Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

The isolation and characterization of inositol polyphosphate 4-phosphatase

We previously identified an alternative pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) in calf brain. The enzyme responsible for the degradation of Ins(1,3,4)P3 was designated as inositol polyphosphate 4-phosphatase (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 9644-9647). We have now purified this enzyme 3390-fold from calf brain-soluble fraction. The isolated enzyme has an apparent molecular mass of 110 kDa as determined by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme migrates as a protein of 105 kDa, suggesting that it is monomeric. Among various 4-phosphate-containing inositol polyphosphates, the enzyme hydrolyzes only Ins(1,3,4)P3 and inositol 3,4-bisphosphate (Ins(3,4)P2), yielding inositol 1,3-bisphosphate and inositol 3-phosphate as products. The inositol polyphosphate 4-phosphatase has apparent Km values of 40 and 25 microM for Ins(1,3,4)P3 and Ins(3,4)P2, respectively. The maximum velocities for these two substrates are 15-20 mumol of product/min/mg protein. Ins(1,3,4)P3 is a competitive inhibitor of Ins(3,4)P2 hydrolysis with an apparent Ki of 27 microM implying that the same active site is involved in hydrolysis of both substrates. The final enzyme preparation retained a small inositol polyphosphate 3-phosphatase activity (less than 2% of rate of inositol polyphosphate 4-phosphatase activity) which most likely reflects a contaminant. The enzyme displays maximum activity between pH 6.5 and 7.5. It is not inhibited by Li+, Ca2+, or Mg2+ except at 10 mM divalent ions. Mn2+ inhibits enzyme at high concentrations IC50 = 1.5 mM.     

Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes

[3H]Inositol hexakisphosphate (InsP6) binds with a heterogeneous distribution to frozen sections of unfixed rat brain and is displaced by unlabelled InsP6. The pattern of binding correlates with binding to neuronal cell bodies. [3H]InsP6 binding to cerebellar membranes has been further characterised, is reversible, and saturable, and exhibits high specificity for inositol polyphosphates. The IC50 for competition by unlabelled InsP6 is approximately 100nM, whereas inositol 1,3,4,5,6 pentakisphosphate (Ins(13456)P5), inositol 1,3,4,5 tetrakisphosphate (Ins(1345)P4), and inositol 1,4,5 trisphosphate (Ins(145)P3) bind with an affinity at least one order of magnitude lower. [3H]InsP6 binding is clearly distinct from previously characterised Ins(145)P3 (ref. 1, 2) and Ins(1345)P4 (ref. 3) binding, both in terms of pharmacology and brain distribution.     

Measuring Brain Uptake and Incorporation into Brain Phosphatidylinositol of Plasma myo-[2H6]Inositol in Unanesthetized Rats: An Approach to Estimate In vivo Brain Phosphatidylinositol Turnover

The in vivo rate of turnover of phosphatidylinositol (PtdIns) in brain is not known. In brain, certain receptor-mediated signal transduction involves metabolism of PtdIns and a method to measure its turnover in awake animals is useful in studying the effect of lithium and other therapeutic agents. In a method described here, rats were infused subcutaneously with myo-[²H₆]inositol (Ins*) using an osmotic pump and, at 1 and 8 weeks, concentrations of free myo-inositol (Ins) and Ins* in plasma and brain were measured by GC-MS (chemical ionization). Also, PtdIns and PtdIns* together in brain were isolated, and Ins and Ins* from their headgroups were released enzymatically and specific activity of incorporated inositol was measured. The specific activity of inositol reached a steady state in plasma within 1 week of infusion, but not in brain even at 8 weeks. However, in brain, the specific activity of phosphatidylinositol was same as that of inositol at both time-points, suggestive of fast turnover of PtdIns. The animal experiment and the analytical methodology described here should be useful for measuring the rate of turnover of brain PtdIns in pathological and drug treatment conditions.     

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.     

Regulation of gene expression by lithium and depletion of inositol in slices of adult rat cortex

Lithium inhibits inositol monophosphatase at therapeutically effective concentrations, and it has been hypothesized that depletion of brain inositol levels is an important chemical alteration for lithium's therapeutic efficacy in bipolar disorder. We have employed adult rat cortical slices as a model to investigate the gene regulatory consequences of inositol depletion effected by lithium using cytidine diphosphoryl-diacylglycerol as a functionally relevant biochemical marker to define treatment conditions. Genes coding for the neuropeptide hormone pituitary adenylate cyclase activating polypeptide (PACAP) and the enzyme that processes PACAP's precursor to the mature form, peptidylglycine alpha-amidating monooxygenase, were upregulated by inositol depletion. Previous work has shown that PACAP can increase tyrosine hydroxylase (TH) activity and dopamine release, and we found that the gene for GTP cyclohydrolase, which effectively regulates TH through synthesis of tetrahydrobiopterin, was also upregulated by inositol depletion. We propose that modulation of brain PACAP signaling might represent a new opportunity in the treatment of bipolar disorder.     

Pharmacologic characterization of cirazoline-activated inositol phospholipid hydrolysis in rat brain cortical slices

Interaction of cirazoline, an imidazoline derivative, with alpha 1-adrenoceptor coupled inositol phospholipid hydrolysis was characterized in rat brain cortical slices. Norepinephrine, a full alpha 1-agonist, and phenylephrine, a partial alpha 1-agonist, on inositol phospholipid hydrolysis were included for comparison. Norepinephrine produced a fourfold stimulation of inositol phospholipid hydrolysis, whereas cirazoline and phenylephrine caused only submaximal responses (40-60%) when compared with norepinephrine. The stimulation of inositol phospholipid hydrolysis by cirazoline was completely blocked by the alpha 1-adrenoceptor antagonist prazosin, but not by selective alpha 2- or beta-adrenoceptor antagonists. Furthermore, the norepinephrine dose-response curve was shifted to the right in the presence of cirazoline, without affecting the maximal response. These results suggest that cirazoline behaves as a partial agonist at brain alpha 1-adrenoceptors linked to inositol phospholipid hydrolysis.     

THE SPECIFICITY AND SULFHYDRYL SENSITIVITY OF THE INOSITOL TRANSPORT SYSTEM OF THE CENTRAL NERVOUS SYSTEM

Abstract— The transport of two cyclohexitol stereoisomers, myo-inositol (inositol) and scyllo-inositol (scyllitol), from blood into the CNS in vivo and into the choroid plexus in vitro was studied. In vitro , the uptake of [³H]scyllitol or [³H]inositol by choroid plexuses, isolated from rabbits and incubated in artificial CSF, was measured. Both scyllitol and inositol inhibited [³H]scyllitol or [³H]inositol accumulation by the choroid plexus. Inositol competitively inhibited [³H]scyllitol accumulation and both isomers had a comparable affinity (K_t= 0.1 m m ) for the single cyclohexitol accumulation system. The other 6 stereoisomers tested had an order of magnitude less affinity for the cyclohexitol accumulation system of choroid plexus. Thiol reagents that penetrate cells inhibited inositol accumulation by choroid plexus more effectively than nonpenetrating thiol reagents. In vivo , in unanesthetized rabbits. the transport of unmetabolized [³H]inositol from blood into CSF, choroid plexus and brain was readily saturated by increasing the plasma levels of myo-inositol but not by the stereoisomer d -chiroinositol. Similarly, the transport of unmetabolized [³H]scyllitol into CSF, brain and choroid plexus was readily saturated by increasing the plasma levels of myo-inositol. Beside documenting the stereospecificity and thiol reagent sensitivity of the inositol transport mechanism of the choroid plexus, these results provide further evidence that the choroid plexus is a locus for cyclohexitol transport between blood and CSF. Moreover, they show that scyllitol, which, like inositol, is present at a higher concentration in brain than plasma, can be transported from blood into CSF and brain by the same system that transports inositol.     

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.     

Effects of Cerebral Ischemia on [3H]Inositol Lipids and [3H]Inositol Phosphates of Gerbil Brain and Subcellular Fractions

Abstract: Intracerebral injection of [³H]inositoi into gerbil brain resulted in labeling of phosphoinositides and inositolphosphates in various subcellular membrane fractions. Phosphatidylinositol (PI) comprised >90% of the radioactivity of inositol lipids. However, the level of labeled poly-PI (with respect to PI) was higher in synaptosomes than in other membrane fractions. Ischemia induced in gerbils by ligation of the common carotid arteries resulted in a 30% decrease in labeled poly-PI in brain homogenates and this decrease was largely attributed to the poly-PI in synaptosomes (50% decrease). Among the inositol phosphates, the ischemia induction resulted in a decrease in labeling of inositol trisphosphate (63%) and inositol bisphosphate (38%), but labeling of inositol phosphate (IP) was increased by 59%. The results suggested a rapid turnover of the inositol phosphates in the gerbil brain. In general, changes in inositol lipids and inositol phosphates due to ischemia were attenuated after pretreatment with lithium (3 meq/kg) injected intraperitoneally 5 h prior to ligation. Surprisingly, lithium treatment alone did not cause an increase in IP labeling in the gerbil brain.     

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.     

Muscarinic Receptors and Hydrolysis of Inositol Phospholipids in Rat Cerebral Cortex and Parotid Gland

Abstract: Exposure of rat brain or parotid gland slices to muscarinic receptor agonists stimulates a phospholipase C that degrades inositol phospholipids. When tissue slices were labelled in vitro with [³H]inositol, this response could be monitored by measuring the formation of [³H]inositol phosphates. Accumulation of inositol 1,4-biphosphate in stimulated brain slices suggests that polyphosphonositides are the primary targets for phospholipase C activity. Li⁺ (10 m M ) in the medium completely blocked the hydrolysis of inositol 1-phosphate, partially inhibited inositol 1,4bisphosphate hydrolysis, but had no effect on the hydrolysis of inositol 1,4,5-trisphosphate by endogenous phosphatases. Muscarinic receptor pharmacology was studied by measuring the accumulation of [³H]inositol 1-phosphate in the presence of 10 m M Li⁺. In experiments on brain slices, the response to carbachol was antagonised by atropine with an affinity constant of approximately 8.79 ± 0.12. Dose-response curves to several muscarinic agonists were constructed using brain and parotid gland slices. The results are consistent with relatively direct coupling of low-affinity muscarinic receptors to inositol phospholipid breakdown in brain slices; full agonists were relatively more potent in the parotid gland compared with the brain. Explanations for these differences are suggested.     

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase.

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2+ channel activity.     

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).     

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.     

Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium

Inositol 1,4,5-trisphosphate is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. We have recently identified high levels of specific binding sites for inositol 1,4,5-trisphosphate in brain membranes (Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161) and have now further characterized these sites. In cerebellar membranes, inositol 1,4,5-trisphosphate binding sites are abundant (20 pmol/mg protein) and display high affinity and selectivity for inositol 1,4,5-trisphosphate (KD approximately equal to 40 nM), whereas other inositol phosphates such as inositol 1,3,4,5-tetrakisphosphate (Ki approximately equal to 10 microM) and inositol 1,4-bisphosphate (Ki approximately equal to 10 microM) exhibit much lower affinity for this site. Submicromolar concentrations of calcium strongly inhibit inositol 1,4,5-trisphosphate binding (IC50 approximately equal to 300 nM). A sharp increase in binding occurs at slightly alkaline pH. These results suggest that actions of inositol 1,4,5-trisphosphate are regulated by physiological alterations in intracellular pH and calcium concentrations.     

Turnover of inositol phosphates in brain during ischemia-induced breakdown of polyphosphoinositides

Using either [³²]ATP or [³H]inositol as precursors which were injected intraventricularly into rat brain, decapitative ischemic treatment resulted in a more rapid loss of labeled phosphatidylinositol 4,5-biphosphates than phosphatidylinositol 4-phosphates in the initial 30 s-1 min. When polyphosphoinositides were labeled with [³H]inositol, the breakdown of these compounds was accompanied by a time-dependent appearance of labeled inositol phosphates. Although the level of radioactivity of inositol trisphosphate was low, a peak labeling activity was shown at 30 s. The radioactivity of inositol bisphosphate showed an increase after a delay of 30 s, and reached a peak at 1 min before declining to the baseline level at 5 min. There was also a lag period of 30 s for the appearance of labeled inositol monophosphate, after which the radioactivity continued to increase in a biphasic manner for the entire 5 min period. Results indicate that decapitative ischemic treatment to rats can serve as an experimental model for assessing in vivo stimulation of the receptor-mediated signal transduction mechanism related to polyphosphoinositide breakdown and subsequent turnover of inositol phosphates in brain.     

Carbachol, but Not Norepinephrine, NMDA, lonomycin, Ouabain, or Phorbol Myristate Acetate, Increases Inositol 1,3,4,5-Tetrakisphosphate Accumulation in Rat Brain Cortical Slices

Abstract: lonomycin, a Ca²⁺ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 m M Ca²⁺, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 μ M ionomycin was equivalent to that seen with 1μ M carbachol. Phorbol 12-myristate 13-acetate or high K⁺ (30 μ M ) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 μ M Ca²⁺. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 μ M carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-tris-phosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase Cβ, in isolated rat brain membranes.     

Putative role of inositol phospholipid metabolism in neurons

Inositol phospholipids play a crucial role in the intracellular signal transduction in most cell types. Activation of an enzyme called phospholipase C or PIP2-phosphodiesterase (PIP2-PDE) leads to the production of two second messenger molecules, diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3). DG activates a kinase called protein kinase C, whereas IP3 mediates the release of Ca2+ from intracellular storage sites. The measurement of IP3 and its degradation products, inositol diphosphate (IP2) and inositol monophosphate (IP1) provides a way of assessing the extent to which this complex system has been activated. In the central nervous system (CNS) most of the studies on the neurotransmitter stimulated formation of inositol phosphates (IPs) have been performed on brain slices, a mixture of mainly neurons and glial cells. The recent development of pure neuronal cultures provides a means of determining which of these responses were of neuronal origin. The purpose of this review is to summarize the results obtained in neurons in primary culture together with a brief appraisal of the possible function of this second messenger system in neurons.