Lithium-induced decrease of brain inositol and increase of brain inositol-1-phosphate is transient

The effects of a single does of LiCl (2.5 or 10 mEq/kg) on brain inositol and inositol-1-phosphate (Ins1P), intermediates of brain phosphoinositude (PI) turnover, were determinated in male Han: Wistar rats. There was a remarkable, 36–58 fold elevation of brain Li⁺ as the single does of LiCl was increased 4-fold. Moreover, the accumulation of brain lithium was slow during repeated administration of LiCl. Brain lithium did not correlate with changes in brain PI turnover either after a single or repeated doses. Thus, after a single does of LiCl the increases in brain Ins1P were much less than the decreases in brain inositol. Also, brain inositol was significantly decreased only with the high dose of LiCl whereas brain Ins1P accumulation was more prominent with the lower dose. Moreover, repeated daily doses of LiCl only transiently increased brain Ins1P at 1 and 7 d whereas inositol remained at control levels throughout the 14 d observation period. Lithium probably caused the transient decrease in brain inositol by inhibiting several enzymes, in addition to the inhibition of myo-inositol mono-phosphates, in the PI cycle. Moreover, a slow dampening down of PI turnover by lithium, possible via an inhibitory action on G-protein-coupling, may also explain the present findings.     

A gas chromatographic method for the determination of inositol monophosphates in rat brain

Regional levels of cerebral inositol-1-phosphate (Ins1P), an intermediate in phosphoinositide (PI) cycle, were readily detected with a new gas chromatographic (GC) method. GC analysis of trimethylsilyated Ins1P and myo-inositol-2-phosphate with a fused silica capillary SE-30 column and flame ionization detection was linear at picomolar range (pmol/l) with a sensitivity to a level of 2 pmol. Also, inositol monophosphates and glucose-6-phosphate are separated in unstimulated brain tissue. The mean recovery of the method is 98±5.2%. Ins 1P levels were higher in frontal than in caudal regions in control brains. Lithium treatment increased the levels of Ins1P throughout the brain but mostly in frontal brain regions and in the hippocampus. The present GC assay to measure the accumulation of Ins1P, an index for the activity of PI signalling, may be suitable for exploring regional differences in cerebral receptor-coupled PI signalling in vivo.     

In Vitro and In Vivo Inhibition of Inositol Monophosphatase by the Bisphosphonate L-690,330

Abstract: We have previously described the synthesis of bis-phosphonate-containing inhibitors of inositol monophosphatase. In the present study, a more detailed examination of the in vitro and in vivo properties of one of these compounds, L-690,330, is described. L-690,330 is a competitive inhibitor of inositol monophosphatase with a K ₁, depending on the source of IMPase, of between 0.2 and 2 μM. Although ∼1,000-fold more potent in vitro than lithium, in muscarinic m1 receptor-transfected Chinese hamster ovary cells prelabelled with [³H]inositol, L-690,330 only produced 40% of the accumulation of [³H]inositol monophosphates achieved by lithium at the same concentration (10 m M ), suggesting that the ability of L-690,330 to cross the cell membrane is limited. Nevertheless, under conditions of cholinergic stimulation (100 mg/kg of pilocarpine s.c.), high doses of L-690,330 were able to increase brain inositol(1)phosphate levels in vivo to three- to fourfold control levels. This effect was dose dependent (ED₅₀= 0.3 mmol/kg s.c.) and was maximal after 1 h. In peripheral tissues, the effects of L-690,330 on inositol(1)phosphate levels mimicked those of lithium both qualitatively and quantitatively. However, in the brain, the effects of L-690,330 were much less than seen with lithium, consistent with the blood-brain barrier restricting access of the polar L-690,330 into the CNS, thereby further limiting entry of compound into cells in the brain. In the future, it may be possible to develop prodrugs of this compound, which circumvent many of the cell permeability problems inherent in bisphosphonate compounds.     

Plasma and erythrocyte choline concentrations in rats following chronic treatment with lithium or choline

Rats were given daily injections of choline, lithium or lithium plus choline for either 11 or 18 days and red cell choline, glycine and glutathione levels were measured using proton nuclear magnetic resonance spectroscopy. In addition, plasma choline, plasma lithium and red cell lithium levels were measured 4 hr after the last dosage. Choline (1 mmol/kg) alone increased plasma but not red cell choline concentrations. Lithium (0.94 mmol/kg) elevated red cell choline levels but did not affect plasma choline concentrations. In contrast, red cell choline levels were not elevated in rats treated with a higher dose of lithium (1.88 mmol/kg). When choline was given in addition to the lower dose of lithium, a similar accumulation of red cell choline was observed suggesting that the lithium-induced choline accumulation was not enhanced by a greater availability of free choline. No differences were detected in red cell glycine or glutathione levels between any of the treatment groups. Therefore, lithium produced a specific (dose-dependent) accumulation of choline in rat erythrocytes. However, the 100% increase observed in rats was not as marked as the increased red cell choline levels reported in patients maintained on lithium (8 to 10-fold). This discrepancy supports the concept that species differences exist in red cell choline transport or metabolism.     

Time-course of malaoxon-induced alterations in brain regional inositol-1-phosphate levels in convulsing and nonconvulsing rats

The potential of a single dose of malaoxon (26.2 or 39.2 mg/kg i.p.) to produce convulsions and to increase cerebral levels of inositol-1-phosphate (Ins1P), an intermediate in phosphoinositide (PI) cycle, was followed for 1, 4, or 72 hr. The lower dose of malaoxon did not produce convulsions whereas the higher dose induced convulsions in 60% of the exposed rats. Malaoxon caused a dosedependent, at most 2-fold, increase in brain regional Ins1P levels in nonconvulsing rats as compared to controls. At the higher dose of malaoxon, in convulsing rats, the Ins1P-levels increased 4-fold above the control Ins1P-levels. In nonconvulsing rats, the Ins1P-levels reached their maximum 1–4 hr after the administration of malaoxon, whereas in convulsing rats the levels increased for 72 hr. The results suggest that PI-signalling is associated with convulsions produced by malaoxon.     

Carbachol- and KCl-induced changes in phosphoinositide metabolism and free calcium in guinea pig cerebral cortex synaptosomes

Phosphoinositide (PI) and calcium metabolism were studied in guinea pig cerebral cortex synaptosomes. Mass amounts of inositol and inositol monophosphates, and the levels of free intrasynaptosomal calcium ([Ca²⁺]_i) were measured after KCl (60 mM), after a direct cholinergic agonist carbachol (CA, 1mM), and after their combination. Inositol, inositol-1-phosphate (Ins1P), inositol-4-phosphate (Ins4P) and [Ca²⁺]_i were measured with and without 10 mM LiCl in the incubation medium. CA-induced cholinergic stimulation elevated synaptosomal Ins4P levels by 40% but did not affect Ins1P or [Ca²⁺]_i. On the contrary, KCl elevated Ins1P by 50% and [Ca²⁺]_i by 40% above the resting level, and decreased inositol by 20%, whereas no alterations in Ins4P occurred. CA did not modify the response of KCl, but KCl abolished the elevation of Ins4P by CA. LiCl attenuated KCl-induced elevation of Ins1P but amplified the CA-induced elevation of Ins4P. The elevation of presynaptic [Ca²⁺]_i was accompanied by accumulation of Ins1P but not that of Ins4P. Hence, the present results suggest that presynaptic cholinergic stimulation and KCl-induced depolarization may activate different degradation pathways of inositolphosphate metabolism.     

Mass assay for inositol 1-phosphate in rat brain by high-performance liquid chromatography and pulsed amperometric detection.

A high-performance liquid chromatographic method for direct mass measurement of inositol 1-phosphate (I(1)P) in rat brain is described. Separation of I(1)P from its isomers and from endogenous components is achieved by polymeric anion-exchange chromatography with a sodium hydroxide/sodium acetate mobile phase. Detection is performed at high pH by pulsed amperometric detection at a gold electrode. Sample preparation involves liquid-liquid extraction and ion-exchange solid-phase extraction, prior to HPLC. The method is sufficiently sensitive and selective to enable facile determination of basal levels of I(1)P in small amounts of brain tissue. The applicability of the method is demonstrated by the in vivo monitoring of I(1)P levels in rat brain after administration of the inositol monophosphatase inhibitor lithium and the cholinergic agonist pilocarpine. The method is a significant improvement over existing published mass assays for I(1)P by virtue of its simplicity, speed, sensitivity, and ruggedness.     

Lithium Effects on Inositol Phospholipids and Inositol Phosphates: Evaluation of an In Vivo Model for Assessing Polyphosphoinositide Turnover in Brain

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.     

Evidence that inositol 1-phosphate in brain of lithium-treated rats results mainly from phosphatidylinositol metabolism.

In cerebral cortex of rats treated with increasing doses of LiCl, the relative concentrations of Ins(1)P, Ins(4)P and Ins(5)P (when InsP is a myo-inositol phosphate) are approx. 10:1:0.2 at all doses. In rats treated with LiCl followed by increasing doses of pilocarpine a similar relationship occurs. myo-Inositol-1-phosphatase (InsP1ase) from bovine brain hydrolyses Ins(1)P, Ins(4)P and Ins(5)P at comparable rates, and these substrates have similar Km values. The hydrolysis of Ins(4)P is inhibited by Li+ to a greater degree than is hydrolysis of Ins(1)P and Ins(5)P. D-Ins(1,4,5)P3 and D-Ins(1,4)P2 are neither substrates nor inhibitors of InsP1ase. A dialysed high-speed supernatant of rat brain showed a greater rate of hydrolysis of Ins(1)P than of D-Ins(1,4)P2 and a lower sensitivity of the bisphosphate hydrolysis to LiCl, as compared with the monophosphate. That enzyme preparation produced Ins(4)P at a greater rate than Ins(1)P when D-Ins(1,4)P2 was the substrate. The amount of D-Ins(3)P [i.e. L-Ins(1)P, possibly from D-Ins(1,3,4)P3] is only 11% of that of D-Ins(1)P on stimulation with pilocarpine in the presence of Li+. DL-Ins(1,4)P2 was hydrolysed by InsP1ase to the extent of about 50%; both Ins(4)P and Ins(1)P are products, the former being produced more rapidly than the latter; apparently L-Ins(1,4)P2 is a substrate for InsP1ase. Li+, but not Ins(2)P, inhibited the hydrolysis of L-Ins(1,4)P2. The following were neither substrates nor inhibitors of InsP1ase; Ins(1,6)P2, Ins(1,2)P2, Ins(1,2,5,6)P4, Ins(1,2,4,5,6)P5, Ins(1,3,4,5,6)P5 and phytic acid. myo-Inositol 1,2-cyclic phosphate was neither substrate nor inhibitor of InsP1ase. We conclude that the 10-fold greater tissue contents of Ins(1)P relative to Ins(4)P in both stimulated and non-stimulated rat brain in vivo are the consequence of a much larger amount of PtdIns metabolism than polyphosphoinositide metabolism under these conditions.     

The role of the phosphatidylinositol cycle in mitosis in sea urchin zygotes. Lithium inhibition is overcome by myo-inositol but not by other cyclitols or sugars

We have investigated the role of the phosphatidylinositol (PI) cycle in cellular events between fertilization and first cleavage in zygotes of the sea urchin Lytechinus pictus. The effects of lithium were studied: The lithium-induced changes due to effects on the PI cycle were reversed by myo-inositol, the next step in the cycle after the lithium block, but were not reversed by scyllo-inositol or other cyclitols or sugars. In this way we implicated the PI cycle in the formation of streak birefringence, in nuclear membrane breakdown, in onset of anaphase, and in cytokinesis. With respect to karyokinesis, mitotic apparatus (MA) structure often was altered when the PI cycle was blocked, and anaphase was blocked when the PI cycle was blocked. For all stages, the effects of 400 mM lithium were overcome by 10-100 microM myo-inositol. Excess myo-inositol potentiated the effect of lithium on MA structure (and on cytokinesis), suggesting that there is a negative feedback loop in the control of the PI cycle.     

Changes in cerebral inositol-1-phosphate concentrations in LiCl-treated rats: Regional and strain differences

LiCl-induced (5 mEq/kg) regional differences in the cerebral phosphoinositide (PI) cycle were studied by measuring inositol-1-phosphate (Ins-1-P), an, intermediate in the PI cycle, in male Sprague Dawley and Han/Wistar rats by gas chromatography/mass spectrometry. Control Ins-1-P levels were higher frontally than caudally in both rat strains. LiCl increased Ins-1-P levels 1.8 to 7.4 fold in different, regions of brain of Sprague Dawley rats but only 1.2 to 1.8 fold in Han/Wistar rats. This strain difference offers a way to compare the effects of lithium on PI metabolism versus receptor-G protein-phospholipase C coupling mechanisms.     

The metabolism of inositol 1,3,4-trisphosphate to inositol 1,3-bisphosphate

We previously demonstrated a pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) to inositol 3,4-bisphosphate (Ins(3,4)P2) in calf brain extracts. Inositol polyphosphate 1-phosphatase, a Mg2+-dependent, lithium ion-inhibited enzyme, specifically hydrolyzes Ins(1,3,4)P3 to Ins(3,4)P2 and Ins(1,4)P2 to Ins 4-P (Inhorn, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). Now we have found an alternative pathway for the metabolism of Ins(1,3,4)P3 in crude calf brain extracts. Along this pathway, Ins(1,3,4)P3 is first converted to Ins(1,3)P2 which is further hydrolyzed to Ins 1-P. This pathway involves a 4-phosphatase and a 3-phosphatase which do not require Mg2+ and are not inhibited by lithium ions. A similar 4-phosphatase also degrades Ins(3,4)P2 to Ins 3-P. Three different inositol bisphosphates formed from calf brain supernatant are each further metabolized by a separate enzyme. The three inositol monophosphates, i.e. Ins 1-P, Ins 3-P, and Ins 4-P, are converted to inositol by inositol monophosphate phosphatase (Ackermann, K. E., Gish, B. G., Honchar, M. P., and Sherman, W. R. (1987) Biochem. J. 242, 517-524).     

Chronic Lithium Treatment Impairs Phosphatidylinositol Hydrolysis in Membranes from Rat Brain Regions

Membranes prepared from rat brain regions were used to measure the receptor-coupled and/or guanine nucleotide-binding protein (G protein)-mediated hydrolysis of exogenous [3H]phosphatidylinositol ([3H]PI). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and NaF (in the presence of AlCl3) caused concentration-dependent stimulations of [3H]PI hydrolysis, supporting the conclusion that G proteins mediating [3H]PI hydrolysis can be activated in this preparation. Neither of these responses was altered by in vitro incubation with 8 mM LiCl, but both were reduced in hippocampal, striatal, and cortical membranes from rats that had been treated with lithium for 4 weeks compared with controls. Two cholinergic agonists, carbachol and pilocarpine, induced no hydrolysis of [3H]PI unless GTP gamma S was also present, in which case each equally stimulated [3H]PI hydrolysis above that obtained with GTP gamma S alone. In the presence of GTP gamma S several excitatory amino acid agonists stimulated [3H]PI hydrolysis to an extent similar to that of carbachol. After chronic lithium treatment, [3H]PI hydrolysis stimulated by carbachol was significantly attenuated, but the response to quisqualate was unaffected. Therefore, lithium added in vitro does not have an effect on cholinergic receptor- or G protein-mediated [3H]PI hydrolysis, but each of these is reduced by chronic lithium treatment. Because exogenous [3H]PI was provided as the substrate, it is evident that the inhibitory effect of chronic lithium treatment cannot be due to substrate depletion. Impaired function of G proteins appears to be the most likely mechanism accounting for attenuated [3H]PI hydrolysis after chronic administration of lithium.     

Effects of lithium on the hypothalamo-pituitary-adrenal axis

The effect of lithium on the hypothalamo-pituitary-adrenal axis was studied in vivo and in vitro. The levels of plasma vasopressin, ACTH and corticosterone increased after the administration of lithium (LiCl 4 mmol/kg BW, 11 days) in rats, while the tissue vasopressin concentration in the median eminence, the rest of the hypothalamus and the posterior pituitary was decreased. The CRF concentration in the posterior pituitary increased markedly, but it did not change significantly in the median eminence or the rest of the hypothalamus. The elevated plasma ACTH level might be at least partly due to the increased vasopression secretion. Lithium stimulated ACTH secretion per se and also enhanced vasopressin-induced ACTH secretion in cultured pituitary cells and in half pituitary incubations, while it did not affect CRF-induced ACTH secretion. Lithium inhibited CRF-induced cAMP accumulation in half pituitary incubations, while lithium and vasopressin did not affect cAMP accumulation per se or even when administered together. The results suggest that lithium-induced ACTH release is via a cAMP-independent mechanism. Thus, it is possible that lithium stimulates ACTH release by acting directly on the corticotroph, stimulating vasopressin release and potentiating vasopressin-induced ACTH release.     

Transport of tryptophan and tyrosine in rat brain slices in the presence of lithium

Slices from rat cerebral cortex, brain stem, and cerebellum were incubated in media in which 1, 10, or 100 mmol/liter NaCl had been replaced by equimolar amounts of LiCl. The initial influx fo tryptophan and tyrosine into the slices diminished in the lithium-containing media. The lithium-induced inhibition was not competitive. The equilibrium accumulation of the amino acids was also less in the presence of LiCl. The incorporation of tryptophan and tyrosine into the proteins of the slices was inhibited by lithium. There were no clear differences between the brain areas studied. It has been suggested earlier that a lithium treatment enhances thesin vivo cerebral uptake of these aromatic amino acids. The present results show that such a possible increase in uptake is not a direct effect of lithium ions on cell membranes.     

Effect of lithium on norepinephrine metabolic pathways

We investigated lithium-induced changes in norepinephrine (NE) catabolism. NE and its major metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenyl glycol (DHPG), ions such as lithium (Li(+)), magnesium (Mg(2+)), and potassium (K(+)) were measured in rat plasma and cerebral cortex using an HPLC method with electrochemical detection for amines. The results obtained with a group of rats treated by lithium chloride (2 mmol/kg/IP) were compared with a control group receiving sodium chloride (2 mmol/kg/IP). Animals were killed at different times over a period of six hours in the morning following salt administration to minimize possible chronobiological effects. There are two pathways leading to MHPG formation: way A, without DHPG, and way B, with DHPG. In plasma and cerebral cortex of lithium treated rats, way A catabolism seems to be preferential. Lithium increases Mg(2+) and K(+) plasma levels. These results suggest that lithium may increase inactivation of NE and decrease NE available for adrenergic receptors.     

Reduced inositol polyphosphate accumulation and inositol supply induced by lithium in stimulated cerebral cortex slices.

The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.     

Purification, characterization and proteinase-inhibitory activity of a Clara-cell secretory protein from the pulmonary extracellular lining of rabbits.

Although activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells results in a linear accumulation of inositol phosphates for up to 60 min in the presence of LiCl [Masters, Quinn & Brown (1985) Mol. Pharmacol. 27, 325-332], activation of H1-histamine receptors resulted in an increase in total inositol phosphate formation that was maintained for less than 5 min. The effects of stimulation of these two receptors on accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were also examined. Incubation of 1321N1 cells with carbachol resulted in a rapid accumulation of all three inositol phosphates, reaching a maximum within 30 s; this elevated value was maintained for up to 60 min. The rate of disappearance of Ins(1,3,4)P3 from carbachol-treated cells after the addition of atropine paralleled or exceeded the rate of disappearance of Ins(1,4,5)P3. Although the initial rates of accumulation of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1,3,4,5)P4 in the presence of histamine were similar to that observed with carbachol, the amounts of these inositol phosphates had returned to control values within 5 min after the addition of histamine. The results indicate that, although the acute effects of muscarinic receptor and H1-histamine receptor stimulation on phosphoinositide hydrolysis are very similar, the histamine receptor is desensitized rapidly, whereas the muscarinic receptor is not. This effect on histamine-receptor function is apparently homologous, since preincubation of 1321N1 cells with histamine did not decrease the subsequent response to carbachol.     

Properties of inositol polyphosphate 1-phosphatase

We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.     

Effects of Chronic Lithium Treatment on Agonist-Enhanced Extracellular Concentrations of Cyclic AMP in the Dorsal Hippocampus of Freely Moving Rats

Abstract: Studies on brain slices and homogenates suggest that chronic lithium treatment affects the activity of adenylate cyclases in the brain. To investigate whether chronic lithium administration influences the cyclic AMP (cAMP) synthesis in vivo, we have used microdialysis to assess lithium-induced alterations in extracellular concentrations of cAMP in the dorsal hippocampus of freely moving rats. Local infusion of noradrenaline or forskolin through the microdialysis probes produced rapid increases in the extracellular concentrations of cAMP in the dorsal hippocampus. Lithium administration for 4 weeks (serum lithium concentration of 0.8 ± 0.11 mmol/L) did not affect the baseline levels of cAMP. However, in rats fed a lithium-supplemented diet, noradrenaline- and forskolin-induced enhancement of cAMP levels was decreased in the dorsal hippocampus. The rats were videotaped 18 min before and 27 min after initiating the introduction of noradrenaline and forskolin into the dorsal hippocampus. The infusion of agonists induced a moderate behavioral excitation. Rats treated with lithium were less active compared with the control rats. Taken together, these data confirm that chronic lithium administration affects the cAMP signaling system in the brain of living animals, presumably by interfering with a site beyond the receptor level.