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PubMed2004

The effect of inositol supplements on the psoriasis of patients taking lithium: a randomized, placebo-controlled trial.

BACKGROUND: Lithium carbonate is the most widely used long-term treatment for bipolar affective disorders, but its ability to trigger and exacerbate psoriasis can become a major problem in patients for whom lithium is the only treatment option. Inositol depletion underlies the action of lithium in bipolar affective disorders and there are good theoretical reasons why the use of inositol supplements might be expected to help this group of patients. OBJECTIVES: To determine whether inositol supplements improve the psoriasis of patients on lithium therapy. METHODS: Fifteen patients with psoriasis, who were taking lithium, took part in a randomized, double-blind, placebo-controlled, crossover clinical trial comparing the effect of inositol supplements with those of a placebo (lactose). Changes in the severity of their psoriasis were measured by Psoriasis Area and Severity Index scores recorded before and after the different courses of treatment. The effect of inositol supplements on the psoriasis of 11 patients who were not taking lithium was evaluated in the same way. RESULTS: The inositol supplements had a significantly beneficial effect on the psoriasis of patients taking lithium. No such effect was detected on the psoriasis of patients not on lithium. CONCLUSIONS: The use of inositol supplements is worth considering for patients with intractable psoriasis who need to continue to take lithium for bipolar affective disorders.

Adult↗
PubMed1982

Yeast mutant defective in synthesis of phosphatidylinositol. Isolation and characterization of a CDPdiacylglycerol--inositol 3-phosphatidyltransferase Km mutant.

1. A Km mutant of Saccharomyces cerevisiae with a lesion in CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated. The mutant required a high concentration of myo-inositol for growth. 2. The CDPdiacylglycerol-inositol 3-phosphatidyltransferase in the mutant cells showed an apparent Km for myo-inositol over 200-times higher than that of the enzyme in wild-type cells. The maximum velocity of the mutant enzyme was comparable to that of the wild-type enzyme. 3. In mutant cells, labelled myo-inositol, phosphate and acetate were incorporated into phosphatidylinositol at much slower rates than in wild-type cells. The phosphatidylinositol content of mutant cells was markedly lower than that observed in wild-type cells. 4. Genetic analysis showed that the growth phenotype of the mutant arose from a single nuclear gene mutation in a gene coding for CDPdiacylglycerol-inositol 3-phosphatidyltransferase. 5. The mutant showed a normal level of phosphatidylserine synthase activity. The phosphatidylserine synthase gene was located between ura3 and hom3 on chromosome V, whereas the CDPdiacylglycerol-inositol 3-phosphatidyltransferase gene showed no linkage with ura3. 6. Labelled acetate was incorporated into various lipids including triacylglycerols, diacylglycerols, sterol esters and phospholipids other than phosphatidylinositol at faster rates in mutant cells than in wild-type cells. Incorporation into both the fatty acid and the sterol moieties was facilitated in the mutant. 7. A striking change in the cell-division process was observed when phosphatidylinositol synthesis was limited. The results showed that phosphatidylinositol synthesis is involved in the cell-division cycle of yeast.

CDP-Diacylglycerol-Inositol 3-Phosphatidyltransfer↗
PubMed1984

The effect of myo-inositol deficiency on phosphatases of yeast.

Activities of several phosphohydrolases are significantly enhanced when cells of the inositol-requiring yeast, Saccharomyces uvarum ATCC 9080, are deprived of inositol. This effect is most pronounced for the external acid phosphatase and cannot be explained simply by limitation of cellular growth, because starvation for vitamins or sulphate has no effect on acid phosphatase activities. Excessive secretion of acid phosphatase by spheroplasts prepared from inositol-deficient cells is greatly reduced when the spheroplast medium is supplemented with inositol and is immediately suppressed by the addition of cycloheximide. These results together with data obtained from experiments with whole cells, employing cycloheximide and actinomycin D, point to a regulatory effect of inositol limitation at the level of transcription. The external enzymes beta-D-fructofuranosidase, alpha-D-galactosidase and L-asparaginase, and the vacuolar enzyme carboxypeptidase Y are not affected by inositol deficiency indicating that inositol deficiency has no general effect on protein secretion.

Acid Phosphatase↗
PubMed1984

Molecular cloning of the gene encoding CDPdiacylglycerol-inositol 3-phosphatidyl transferase in Saccharomyces cerevisiae.

The structure gene encoding CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated by complementation in a Saccharomyces cerevisiae mutant after transformation with a comprehensive gene library containing Sau3AI partial restriction fragments of wild-type yeast DNA inserted into the YEp13 shuttle vector. Introduction of the cloned plasmid into the mutant restored both growth of cells and CDPdiacylglycerol-inositol 3-phosphatidyltransferase activity. A subcloning study indicated that the complementing gene was contained within a 1700-base-pair fragment of DNA. Expression of the cloned sequence was independent of its orientation in the vector. CDPdiacylglycerol-inositol 3-phosphatidyltransferase activity in the transformant was eight fold higher than that in the wild-type strain. Although the mutant enzyme had a greatly increased Km for myo-inositol, the enzyme in the transformant had an apparent Km for myo-inositol equal to that of the wild-type enzyme, indicating the presence of the wild-type structure gene on the recombinant plasmid. The elevated level of enzyme activity in the transformant did not lead to an increase in the content of phosphatidylinositol. In contrast, supplementation of the culture medium with increasing concentrations of myo-inositol resulted in an increase in the phosphatidylinositol content. Thus, the availability of myo-inositol is a critical regulatory factor in yeast phosphatidylinositol synthesis.

Base Composition↗
PubMed1988

Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver.

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by alpha-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 microM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.

Animals↗
PubMed1981

Myo-inositol turnover in the intact rat brain: increased production after d-amphetamine.

Apparent turnover of myo-inositol in the brain of urethane-anesthetized rats was estimated in vivo from the rate of appearance of endogenous myo-inositol in the cerebroventricular compartment. Ventricular-cisternal perfusion technique combined with isotope dilution of [14C]myo-inositol was used to determine the rate of appearance of brain-produced myo-inositol and its modification by d-amphetamine. A mean value of 0.75 nmol/min was obtained for the rate of appearance in the cerebroventricular system. A dose-dependent increase in this rate was seen after the administration of d-amphetamine. The endogenous removal of myo-inositol from the perfusate was also studied and found to be mediated in part by a saturable transport system that was not influenced by d-amphetamine. The rate of entry of myo-inositol from blood to the cerebroventricular system was very low and accounted for only 2% of the total rate of appearance, indicating that the majority of myo-inositol in the rat cerebroventricular fluid originates in the brain.

Animals↗
PubMed1984

Inositol phospholipid hydrolysis in rat cerebral cortical slices: I. Receptor characterisation.

Characterisation of receptor-mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5-hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, alpha 1-adrenoceptor, and histamine H1 receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose-response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline adrenaline were full agonists at alpha 1-adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.

Animals↗
PubMed1985

Manganese stimulates the incorporation of [3H]inositol into a pool of phosphatidylinositol in brain that is not coupled to agonist-induced hydrolysis.

Mn2+ greatly increases the incorporation of myo-[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI-myo-inositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+-stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo-[3H]inositol for 60 min in an N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo-inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist-stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight- and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate [( 3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+-treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo-[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist-induced hydrolysis.

Animals↗
PubMed1988

Effect of sorbinil on myo-inositol metabolism in cultured neuroblastoma cells exposed to increased glucose levels.

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.

Animals↗
PubMed1988

Stimulation of formation of inositol phosphates in primary cultures of bovine adrenal chromaffin cells by angiotensin II, histamine, bradykinin, and carbachol.

Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met-Lys-bradykinin, and [Des-Arg9]-bradykinin was relatively inactive, implicating a BK-2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion-exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4-trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.

Adrenal Medulla↗
PubMed1992

Stimulus-induced accumulation of inositol tetrakis-, pentakis-, and hexakisphosphates in N1E-115 neuroblastoma cells.

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.

Alprostadil↗
PubMed1993

Lithium enhances muscarinic receptor-stimulated CDP-diacylglycerol formation in inositol-depleted SK-N-SH neuroblastoma cells.

The psychotherapeutic action of Li+ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li+ on muscarinic receptor-stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 +/- 4 nmol/mg of protein. The addition of carbachol to [3H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [3H]CDP-DAG was completely blocked by the addition of 10 microM atropine, was not dependent on the presence of Li+, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [3H]CDP-DAG was potentiated approximately 10-fold by Li+ and substantially reduced by coincubation with inositol. The formation of [3H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [3H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol > or = oxotremorine-M > bethanecol > or = arecoline > oxotremorine > pilocarpine.(ABSTRACT TRUNCATED AT 250 WORDS)

Atropine↗
PubMed1985

Histamine-induced inositol phospholipid breakdown in the longitudinal smooth muscle of guinea-pig ileum.

The characteristics of histamine-stimulated inositol phospholipid breakdown in slices of guinea-pig ileal smooth muscle and cerebellum have been investigated. In cerebellar slices the inhibition of the inositol phospholipid response to histamine by mepyramine was consistent with competitive antagonism of histamine H1-receptors. In slices of the longitudinal smooth muscle of guinea-pig ileum, mepyramine produced only a weak inhibition of the response to histamine, at concentrations up to 1 microM. This was in striking contrast to the potent competitive antagonism of the H1-mediated contractile responses obtained with mepyramine in this tissue. The H1-receptor antagonists (+)-chlorpheniramine and promethazine similarly had no effect on the EC50 value for histamine in guinea-pig ileum, while promethazine competitively antagonized the muscarinic receptor-mediated inositol phospholipid response in this tissue (Ka 3.6 X 10(7)M-1). Cimetidine, on its own, did not significantly inhibit the inositol phosphate accumulation elicited by histamine in ileum. In the presence of 0.2 microM mepyramine, cimetidine (0.1 mM) produced a small parallel shift of the histamine concentration-response curve (Ka 3 X 10(4) M-1). This inhibition, however, was not consistent with antagonism of an H2-receptor-mediated response. The effect of a range of histamine analogues on inositol phospholipid breakdown was determined. Dose-response curves were constructed and characterized in terms of the EC50, slope and maximal response attainable relative to histamine. The H1-agonists, N alpha,N alpha-dimethylhistamine, N alpha-methylhistamine, 2-pyridylethylamine and 2-thiazolyethylamine produced the largest accumulations of [3H]-inositol-1-phosphate. A very weak response was produced by the H2-selective agonist impromidine, while dimaprit (also H2-selective) was without significant effect. Mepyramine appeared to antagonize competitively the response to the H1-selective agonist 2-pyridylethylamine. This was in contrast to the data obtained with other H1-agonists, where mepyramine produced only a small dextral shift of the agonist curves at low agonist concentrations and an increase in the Hill coefficient. This was particularly striking in the case of 2-methylhistamine. The results suggest that an H1-receptor component in guinea-pig ileum, may coexist with a larger inositol phospholipid response to histamine which is independent of the activation of H1- or H2-receptors.

Animals↗
PubMed1987

Stimulation of alpha 1-adrenoceptors in rat kidney mediates increased inositol phospholipid hydrolysis.

The molecular events which follow activation of alpha 1-adrenoceptors in rat kidney were investigated by measuring inositol phospholipid hydrolysis. Slices were labelled with [3H]-inositol (0.25 microM) and the accumulation of [3H]-inositol phosphates ([3H]-IP's) was measured after stimulation with alpha-adrenoceptor agonists. Phospholipid labelling was both time- and Ca2+-dependent. In kidney, Ca2+ (1 mM) increased the incorporation of [3H]-inositol by 49% and in cerebral cortex reduced it by 46%. Following addition of noradrenaline (NA, 1 mM), accumulation of [3H]-IP's increased linearly for at least 60 min. In Ca2+-free buffers a 2.1 fold increase in [3H]-IP accumulation was observed and further increases in stimulated and control levels were produced in the presence of Ca2+ (2.5 mM). These responses were attenuated by the inclusion of indomethacin (10 microM) and abolished in the presence of EGTA (0.5 mM). Responses to (-)-NA were more than 4 fold higher in the renal cortex than in the medulla. Separation of the IP's which accumulate after alpha-adrenoceptor agonists showed that after 60 min stimulation the major products were glycerophosphoinositol and inositol-phosphate with smaller amounts of inositol-bisphosphate and inositol-trisphosphate. The most effective agonists tested for stimulation of accumulation of [3H]-IP's were (-)-NA greater than phenylephrine greater than methoxamine, (+)-NA. Clonidine and (-)-isoprenaline were ineffective at concentrations up to 100 microM. The order of effectiveness of alpha-adrenoceptor antagonists was prazosin greater than BE2254 greater than phentolamine greater than idazoxan greater than rauwolscine. The results indicate that alpha 1-adrenoceptors in rat kidney are linked to phosphoinositide hydrolysis and that this response is localized mainly to the renal cortex.

Adrenergic alpha-Agonists↗
PubMed1991

Histamine-induced inositol phosphate accumulation in HeLa cells: lithium sensitivity.

1. In the presence of 10 mM Li+ the histamine-stimulated accumulation of [3H]-inositol monophosphates [( 3H]-IP1) in HeLa cells prelabelled with [3H]-inositol increased over 10-20 min to a plateau level, which was normally maintained up to 60 min. Levels of [3H]-inositol bis- and trisphosphates [( 3H]-IP2 and [3H]-IP3) initially increased rapidly but declined to near basal levels by 20 min. 2. The same pattern of histamine-induced [3H]-IP1 accumulation was observed in cells in which [3H]-inositol was present 30 min before and during the incubation with histamine. Concentration-response curves for histamine measured in the presence of 10 mM Li+ were closely similar in cells prelabelled for 24 h with [3H]-inositol and in cells exposed to [3H]-inositol for only 30 min before addition of histamine, without removing the [3H]-inositol. The EC50 for histamine was 1.6 +/- 0.2 microM. 3. [3H]-IP1 accumulation induced by a sub-maximal concentration of histamine, 1 microM, also reached a plateau, but at a lower level than with 1 mM histamine. 4. Addition of 10 mM NaF at the plateau phase of [3H]-IP1 accumulation induced by 1 mM histamine resulted in a further increase in the level of [3H]-IP1. The level of [3H]-IP1 in the presence of histamine + NaF was 1.4 +/- 0.2 fold of that of the sum of the responses to histamine and NaF acting alone (basal levels subtracted). 5. Addition of 1 microM mepyramine at the plateau phase of [3H]-IP1 accumulation induced by 1 mM histamine in the presence of 10mM Li + resulted in a decline in the level of [3H]-IP1, implying that the metabolism of [3H]-IP1 is not completely blocked by 10mM Li' in HeLa cells. 6. Omission of the 15 min preincubation period with 10mM Li+ before stimulation with 100 microM histamine for 15 min resulted in an approximate halving of the level of [3H]-IP1, without any significant change in basal accumulation. Periods of preincubation with 10mM Li' longer than 15min did not produce any further increase in the level of [3H]-IP1 induced by histamine. 7. Basal and histamine-induced levels of [3H]-IP, increased as the concentration of Li+ was increased from 0 to 60mm, but the effect on the histamine-induced response was greater than on the basal level. Increasing the concentration of Li+ from 0 to 60 mm had only a small and mostly statistically insignificant effects on the levels of [3H]-1P2 and [3H]-1P3. The EC50 for histamine-induced [3H]-4P1 accumulation in the presence of 30mm Li + was 2.4 +/- 0.4 microM. 8. The results indicate that IP1 metabolism in HeLa cells is much less sensitive to Li+ than in mammalian brain. The plateau phase of histamine-induced [3H]-IP1 accumulation in the presence of 10mM Li+ thus represents a steady-state level. On a simple model the plateau level will be proportional to the rate of [3H]-IP1 formation at that time. The lower the concentration of Li' present, the better the approximation will be. The information obtained is thus not the same as when [3H]-1P, metabolism is completely blocked, in which case [3H]-IP1 accumulated can be used to calculate a mean rate of formation over the whole of the incubation period.

Fluorides↗
PubMed1995

Effects of GTP gamma S on muscarinic receptor-stimulated inositol phospholipid hydrolysis in permeabilized smooth muscle from the small intestine.

1. Smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig were permeabilized with Staphylococcus aureus alpha toxin (alpha-toxin) and used to investigate the role of G-protein activation in the regulation of muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis. 2. The efficiency of alpha-toxin permeabilization was estimated by the release of [3H]-2-deoxyglucose ([3H]-2DG) after prior loading or lactate dehydrogenase (LDH) enzyme release from the smooth muscle fragments. 3. In alpha-toxin-permeabilized smooth muscle, but not in non-permeabilized muscle, GTP gamma S induced time- and concentration-dependent increases in labelled inositol phosphates. Carbachol (CCh) increased labelled inositol phosphates in both permeabilized and non-permeabilized muscle, although the increases were greater in non-permeabilized smooth muscle. The response to 100 microM CCh was severely reduced by 0.5 microM atropine. 4. In permeabilized muscle the effects of GTP gamma S or CCh on inositol phosphate levels were reduced by treatment with pertussis toxin (PTX) and completely inhibited by GDP beta S. 5. GTP gamma S caused a concentration-dependent inhibition of the CCh-induced increases in the levels of labelled inositol phosphates. Dibutyryl cyclic AMP or Sp-cAMPs (adenosine-3',5'-cyclic phosphorothiolate-Sp) reduced the effects of CCh on inositol phosphate levels. 6. The results suggest that muscarinic AChR activation induces inositol phospholipid hydrolysis via more than one G-protein in this smooth muscle and that several mechanisms may contribute to the modulation of both stimulatory and inhibitory responses observed.

Animals↗
PubMed1996

Inositol hexakisphosphate binding sites in rat heart and brain.

1. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol hexakisphosphate (InsP6) are produced in response to stimulation of cardiac alpha 1-adrenoceptors. While the role of Ins(1,4,5)P3 and Ins(1,4,5)P3 receptors is well-defined in many tissues including brain, the functional role of the putative InsP6-InsP6 receptor system in cardiac function is less clear. Using quantitative autoradiography, this study examined the characteristics and regional localization of [3H]-InsP6 binding sites in rat heart and compared the affinity of a range of inositol polyphosphates for [3H]-InsP6 and [3H]-Ins(1,4,5)P3 binding sites in heart and brain. 2. [3H]-InsP6 bound to a single, high affinity site in sections of rat heart (KD ranging from 22 +/- 1.9 nM in right atria to 35 +/- 2.6 nM in the interventricular septum, n = 7). The maximal number of binding sites (Bmax) ranged from 5.1 +/- 0.48 to 12 +/- 1.8 pmol mg-1 protein in left atrium and left ventricle, respectively. Inositol phosphates inhibited binding of [3H]-InsP6 with the order of potency: InsP6 > Ins(1,4,5)PS3 > inositol 1,3,4,5-tetrakisphosphate > or = inositol pentakisphosphate > Ins(1,4,5)P3 > > inositol mono- and bisphosphates, consistent with the labelling of an InsP6 binding site. 3. The Ins(1,4,5)P3 analogue, Ins(1,4,5)PS3, originally investigated as a putative selective radioligand for the Ins(1,4,5)P3 receptor, was a potent inhibitor of [3H]-InsP6 binding in all heart regions (K1 = 170-260 nM). The K1 of Ins(1,4,5)PS3 for the inhibition of [3H]-Ins(1,4,5)P3 binding in rat brain (60-220 nM) was similar to that observed for the inhibition of [3H]-InsP6 binding in heart, suggesting that Ins(1,4,5)PS3 is not a specific ligand for either Ins(1,4,5)P3 or InsP6 receptor binding sites. 4. Previous studies have detected [3H]-InsP6 binding in mitochondrial and sarcoplasmic reticulum fractions of heart and links between InsP6 and cardiac mitochondrial Ca2+ regulation have been proposed, suggesting further studies are warranted to determine the functional role(s) of InsP6 and InsP6 receptor binding sites in cardiac tissue.

Animals↗
PubMed1991

Effects of ethanol on inositol 1,3,4,5-tetrakisphosphate metabolism by rat brain homogenates.

The hydrolysis of membrane phosphoinositides is widely recognized as an important signal transduction pathway in brain. One of the products of phosphoinositide hydrolysis, Ins(1,4,5)P3, is thought to participate in signal transduction by mobilizing intracellular calcium and it is now clear that Ins(1,4,5)P3 metabolism is a complicated process that may be highly regulated. In addition to being dephosphorylated by the action of a 5-phosphatase, Ins(1,4,5)P3 can be phosphorylated by a 3-kinase to Ins(1,3,4,5)P4. Although the physiological significance of the higher inositol polyphosphates is not clear, recent evidence suggests that Ins(1,3,4,5)P4 may also have important second messenger function. Since ethanol is known to have potent effects on synaptic transmission, we investigated the in vitro effects of ethanol on [3H]Ins(1,3,4,5)P4 metabolism by rat whole brain homogenates. Ins(1,3,4,5)P4 was rapidly hydrolyzed to Ins(1,3,4)P3, inositol bisphosphates [Ins(3,4)P2 and Ins(1,3)P2], inositol monophosphates [Ins(1)P/Ins(3)P and Ins(4)P], and to inositol by sequential dephosphorylation. No [3H]Ins(1,4,5)P3 was detected. Ethanol (500 mM), significantly accelerated the dephosphorylation of Ins(1,4,5)P3, resulting in a more rapid formation of inositol bisphosphates, monophosphates and inositol. However, intoxicating and sedative-hypnotic concentrations of ethanol (30-100 mM) had no effect upon Ins(1,3,4)P3 dephosphorylation, suggesting that pharmacologically relevant concentrations of ethanol do not directly effect the enzymes involved in the dephosphorylation of Ins(1,3,4,5)P4 to free inositol in brain.

Animals↗