sα, suggesting that lithium's effect may be achieved at the level of ADP-ribosyltransferases and not specific substrate proteins. In contrast to lithium, chloride salts of sodium and potassium do not alter endogenous ADP-ribosylation activity in frontal cortex. To assess the possible in vivo relevance of this in vitro action of lithium, we studied the effect of chronic lithium administration on levels of endogenous ADP-ribosylation in frontal cortex. It was found that chronic lithium treatment, in contrast to the inhibitory effect of the drug in vitro, produced a >35% increase in endogenous ADP-ribosylation activity. A similar degree of increase was observed for all of the substrate proteins identified. These novel findings raise the possibility that certain endogenous ADP-ribosyltransferases are among the acute targets of lithium in the brain and that adaptations in these enzymes may be part of the mechanisms underlying lithium's long-term effects on brain function.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

Potassium Ion Facilitation of Phosphoinositide Turnover Activation by Muscarinic Receptor Agonists in Rat Brain

In rat hippocampal slices kept in Krebs-Henseleit medium, an increase of K+ ions to 12 mM potentiates the stimulation of phosphoinositide turnover elicited by carbachol and (+/-)-cis-methyldioxolane. Oxotremorine is inactive if tested in Krebs-Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+-deprived Krebs-Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.     

The Phosphoinositide Signal Transduction System Is Impaired in Bipolar Affective Disorder Brain

Abstract: The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [³H]phosphatidylinositol ([³H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca²⁺ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration-dependently activated by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and by NaF. GTPγS-stimulated [³H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS-stimulated [³H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans-1-aminocyclopentyl-1,3-dicarboxylic acid, serotonin, and ATP each activated [³H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein-stimulated [³H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.     

Effectiveness of Zinc in Modulating Lithium Induced Biochemical and Behavioral Changes in Rat Brain

1. The purpose of the present study was to determine the effect of zinc on the status of various neurotransmitters as well as behavioral patterns of lithium-treated rats. The study was designed with a view to find out whether zinc affords protection to brain against lithium toxicity. 2. Animals were segregated into four different groups. Animals in group I were fed with standard laboratory feed and water ad libitum and served as normal controls. Animals in group II and IV were given lithium in the form of lithium carbonate in their diet at a dose level of 1.1 g/Kg diet. Animals in group III and IV were given zinc treatment in the form of zinc sulfate at a dose level of 227 mg/L mixed in drinking water of animals. 3. The effects of all the treatments were studied for a duration of 1, 2, and 4 months with regard to the parameters, which included estimation of serotonin and dopamine concentrations as well as the activity of acetylcholinesterase in cerebral cortex of rat brain. Further, passive avoidance, active avoidance, and behavior despair tests were conducted to assess the short-term memory, cognitive behavior, and psychomotor dysfunction of the animals, respectively. 4. Initially, a decrease in the acetylcholinesterase activity was reported in cerebral cortex followed by an increase in the enzyme activity after 2 and 4 months of lithium treatment. Serotonin concentration significantly decreased after 2 and 4 months of lithium treatment, whereas dopamine concentration increased significantly after 4 months of lithium treatment. Zinc administration to the lithium-treated group significantly improved the acetylcholinesterase activity as well as the concentration of dopamine and serotonin. Further, lithium-treated rats showed an increase in depression time as compared to normal controls both after 1 and 4 months of treatment. Short-term memory significantly improved in lithium-treated rats in all treatment groups. However, no change in the cognitive behavior of the animals was reported after lithium treatment. Zinc co-administration with lithium significantly improved the short-term memory and cognitive functions of the animals. From the above results it can be concluded that zinc proved beneficial in altering the status of neurotransmitters as well as the behavior patters of the animals treated with both short and long-term lithium therapy.     

Asymmetry of Diacylglycerol Metabolism in Rat Cerebral Hemispheres

Diacylglycerols (DGs) were found to be asymmetrically distributed between the two cerebral hemispheres of rat brain. The left cerebral hemisphere (LCH) contained 100% more DG than the right cerebral hemisphere (RCH). The lateralization was enhanced in animals subjected to depolarization induced by a single electroconvulsive shock (ECS). During the acute phase of the convulsion, the DG pool increased in both hemispheres, with the LCH attaining a concentration 180% higher than the RCH. Stearate and arachidonate were the principal DG-acyl groups accumulated in the RCH, whereas in the LCH stearate and palmitate were mainly involved. After the last of a series of five shocks (one per day) the lateralization of the "DG response" was less accentuated during the acute phase of the ECS. Whereas DG release was drastically reduced in the LCH, in the RCH it was minimally affected. The DG sidedness after five shocks was nevertheless maintained at the level of arachidonate-containing DGs, which showed a higher accumulation in the LCH than in the RCH. The kinetics of DG removal showed a rapid phase during the first minute following a single or five ECSs. Total DG levels returned to basal values in the RCH, whereas in the LCH they remained slightly increased with respect to the initial levels 1 min after the convulsive episode. Minimal changes occurred in the subsequent 4 min. Chronic ECS altered the endogenous DG content and composition. Thus, 24 h after the last of four ECSs, total levels of DGs diminished by 40% in the RCH, whereas they remained unchanged in the LCH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoinositide Breakdown in Rat Hippocampal Slices: Sensitivity to Glutamate Induced by In Vitro Anoxia

We examined the effects of in vitro anoxia on phosphoinositide (PI) breakdown in rat hippocampal slices stimulated by glutamate and quisqualate. In addition to assays of accumulations of 3H-inositol phosphates (3H-IPs) degraded from prelabeled PI, we adopted direct assay procedures of inositol 1,4,5-triphosphate (1,4,5-IP3) using 1,4,5-IP3-specific binding protein to determine the formation of 1,4,5-IP3. The first effect, observed with anoxic incubation by itself, was the diminished quisqualate (10(-5) M)-stimulated accumulation of 3H-IPs degraded from prelabeled PI under prolonged anoxia. Quisqualate caused a transient increase in 1,4,5-IP3 formation in the early phase of anoxia, similar to that under oxygenated conditions. Glutamate (10(-5) M), under normal conditions, influenced neither the accumulation of 3H-IPs nor the formation of 1,4,5-IP3. Also, the accumulation of 3H-IPs under prolonged anoxia was unaffected. The same concentration of glutamate, however, gave rise to a transient increase in 1,4,5-IP3 content in the early phase of anoxia, similar to that caused by quisqualate. The second effect, observed by oxygenation following anoxia, was the induction of glutamate-stimulated accumulation of 3H-IPs. When the hippocampal slices were oxygenated following a sufficiently long (greater than 30-min) exposure to anoxia, glutamate (10(-5) M) caused a significant increase in accumulation of 3H-IPs degraded from prelabeled PI. Quisqualate-stimulated accumulation of 3H-IPs under oxygenated incubations was also increased by prior exposure of slices to anoxia. These results support the hypothesis that an exposure of hippocampal slices to anoxia induces a sensitivity of the PI breakdown pathway to glutamate and that, given an oxygen supply following sufficiently long exposure to anoxia, the slices maintain their sensitivity to glutamate with an apparent increase in the accumulation of 3H-IPs.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Evidence that Lithium Alters Phosphoinositide Metabolism: Chronic Administration Elevates Primarily d-myo-Inositol-1-Phosphate in Cerebral Cortex of the Rat

The administration of LiCl (3.6 mequiv./kg/day) to adult male rats for 9 days results in an increase in the cerebral cortex level of myo-inositol-1-phosphate (M1P) to 4.43 +/- 0.52 mmol/kg (dry weight) compared with a control level of 0.24 +/- 0.02 mmol/kg. This establishes that the previously observed acute effect of lithium on M1P (Allison et al., 1976) is both prolonged and augmented by repeated doses of lithium. Larger doses of LiCl over a 3-5 day period result in even larger increases in M1P and a 35% decrease in myo-inositol. In each case, 90% of the increase is due to the D-enantiomer, evidence that lithium is largely producing this effect via phospholipase C-mediated phosphoinositide metabolism. Data are presented showing that lithium is an uncompetitive inhibitor of the hydrolysis of both D- and L-M1P by M1P'ase.     

High-affinity Neurotensin Receptor is Involved in Phosphoinositide Turnover Increase by Inhibition of Sodium Pump in Neonatal Rat Brain

Phosphoinositide (PI) metabolism is enhanced in neonatal brain by activation of neurotransmitter receptors and by inhibition of the sodium pump with ouabain or endogenous inhibitor termed endobain E. Peptide neurotensin inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity, an effect blocked by SR 48692, a selective antagonist for high-affinity neurotensin receptor (NTS1). The purpose of this study was to evaluate potential participation of NTS1 receptor on PI hydrolysis enhancement by sodium pump inhibition. Cerebral cortex miniprisms from neonatal Wistar rats were preloaded with [³H]myoinositol in buffer during 60 min and further preincubated for 0 min or 30 min in the absence or presence of SR 48692. Then, ouabain or endobain E were added and incubation proceeded during 20 or 60 min. Reaction was stopped with chloroform/methanol and [³H]inositol-phosphates (IPs) accumulation was quantified in the water phase. After 60-min incubation with ouabain, IPs accumulation values reached roughly 500% or 860% in comparison with basal values (100%), if the preincubation was omitted or lasted 30 min, respectively. Values were reduced 50% in the presence of SR 48692. In 20-min incubation experiments, IPs accumulation by ouabain versus basal was 300% or 410% if preincubation was 0 min or 30 min, respectively, an effect blocked 23% or 32% with SR 48692. PI hydrolysis enhancement by endobain E was similarly blocked by SR 48692, being this effect higher when sample incubation with the endogenous inhibitor lasted 60 min versus 20 min. Present results indicate that PI hydrolysis increase by sodium pump inhibition with ouabain or endobain E is partially diminished by SR 48692. It is therefore suggested that NTS1 receptor may be involved in cell signaling system mediated by PI turnover.