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PubMed2005

Thyrotropin stimulates the generation of inositol 1,4,5-trisphosphate in human thyroid cells.

CONTEXT: Dual activation by TSH of the phospholipase C and cAMP cascades has been reported in human thyroid cells. In contrast, Singh et al. reported convincing data in FRTL-5 thyrocytes arguing against such an effect in this model. Their data in FRTL-5 cells indicated no increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to TSH. Therefore, the authors questioned results previously obtained on human cells by cruder methodology. OBJECTIVE: We investigated the formation of inositol phosphates by HPLC techniques in human thyroid slices to separate the inositol phosphate isomers. RESULTS: Ins(1,4,5)P3, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate were increased after TSH stimulation. The effect of TSH in human thyroid cells was reproduced by recombinant TSH and prevented by antibodies blocking the TSH receptor. Thyroid-stimulating antibodies at concentrations eliciting a cAMP response equivalent to TSH failed to stimulate inositol phosphate generation. CONCLUSIONS: TSH, but not thyroid-stimulating antibodies, activates both cAMP and the phospholipase C cascade in human thyroid as now demonstrated by an increase in Ins(1,4,5)P3 and its inositol phosphate metabolites. Therefore, this effect cannot be extrapolated to the FRTL-5 cell line. The apparent discrepancy may be due to a difference between species (human vs. rat) or to the loss of the fresh tissue properties in a cell line. The dual effect of TSH in human cells, through cAMP on secretion of thyroid hormones and through the diacylglycerol, Ins(1,4,5)P3 Ca2+ pathway on thyroid hormone synthesis, implies the possible separation of these effects in thyroid disease.

Animals↗
PubMed1998

Lithium chloride does not inhibit the proliferation of L6 myoblasts by decreasing intracellular free inositol.

We conducted a series of experiments to determine whether lithium chloride (LiCl) inhibited the proliferation of L6 myoblasts by reducing the availability of intracellular free inositol. After the myoblasts were plated in DMEM + 10% fetal bovine serum (FBS) for 24 h, medium was replaced with DMEM + 10% FBS containing 0 (control), 5, 10, or 20 mM LiCl. Cell number, protein content, and [3H]thymidine incorporation into DNA were determined at 24-h intervals. Control cells exhibited a 3.8-fold increase in cell number by 96 h in culture. Although 5 mM LiCl did not affect the rate or extent of proliferation, 10 and 20 mM LiCl caused 36 and 86% decreases, respectively (P < .05), in cell number by 96 h in culture. The effects of LiCl could not be overcome by the addition of free inositol (up to 20 mM) to the medium. Lithium chloride caused 4.6- and 7.3-fold increases (P < .05) in lactate dehydrogenase activity in culture media after 96 h of exposure to 10 and 20 mM LiCl, respectively, indicating loss of viability after chronic treatment. However, the acute effects of LiCl after 24 h of treatment were reversible, as indicated by a rapid resumption of proliferation following removal of LiCl. Concentrations of 5, 10, and 20 mM LiCl caused 4.7-, 8.2-, and 9.1-fold increases (P < .05), respectively, in the accumulation of [3H]inositol within the inositol monophosphate pool. Treatment of cells with 10 and 20 mM LiCl also increased (P < .05) label recovered as inositol bisphosphate. Rather than depress phosphoinositide synthesis, the addition of 10 and 20 mM LiCl dose-dependently increased (P < . 05) the incorporation of [3H]inositol into phosphatidylinositol and phosphatidylinositol-4-phosphate. These results indicate that LiCl does not decrease proliferation of L6 myoblasts via a depletion in the intracellular free inositol pool. Instead, LiCl may block the hydrolysis of phosphatidyl inositides.

Animals↗
PubMed1988

Angiotensin II and guanine nucleotides stimulate formation of inositol 1,4,5-trisphosphate and its metabolites in permeabilized adrenal glomerulosa cells.

Angiotensin II (AII) interacts with specific receptors in the adrenal glomerulosa cell and stimulates the hydrolysis of plasma membrane phosphoinositides by phospholipase C, with production of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and subsequent mobilization of intracellular Ca2+. In electrically permeabilized, [3H]inositol-labeled glomerulosa cells, AII stimulated Ins-1,4,5-P3 production within 15 s with half-maximal potency of 10(-9) M. The nonhydrolyzable GTP analog, guanosine 5'-O-thiotriphosphate (GTP gamma S), stimulated Ins-1,4,5-P3 formation in a dose-dependent manner with half-maximal effect at 10(-7) M. AII-activated Ins-1,4,5-P3 production was further increased by guanine nucleotides. The rate at which GTP gamma S-stimulated inositol polyphosphate production was consistently slower than that of AII. In adrenal membrane preparations, GTP gamma S-stimulated polyphosphoinositide hydrolysis was enhanced by Ca2+, with half-maximal activity at 300 nM free Ca2+. Ins-1,4,5-P3 formation was also increased by NaF, further indicating the involvement of a guanine nucleotide regulatory protein. In addition to Ins-1,4,5-P3 and its metabolites formed during degradation via the 4-monophosphate pathway, AII and GTP gamma S stimulated the formation of the phosphorylated metabolite inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in permeabilized cells. The absence of a significant rise in inositol 1-monophosphate indicated that phosphatidylinositol hydrolysis was not stimulated by AII or GTP gamma S. Pretreatment of glomerulosa cells with pertussis toxin for 12 h before permeabilization did not inhibit AII- or GTP gamma S-stimulated inositol polyphosphate formation. However, treatment with cholera toxin, forskolin, or 8-Br-cAMP for 12 h enhanced both basal and ligand-stimulated Ins-1,4,5-P3 production. These observations suggest that agonist binding to the AII receptor activates a polyphosphoinositide-specific phospholipase C in the adrenal glomerulosa cell, and that a distinctive guanine regulatory protein is involved in this mechanism.

Adrenal Cortex↗
PubMed1986

Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells.

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.

Animals↗
PubMed1984

The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin.

The biochemical events underlying the ability of thrombin to enhance the metabolism of inositol phospholipids in human platelets have been investigated using platelets prelabeled with [3H]inositol. Thrombin treatment caused rapid formation of radioactive inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) with less marked and more variable changes in the levels of radioactive inositol phospholipids. Formation of IP2 and IP3 could be detected 5 s after exposure to thrombin and before IP levels increased. Low doses of thrombin which produced only shape change in human platelets also caused significant formation of IP2 and IP3 but not IP. These results suggest that thrombin-induced platelet activation may be mediated through hydrolysis of polyphosphoinositides. The majority of IP formed presumably arises from the hydrolysis of IP2. Prostacyclin inhibited thrombin-induced formation of all three inositol phosphates.

Blood Platelets↗
PubMed1984

Inositol 1,4,5-trisphosphate stimulates phosphorylation of a 62,000-dalton protein in monkey fibroblast and bovine brain cell lysates.

Inositol 1,4,5-trisphosphate (IP3) is produced in cells as a breakdown product of the diphosphorylated form of phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate. Stimulated breakdown of phosphoinositides has been correlated with a wide variety of hormonal stimuli which mobilize intracellular calcium, and IP3 has recently been found to cause calcium release from intracellular stores, thus implicating it as a second messenger in hormonal stimulation. In this paper we have examined the effect of IP3 on protein phosphorylation, and have found that IP3 stimulates phosphorylation of a 62-kDa protein in cell lysates made from cultured monkey fibroblasts and from bovine brain. Fifty per cent maximal stimulation of phosphorylation of this protein occurred at 2.5 X 10(-7) M IP3. Other inositol phosphates (inositol 1,4-bisphosphate, inositol 1-phosphate, inositol hexaphosphate, and myo-inositol) had no effect on protein phosphorylation at 10(-6)M, although inositol 1,4-bisphosphate at higher concentrations enhanced phosphorylation of the 62-kDa protein in brain lysates. The IP3-stimulated phosphorylation was calcium-independent and did not appear to result from inhibition of an endogenous protein phosphatase. We suggest that IP3, like other second messengers, acts as a protein kinase regulator.

Animals↗
PubMed1993

Transient temporal relationship between 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated synthesis and hydrolysis of polyphosphoinositides: desensitization of phospholipase C and the inositol lipid kinases upon long-term treatment of ascites cells by exogenous OAG.

In ascites tumor cells, phosphoinositide metabolism can be activated by short-term treatment with exogenously added 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the membrane-permeable analog of diacylglycerides (DAG). Quiescent cells prelabeled with D-myo-2-[3H]inositol and then stimulated with OAG (20 micrograms/ml of medium) reveal transient increases in the liberation of inositol 1,4-bis- and inositol 1,4,5-trisphosphate with peaks at 30 min, and a sustained accumulation of inositol phosphate 30 min after stimulation. The labeling patterns of the corresponding inositol lipids show transient activity profiles for phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and a sustained high activity level for PtdIns 30 min after OAG treatment. These data demonstrate a temporal relationship between synthesis and phospholipase C (PLC)-induced hydrolysis of these lipids. Simultaneous labeling of the cellular inositol phospholipids with [1-14C]arachidonic acid reveals modest accumulations after OAG stimulation. The relative 3H radioactivity distribution between the lipids and their inositol metabolites show that about 10% of the polyphosphoinositide pools are metabolically active. Long-term culturing of the cells (> 24 h) under OAG supplementation produces significant reductions in the catalytic activities of PLC and the PtdIns and PtdIns(4)P-specific kinases which is paralleled by a reduced radioactive labeling of PtdIns(4)P and PtdIns(4,5)P2 under these conditions. These data suggest that diglycerides affect the phosphoinositide metabolism by controlling PLC and phosphoinositide kinase activities probably via modification of membrane properties, and by functioning as modulator of other events.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

We determined the temporal relationship between the formation of inositol phosphates and increase in cytosolic [Ca2+] elicited by bombesin, vasopressin and platelet-derived growth factor (PDGF) in quiescent Swiss 3T3 cells. These responses were measured under identical conditions. Bombesin caused a rapid increase in inositol 1,4,5-trisphosphate which coincided with the increase in cytosolic [Ca2+]. This was followed by a slower but marked increase in inositol 1,3,4-trisphosphate and inositol-bisphosphate. Vasopressin elicited a similar sequence of events. In sharp contrast, highly purified porcine PDGF induced increases in cytosolic [Ca2+] and inositol 1,4,5-trisphosphate that were temporally uncoupled: detectable inositol polyphosphate formation occurred after Ca2+ mobilization from intracellular stores. The same temporal dissociation was observed when a recombinant v-sis product was used instead of porcine PDGF. However, PDGF was as effective as bombesin in stimulating the formation of inositol phosphates after 5-10 min of incubation. The data suggest that PDGF increases cytosolic [Ca2+] via a different signal transduction pathway from that utilized by bombesin and vasopressin. These findings have important implications for understanding the signal transduction pathway activated by PDGF.

Animals↗
PubMed1990

Phorbol esters modulate the high Ca2(+)-stimulated accumulation of inositol phosphates in bovine parathyroid cells.

We examined the effects of TPA on the high Ca2(+)-stimulated accumulation of inositol phosphates in bovine parathyroid cells to determine whether protein kinase C modulates phosphoinositide turnover in a fashion similar to that observed in other cell types stimulated by more classic Ca2+ mobilizing hormones. Following exposure of parathyroid cells to TPA (10(-6) M) for 10 or 30 minutes, there was a time- and dose-dependent inhibition of the accumulation of inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) stimulated by 3 mM Ca2+. Half the maximal observed inhibition took place at 1-10 nM TPA, with 50-60% inhibition of high Ca2(+)-stimulated accumulation of inositol phosphates at 10(-6) M TPA. The active phorbol ester, 4 beta-phorbol didecanoate, produced similar effects; the inactive derivative, 4 alpha-phorbol didecanoate, was without effect. When parathyroid cells were exposed to TPA (10(-6) M) for varying times and were then incubated with high (3 mM) Ca2+, inhibition of inositol phosphate accumulation was observed with 10 or 30 minutes preincubation. In contrast, preincubation of cells with TPA for 3 or 18 h markedly enhanced the high (3 mM) Ca2(+)-induced increase in inositol phosphates. In cells preincubated with TPA for 18 h, binding sites for [3H]phorbol dibutyrate and total protein kinase C (PKC) activity were reduced by greater than 95% and by 71%, respectively, consistent with downregulation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Inositol is a required nutrient for keratinocyte growth.

Bovine hypothalamus is known to contain a growth-promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo-inositol. The identity of the substance as myo-inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 microM (10 micrograms/ml), myo-inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 microM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth-promoting activities in the tissue extract. No other skin-derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain-derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.

Cells, Cultured↗
PubMed2003

Structural distinction among inositol phosphate isomers using high-energy and low-energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.

Electrospray (ESI) collisional-activated dissociation (CAD) tandem mass spectrometric methods for the structural characterization of inositol phosphates (InsPs) using both quadrupole and sector mass spectrometers are described. Under low-energy CAD, the [M + H](+) ions of the positional isomers of inositol phosphates, including inositol mono-, bis- and trisphosphates, yield distinguishable product-ion spectra, which are readily applicable for isomer differentiation. In contrast, the product-ion spectra arising from high-energy CAD (2 keV collision energy, floating at 50%) tandem sector mass spectrometry are less applicable for isomer identification. The differences in the product-ion spectrum profiles among the aforementioned InsP isomers become more substantial and differentiation of positional isomers can be achieved when the collison energy is reduced to 1 keV (floating at 75%). These results demonstrate that the applied collision energies play a pivotal role in the fragmentations upon CAD. The product-ion spectra are similar among the positional isomers of inositol tetrakisphosphates and of inositol pentakisphosphates. Thus, isomeric distinction for these two inositol polyphosphate classes could not be established by the tandem mass spectrometric methods that have achieved such distinctions for the less highly phosphorylated inositol phosphate classes. Under both high- and low-energy CAD, the protonated molecular species of all InsPs undergo similar fragmentation pathways, which are dominated by the consecutive losses of H(2)O, HPO(3) and H(3)PO(4).

Animals↗
PubMed2005

Scyllo-inositol in normal aging human brain: 1H magnetic resonance spectroscopy study at 4 Tesla.

The scyllo-inositol and myo-inositol concentrations of 24 normal human subjects were measured in vivo using 1H magnetic resonance spectroscopy at 4 T. Single-voxel short-echo (TE = 15 ms) metabolite spectra were collected from the white matter region of the corona radiata. Test-retest studies performed on 10 normal subjects demonstrated coefficient of variation for scyllo-inositol measurement of 37%, compared with 6% for N-acetyl aspartate. Comparisons between old and young subjects showed higher concentration of scyllo-inositol and myo-inositol in older subjects and a trend for a correlation between scyllo-inositol and myo-inositol levels across subjects.

Adaptation, Physiological↗
PubMed1992

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.

Animals↗
PubMed1993

Glucose and scyllo-inositol impair phosphoinositide hydrolysis in the 10.5-day cultured rat conceptus: a role in dysmorphogenesis?

Culture of the postimplantation rat conceptus from gestational day 9.5-10.5 in media supplemented with d-glucose or scyllo-inositol decreases tissue myo-inositol and phosphoinositides with a concomitant increase in dysmorphogenesis. A number of mitogenic agents initiate cellular proliferation and differentiation through receptors coupled to phosphoinositide hydrolysis. To test whether the decrease in conceptus phosphoinositides is associated with a reduced phosphoinositide hydrolytic response, we developed a protocol to stimulate phosphoinositide hydrolysis. Phosphoinositide hydrolysis was monitored by measurement of [3H]inositol phosphates after preincubation in serum free media. We examined the ability of serum, platelet-derived growth factor (PDGF), epidermal-derived growth factor (EGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), endothelin-1 (ET-1), and endothelin-2 (ET-2), to stimulate phosphoinositide hydrolysis. As measured by [3H]inositol monophosphate ([3H]InsP1) accumulation, normal rat seru, ET-1, and ET-2 stimulated phosphoinositide hydrolysis 47%, 420%, and 154% above the basal rate observed in serum free controls. EGF stimulated a statistically insignificant 15% increase while PDGF, IGF-1, or IGF-2 were without effect. We further characterized ET-1 stimulated phosphoinositide hydrolysis. Dose-response studies disclosed that incremental increases in [3H]InsP1 (129-420%) are observed over a concentration range of 10-1,000 nM. Maximal stimulation was not reached even at 1,000 nM. Temporally [3H]InsP1 and [3H]InsP3 levels increased linearly during incubation periods of 15-60 min. We further analyzed ET-1 stimulated phosphoinositide hydrolysis in 10.5-day conceptuses cultured for 24 hr in media containing high concentrations of glucose (23.3-56.6 mM) or scyllo-inositol (0.55, 5.5 mM). Under these dysmorphogenic conditions that concomitantly decrease the phosphoinositide precursor pool the response to ET-1 was blunted 28-76% for glucose and 29-65% for scyllo-inositol. This suggests that the effect of glucose and scyllo-inositol on lowering phosphoinositide precursor pools also results in a decrease in the response to agonists using the inositol/lipid intracellular pathway. This impaired signaling response may contribute to initiating dysmorphogenic events in diabetic embryopathy.

Animals↗
PubMed2002

A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.

Animals↗
PubMed1997

Inositol-1,3,4,5-tetrakisphosphate binding sites in control and ras-transformed NIH/3T3 fibroblasts.

Inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) binding properties were investigated in NIH/3T3 fibroblasts and its ras-transformant (DT cells), in which inositol tetrakisphosphates induce Ca2+ influx. [3H]-Ins(1,3,4,5)P4 bound to membranes of both types of cells with Kd values of 10.6 and 8.6 nM, respectively. The rank order of inositol polyphosphates for displacing [3H]Ins(1,3,4,5)P4 in DT cells was Ins(1,3,4,5)P4 > inositol-1,3,4,5,6-pentakisphosphate > inositol hexakisphosphate > inositol-1,4,5-trisphosphate. This order is similar to that reported in two Ras-GTPase-activating proteins, GAP1IP4BP and GAP1m, which are also the Ins(1,3,4,5)P4 binding proteins. Northern blot analysis revealed that NIH/3T3 and DT cells expressed mRNA species that were hybridizable with GAP1m cDNA. These results suggest that parental and ras-transformed NIH/3T3 fibroblasts possess GAP1-like proteins, which may be responsible for triggering inositol tetrakisphosphate-dependent Ca2 influx.

3T3 Cells↗
PubMed1987

Interactions between intracellular cyclic AMP and agonist-induced inositol phospholipid breakdown in isolated gastric mucosal cells of the rat.

The interactions between putative second effector mechanisms for hydrogen ion secretion were studied in isolated gastric cell preparations of the rat containing 60-70% parietal cells. Dibutyryl-cAMP and the compounds which increased the level of cAMP (histamine plus rolipram and forskolin plus rolipram) inhibited the carbachol-induced accumulation of [3H]inositol tris-, bis- and monophosphate. There was both a temporal and quantitative correlation between the increase in cAMP and the inhibition of the accumulation of [3H]inositol phosphates. Cimetidine attenuated the inhibitory effect of histamine on the formation of [3H]inositol phosphates. The enhancement of the accumulation of [3H]inositol phosphates by various concentrations of carbachol affected neither the basal nor the histamine-stimulated cAMP levels. In contrast to dibutyryl-cAMP, dibutyryl-cGMP did not modify the carbachol-induced formation of [3H]inositol phosphates. The biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C, inhibited both the basal and carbachol-induced accumulation of [3H]inositol phosphates. We suggest that the inhibition of the formation of inositol trisphosphate by the increase in the intracellular level of cAMP and by the activation of protein kinase C might be intracellular negative feedback systems which prevent the overreaction of the acid-secreting parietal cells under the simultaneous influence of the physiological gastric secretagogues.

Animals↗
PubMed1983

Myo-inositol and sorbitol metabolism in relation to peripheral nerve function in experimental diabetes in the rat: the effect of aldose reductase inhibition.

A possible relationship between increased sorbitol concentration and decreased myo-inositol concentration in peripheral nerves of diabetic rats has been examined. To this end, sorbinil, an aldose reductase inhibitor, was used either to prevent or reverse elevation of nerve sorbitol concentration in diabetic rats. Sorbinil treatment at 20 mg . kg-1 . day-1 prevented elevation of nerve sorbitol levels in early diabetes and reduced sorbitol concentration from 2.38 to 0.51 mumol/g in rats diabetic for 10 weeks. This treatment reduced the increase in nerve fructose concentration and prevented the reduced myo-inositol concentration found in diabetic rat nerve (control 3.63, diabetic 2.40, diabetic/sorbinil, 3.56 mumol/g). Sorbinil treatment did not prevent a significant slowing of motor-nerve conduction velocity at 10 weeks although treatment reduced the extent of slowing. Sorbinil treatment at 25 mg . kg-1 . day-1 reduced elevated sorbitol and fructose concentrations in diabetic in diabetic rat nerve and normalised myo-inositol concentration. Myo-Inositol treatment at 650 mg . kg-1 . day-1 did not affect the elevated concentrations of sorbitol, fructose or glucose in peripheral nerves of diabetic rats, but it did restore reduced myo-inositol concentration. Both sorbinil and myo-inositol treatment partially reversed the slowing of motor-nerve conduction velocity in diabetic rats. These results are discussed in relation to the involvement of sorbitol and myo-inositol metabolism in the aetiology of diabetic neuropathy.

Aldehyde Reductase↗