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PubMed1992

Activation of beta-adrenergic receptors inhibits Ca2+ entry-mediated generation of inositol phosphates in the guinea pig myometrium, a cyclic AMP-independent event.

In the guinea pig myometrium, carbachol, oxytocin, and fluoroaluminates stimulated the breakdown of phosphatidylinositol 4,5-bisphosphate, which was insensitive to pertussis toxin [Biochem. J. 255:705-713 (1988)]. We now demonstrate that an increased accumulation of inositol phosphates, with an early production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], could also be obtained with K+ (30 mM) and the Ca2+ ionophore ionomycin. Removal of extracellular Ca2+ or addition of the Ca2+ channel antagonists nifedipine and verapamil almost totally abolished stimulations elicited by high K+ and partially attenuated receptor- and fluoroaluminate-mediated increases in inositol phosphates. Isoproterenol similarly attenuated the accumulation of inositol phosphates elicited by carbachol, oxytocin, and fluoroaluminates (maximal inhibition, 35%; EC50, 0.5 nM), with no change in the rate of Ins(1,4,5)P3, inositol bisphosphate, and inositol monophosphate generation. The beta-adrenergic receptor-induced inhibition was prevented by pertussis toxin and could not be reproduced by forskolin, indicating that cAMP was not involved. Experimental findings were, rather, consistent with a predominant role for Ca2+. Thus, inhibition due to isoproterenol was lost in a Ca(2+)-depleted medium and was not additive with that caused by nifedipine. Accumulation of inositol phosphates triggered by high K+ was insensitive to the beta-adrenergic receptor inhibition. The inhibitory effect of isoproterenol, similar to that of nifedipine, was counteracted by ionomycin and also by the Ca2+ channel agonist Bay K 8644. These data indicate that in the myometrium 1) phospholipase C can be activated through a voltage-gated Ca2+ entry-dependent process that contributes at least partially to the stimulations triggered by receptor- and/or guanine nucleotide-binding protein-mediated activation and 2) beta-adrenergic receptor activation is linked via a cAMP-independent, pertussis toxin-sensitive pathway to an inhibition of voltage-gated Ca2+ channels, resulting in an attenuation of the Ca(2+)-associated generation of inositol phosphates.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1990

Dual mechanisms of inhibition by dopamine of basal and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells. Evidence for an inhibition not mediated by voltage-dependent Ca2+ channels.

In primary cultures of anterior pituitary cells, dopamine inhibited basal and thyrotropin-releasing hormone (TRH)-stimulated inositol monophosphate, bisphosphate, and trisphosphate production. This inhibition by dopamine can be resolved into two distinct components. One of the components was rapid and already present after 10 s. The other was slower, starting after 1 min, and was mimicked by nimodipine, a dihydropyridine calcium channel antagonist. The effects of dopamine and nimodipine were not additive on both basal and TRH-stimulated inositol phosphate production. Furthermore, the dopamine inhibition in the presence of TRH was much higher than the inhibition induced by nimodipine. It is thus likely that calcium entry through voltage-dependent calcium channels triggers a positive feedback on TRH stimulation of phospholipase C. However, depolarizing concentrations of K+ or BAY-K-8644, a voltage-dependent calcium channel agonist, had no effect on inositol monophosphate and bisphosphate accumulation. Ionomycin, even at a very high concentration (10 microM), had only a slight and transient effect on inositol phosphate formation. In addition, these agents did not affect the TRH dose-dependent stimulation of inositol phosphate production. These results suggest that the intracellular calcium concentrations that we measured under basal and TRH-stimulated conditions are sufficient to allow the maximal activity of phospholipase C which can be obtained under these two experimental conditions. In contrast, any decrease in the intracellular calcium concentration by a dihydropyridine antagonist, suppression of extracellular calcium, or inactivation of a voltage-dependent calcium channel by long term depolarization with K+ decreased the phospholipase C activities measured under basal and TRH-stimulated conditions. From these data it can be concluded that dopamine inhibits inositol phosphate production by two distinct mechanisms. The slow dopamine-induced inhibition of TRH-stimulated inositol phosphate production which is mimicked by nimodipine is likely because of an inhibition of a voltage-dependent calcium channel. This is substantiated further by the fact that ionomycin (10 microM) was able to reverse the nimodipine inhibitions as well as this slow component of dopamine inhibition. The nature of the rapid inhibition of TRH-stimulated inositol phosphate production induced by dopamine, but not by nimodipine, remains to be determined. It is suppressed in the absence of extracellular Ca2+. This may suggest that this inhibition is related to blockade of non-dihydropyridine-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1991

Myo-inositol attenuates two specific behavioral effects of acute lithium in rats.

Acute and chronic lithium treatment reduces levels of brain myo-inositol in rats. Several biological effects of lithium can be reversed in vitro by addition of myo-inositol. The ability of myo-inositol to reverse behavioral effects of lithium was tested using chronic inositol administration or acute intracerebroventricular (i.c.v.) injections. Chronic myoinositol elevated activity during the first 10 min in an open field, but did not reverse lithium-induced hypokinesia. Myo-inositol (i.c.v.) reversed the suppression of rearing behavior 24 hrs after an acute dose of lithium (5 mEq/kg) but did not attenuate hypokinesia 24 hrs after a high dose of lithium (10 mEq/kg). Myo-inositol, but not the inactive isomer chiro-inositol (i.c.v.), also significantly prolonged the latency to clonus in the lithium pilocarpine seizure model. These studies suggest that reduction of brain myo-inositol may be a critical mechanism for the behavioral effects of lithium.

Animals↗

PubMed1991

Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells.

Pasteurella multocida toxin, both native and recombinant, is an extremely potent mitogen for Swiss 3T3 cells and acts to enhance the formation of total inositol phosphates (Rozengurt, E., Higgins, T., Changer, N., Lax, A.J., and Staddon, J.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 123-127). P. multocida toxin also stimulates diacylglycerol production and activates protein kinase C (Staddon, J.M., Chanter, N., Lax, A.J., Higgins, T.E., and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848). Here we analyze, by [3H]inositol labeling and high performance liquid chromatography, the inositol phosphates in recombinant P. multocida toxin-treated cells. Recombinant P. multocida toxin stimulated increases in [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and its metabolic products, including Ins(1,3,4,5)P4, Ins(1,3,4)P3, Ins(1,4)P2, Ins(4/5)P, and Ins(1/3)P. The profile of the increase in the cellular content of these distinct inositol phosphates was very similar to that elicited by bombesin. Furthermore, recombinant P. multocida toxin, like bombesin, mobilizes an intracellular pool of Ca2+. Recombinant P. multocida toxin pretreatment greatly reduces the Ca2(+)-mobilizing action of bombesin, consistent with Ca2+ mobilization from a common pool by the two agents. The enhancement of inositol phosphates and mobilization of Ca2+ by recombinant P. multocida toxin were blocked by the lysosomotrophic agents methylamine, ammonium chloride, and chloroquine and occurred after a dose-dependent lag period. The stimulation of inositol phosphate production by recombinant P. multocida toxin persisted after removal of extracellular toxin, in contrast to the reversibility of the action of bombesin. Recombinant P. multocida toxin, unlike bombesin and guanosine 5'-O-(gamma-thiotriphosphate), did not cause the release of inositol phosphates in permeabilized cells. These data demonstrate that recombinant P. multocida toxin, acting intracellularly, stimulates the phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Ammonium Chloride↗

PubMed1989

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets.

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.

Blood Platelets↗

PubMed1989

Inositol hexaphosphate and its Cu(II) coordinate complex as inhibitors of intestinal alkaline phosphatase.

Myo-Inositol hexaphosphate (phytic acid), a naturally occurring plant constituent capable of forming coordinate complexes with polyvalent cations, reversibly inhibits the hydrolysis of p-nitrophenyl phosphate by alkaline phosphatase. Kinetics of the inhibition is of the strictly competitive type with Ki equal to 260 microM at pH 8.0 and 25 degrees. A similar result is also obtained in the presence of an inositol hexaphosphate-cupric ion coordinate complex. This inhibition of substrate hydrolysis by either inositol hexaphosphate or its Cu(II) complex is to be differentiated from the effect of the latter on alkaline phosphatase in the absence of substrate. In this case total inhibition of the enzyme is obtained in a time-dependent process which apparently involves a metal ion exchange reaction, i.e., substitution of the enzyme's zinc atoms for copper atoms. The effect of inositol hexaphosphate on alkaline phosphatase contrasts with that of inorganic phosphate which gives inhibition kinetics of the partially competitive type with Ki (for its effect on KM) 17.1 microM; a value close to that of KM for the substrate (15.2 microM). At a low phosphate concentration relative to that of inositol hexaphosphate in the assay solution the observed inhibition was partially additive. At a higher phosphate to inositol hexaphosphate ratio, the inhibition was essentially a phosphate effect. This provides additional support for the conclusion that Inositol hexaphosphate and, by extension, an inositol hexaphosphate-Cu(II) complex inhibits alkaline phosphatase by interaction at the active sites in competition with the substrate.

Alkaline Phosphatase↗

PubMed1987

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides.

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.

Animals↗

PubMed1987

Receptor-mediated inositol phosphate formation in relation to calcium mobilization: a comparison of two cell lines.

Previous studies indicated that activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells (S. K. Ambler and P. Taylor, J. Biol. Chem. 261:5866-5871, 1986) and muscarinic receptors in 1321N1 astrocytoma cells (S. B. Masters, T. K. Harden, and J. H. Brown, Mol. Pharmacol. 27:325-332, 1985) resulted in the rapid mobilization of Ca2+ from internal stores of both cell types. Paradoxically, alpha 1-adrenergic agonists did not rapidly increase inositol trisphosphate (Ins-P3) formation in BC3H-1 cells, in distinction to the rapid increase in Ins-P3 accumulation observed in 1321N1 cells after muscarinic stimulation. To determine whether the variations observed in the Ins-P3 response could be ascribed to differences in the relative amounts of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate (respectively, Ins-1,4,5-P3, Ins-1,3,4-P3, and Ins-P4), we have separated the individual inositol phosphates by high-performance liquid chromatography and examined the rates of conversion of individual inositol phosphates in the two types of cells. Muscarinic stimulation of 1321N1 cells resulted in increased Ins-1,4,5-P3 production, as well as the rapid production of Ins-1,3,4-P3 and Ins-P4. Application of alpha 1-agonist to BC3H-1 cells produced a modest but delayed increase in accumulation of Ins-1,4,5-P3. Adrenergic stimulation also resulted in a smaller and even slower production of Ins-1,3,4-P3, and Ins-P4 could not be detected in BC3H-1 cells under any conditions employed. Thus, over a 30-sec interval in which Ca2+ is mobilized to a maximum extent, increases in Ins-1,4,5-P3, Ins-1,3,4-P3, or Ins-P4 amounted to less than 10% over basal values in BC3H-1 cells. These results indicate that the regulation of Ins-P3 isomer formation and conversion may vary substantially between different cell types. In addition, if inositol 1,4,5-trisphosphate is the sole mediator of intracellular Ca2+ release, it is necessary to propose that an increase in Ins-1,4,5-P3 sufficient to mobilize Ca2+ rapidly may occur only within discrete cellular localities in some cell types. According, it may not be possible to detect the increases in Ins-1,4,5-P3 over basal concentrations when measuring total cellular inositol phosphates.

Animals↗

PubMed1988

Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C.

The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) contains a novel inositol phospholipid which in this and the accompanying paper (Roberts, W.L., Santikarn, S., Reinhold, V.N., and Rosenberry, T.L. (1988) J. Biol. Chem 263, 18776-18784) is shown to be a plasmanylinositol that is palmitoylated on the inositol ring. The inositol phospholipid was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I] iodophenyl)diazirine and characterized by various chemical and enzymatic cleavage procedures whose products were analyzed by thin layer chromatography and autoradiography or gas chromatography. Acidic methanolysis of human erythrocyte acetylcholinesterase (Ehu AChE) revealed 18:0 and 18:1 alkylglycerols (0.55 and 0.20 mol/mol AChE, respectively). Acetolysis was shown by TLC to release alkylacylglycerol acetates from Ehu AChE. Analysis by gas chromatography revealed that 83% of the alkylacylglycerol acetates contained an 18:0 or 18:1 1-alkyl group and a 22:4 (n - 6), 22:5 (n - 3), or 22:6 (n - 3) 2-acyl group. The inositol phospholipid is linked to the anchor by a glucosamine in glycosidic linkage, and deamination with nitrous acid cleaved the glycosidic linkage and released the phospholipid. The deamination and acetolysis products from Ehu AChE were purified by high performance liquid chromatography, and fatty acid analysis following acidic methanolysis of the purified products revealed that 2 fatty acid residues were associated with the deamination product and only one with the alkylacylglycerol acetolysis product. The other fatty acid residue was primarily palmitate and was indicated to be in ester linkage to an inositol hydroxyl(s). This linkage was shown to be responsible for the resistance of the inositol phospholipid to cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase. Deacylation of the inositol phospholipid deamination product by treatment with base removed this palmitoyl group and facilitated release of alkyl- and alkylacylglycerol species by phosphatidylinositol-specific phospholipase C with concomitant formation of inositol 1-phosphate. In contrast, digestion of Ehu AChE with a recently reported anchor-specific phospholipase D resulted in release of plasmanic acids from the intact palmitoylated plasmanylinositol.

Acetylcholinesterase↗

PubMed1984

Characterization of alpha-1 adrenergic receptors linked to [3H]inositol metabolism in rat cerebral cortex.

The properties of alpha-1 adrenergic receptors in rat cerebral cortex were examined by measuring increases in [3H]inositol metabolism in brain slices. Slices of rat cerebral cortex were incubated in the presence of 0.23 microM [3H]inositol in Krebs-Ringer-bicarbonate buffer containing 10 mM lithium chloride, and the production of water-soluble [3H]inositol phosphates was monitored after extraction and anion-exchange chromatography. Norepinephrine caused a 4- to 6-fold increase in [3H]inositol metabolism in cerebral cortical slices, and this response was blocked much more potently by the alpha-1-selective antagonist prazosin than by the alpha-2-selective antagonist yohimbine. Epinephrine and norepinephrine were both full agonists and stimulated [3H]inositol metabolism to the same extent in this system. The synthetic drugs phenylephrine and methoxamine were both partial agonists at these receptors, with intrinsic activities only 56 to 58% of epinephrine and norepinephrine. A variety of imidazoline and other partial agonists caused no measurable stimulation of [3H]inositol metabolism in this preparation. The response to norepinephrine was completely blocked by alpha adrenergic receptor antagonists with the potency order prazosin greater than BE 2254 greater than indoramin = phentolamine greater than azapetine greater than piperoxan greater than yohimbine. In the absence of calcium, basal [3H]inositol metabolism was increased, but norepinephrine caused the same 5-fold stimulation as in the presence of 2.5 mM CaCl2. The potencies of both antagonists and agonists in inhibiting or activating [3H]inositol metabolism in slices of rat cerebral cortex were highly correlated with their ability to displace the alpha-1 adrenergic receptor selective radioligand [125I]BE 2254 from specific binding sites in membrane preparations of rat cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗

PubMed1980

The effects of lithium ion and other agents on the activity of myo-inositol-1-phosphatase from bovine brain.

myo-Inositol-1-phosphatase has been partially purified from bovine brain. The enzyme has a molecular weight of about 58,000. Both L-myo-inositol 1-phosphate and D-myo-inositol 1-phosphate are hydrolyzed by the enzyme as well as (-)-chiro-inositol 3-phosphate and 2'-AMP. Triphosphoinositide is not a substrate. The phosphatase is completely dependent on Mg2+, which has a Km of 1 mM. Calcium and manganese ions are competitive inhibitors of Mg2+ binding with Ki values of 18 microM and 2 microM, respectively. Lithium chloride inhibits the hydrolysis of both L- and D-myo-inositol 1-phosphate to the extent of 50% at a concentration of 0.8 mM. The phosphatase from testis is similarly inhibited by lithium. Lithium ion is a noncompetitive inhibitor of Mg2+ binding and an uncompetitive inhibitor of myo-inositol 1-phosphate binding. Because lithium chloride administration elicits both an increase in the levels of myo-inositol 1-phosphate and a decrease in the levels of myo-inositol in rat brain (Allison, 1978), and because these actions are blocked by anticholinergic agents, we examined the effects of cholinergic agonists and antagonists on the enzyme and found none. The possibility that the inhibition of this enzyme by lithium ion is related to the pharmacological actions of lithium is discussed.

Animals↗

PubMed1995

Inositol treatment in psychiatry.

Inositol, a naturally occurring isomer of glucose, is a key intermediate of the phosphatidyl-inositol (PI) cycle, a second-messenger system used by several noradrenergic, serotonergic and cholinergic receptors. The suggestion that lithium might treat mania via its reduction of inositol levels led to experiments showing that pharmacological doses of peripheral inositol reverse behavioral effects of lithium in animals and side effects of lithium in man. Cerebrospinal fluid (CSF) levels of inositol are low in depression. An open-label, add-on trial of inositol in depression suggested a beneficial effect. In a subsequent 1-month, parallel-groups, double-blind, placebo-controlled study of 28 patients, inositol was effective as sole therapy for depression (p = .043). Inositol was also effective for panic disorder in a double-blind, random-assignment, placebo-controlled crossover study of 21 patients, with 4 weeks in each phase (p = .02); the effect was comparable to that of imipramine in recent studies.

Clinical Trials as Topic↗

PubMed1995

Inhibition of clathrin assembly by high affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP-3.

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 microM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 microM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 microM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 microM. Moreover, 10 microM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.

Adaptor Proteins, Vesicular Transport↗

PubMed1993

The effect of peripheral inositol injection on rat motor activity models of depression.

Myo-inositol is an important precursor in cellular second-messenger synthesis. It has been reported to be reduced in the cerebrospinal fluid of depressed patients and to reverse a specific effect of Li on rat behavior when given intracerebroventricularly (i.c.v.). However, myo-inositol enters the brain poorly when given peripherally, and its effects on normal rat brain have not been well studied. A series of experiments examined the effect of intraperitoneal inositol in high doses on locomotor activity, on amphetamine-induced hyperactivity, on apomorphine-induced stereotyped behavior, and on pilocarpine-induced behaviors in rats. In addition, the behavioral effect of i.c.v. inositol was compared to that of a control stereoisomer in untreated and lithium-treated rats. Few effects of peripheral myo-inositol were found in these models. However, peripheral inositol in high doses given to rats exhibiting Li-pilocarpine seizures showed that i.p. inositol could prevent these behaviors, confirming that peripheral inositol may have antidepressant potential in appropriate models.

Amphetamine↗

PubMed1993

Triads and transverse tubules isolated from skeletal muscle contain high levels of inositol 1,4,5-trisphosphate.

We measured the content of inositol 1,4,5-trisphosphate in sarcoplasmic reticulum, transverse tubules, and triads isolated from frog skeletal muscle, as well as in triads isolated from rabbit skeletal muscle. We found that acid extracts of both transverse tubules and triads contained significant amounts of inositol 1,4,5-trisphosphate, in the range of 300-400 pmol/mg of protein as determined by a radioreceptor assay, whereas no detectable amounts were found in sarcoplasmic reticulum vesicles. The identity of inositol 1,4,5-trisphosphate in the extracts was confirmed by comigration with [3H]inositol 1,4,5-trisphosphate on polyethyleneimine-cellulose plates, and by phosphorylation to inositol 1,3,4,5-tetrakisphosphate using the inositol 1,4,5-trisphosphate 3-kinase present in muscle extracts. These findings may have important physiological implications. First, the results indicate that the muscle plasma membrane regions differentiated as transverse tubules contain high amounts of inositol 1,4,5-trisphosphate, suggesting that they might possess a high density of binding sites for this compound. Second, since inositol 1,4,5-trisphosphate has been proposed as a chemical transmitter in excitation-contraction coupling in skeletal muscle, our finding that this second messenger is present in high density at the site of coupling may contribute to the understanding of its role in this process.

Animals↗

PubMed1993

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells.

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.

Animals↗

PubMed1976

High uptake of myo-inositol by rat pancreatic tissue in vitro stimulates secretion.

A recent study by Hokin-Neaverson, M., Sadeghian, K., Majumder, A.L., and Eisenberg, F. (1975) Biochem. Biophys. Res. Commun. 67, 1537-1544, demonstrates that free myo-inositol in the pancreas is significantly increased during intense cholinergic stimulation of secretion. Incubation of rat pancreatic tissue in medium with 100 mM myo-inositol increases 10-fold the endogenous content of free myo-inositol and elicits a prompt and sustained 50% increase in the rate of release of amylase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the electrophoretic pattern of the protein mixture released in the presence of 100 mM myo-inositol is the same as that of the secretory output released in the presence of 10 muM carbamylcholine. Microscopic examination of tissue pieces indicates that there is no significant decrease in zymogen granule content of the pancreatic acinar cells during incubation in medium with 100 mM myo-inositol. Jamieson, J.D., and Palade, G.E. (1967) J. Cell Biol. 34, 597-615, have shown that pulse-labeled secretory proteins in guinea pig pancreas first appear in zymogen granules 1 hour postpulse, becoming maximally accumulated in these storage sites by 2 hours postpulse. myo-Inositol (100 mM) stimulates release of pulse-labeled secretory proteins only if incubation in medium with 100 mM myo-inositol is initiated anytime during the first 80 min postpulse. The findings thus indicate that a high uptake of myo-inositol by rat pancreatic tissue in vitro selectively stimulates the release of just those secretory proteins being packaged in newly forming zymogen granules.

Amylases↗

PubMed1993

Evidence for receptor-specific regulation of the metabolism of the second messenger, inositol trisphosphate, in human endothelial cells.

Primary cultures (1-2 weeks-old, no passage) of endothelial cells from the human umbilical vein were pre-labelled with [3H]inositol, stimulated for varying times (15 seconds to 5 minutes) in the presence of LiCl with either thrombin or histamine, and the extracted radiolabelled isomers of inositol phosphates were analysed by anion-exchange chromatography. The doses of each receptor-agonist were chosen to stimulate a large accumulation of inositol phosphates at approximately the same rate. At all times of stimulation the relative accumulation of inositol trisphosphate isomers and inositol tetrakisphosphate was different upon stimulation with these agonists, particularly at earlier times. These results argue for the receptor-specific regulation of the inositol trisphosphate 3-kinase. The significance of this observation is discussed with respect not only to the current ideas on the control of calcium homoeostasis by inositol phosphates, but also in consideration of their role as second messengers controlling different end-points of vascular function.

Cells, Cultured↗