FSH receptor binding inhibitor influences estrogen production, receptor expression and signal pathway during in vitro maturation of sheep COCs.

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 µg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERß of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERß mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERß protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERß mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs.

Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers.

Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62 µmol/g in the control to 0.7 µmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.

Quantitative analysis of ³¹P NMR spectra of soil extracts--dealing with overlap of broad and sharp signals.

Solution (31)P NMR analysis following extraction with a mixture of sodium hydroxide and ethylenediaminetetraacetic acid is the most widely used method for detailed characterization of soil organic P. However, quantitative analysis of the (31)P NMR spectra is complicated by severe spectral overlap in the monoester region. Various deconvolution procedures have been developed for the task, yet none of these are widely accepted or implemented. In this mini-review, we first describe and compare these varying approaches. We then review approaches to similar issues of spectral overlap in biomedical science applications including NMR-based metabolic profiling and analyzing (31)P magnetic resonance spectra of ex vivo and in vivo intact tissues. The greater maturity and resourcing of this biomedical research means that a wider variety of approaches has been developed. Of particular relevance are approaches to dealing with overlap of broad and sharp signals. Although the existence of this problem is still debated in the context of soil analyses, not only is it well-recognized in biomedical applications, but multiple approaches have been developed to deal with it, including T2 editing and time-domain fitting. Perhaps the most transferable concept is the incorporation of 'prior knowledge' in the fitting of spectra. This is well established in biomedical applications but barely touched in soil analyses. We argue that shortcuts to dealing with overlap in the monoester region (31)P NMR soil spectra are likely to be found in the biomedical literature, although some degree of adaptation will be necessary.

Iron absorption from NaFeEDTA-fortified oat beverages with or without added vitamin C.

BACKGROUND: Food fortification is the best long-term approach for reducing the incidence of iron deficiency. OBJECTIVE: To determine iron absorption from NaFeEDTA-fortified oat beverages without and with vitamin C. MATERIALS AND METHODS: Iron absorption in 19 apparently healthy 6-year-old children was studied. Two oat beverages fortified with iron (labeled with stable isotopes of NaFeEDTA), zinc, and vitamin A, without and with vitamin C was consumed in two consecutive days in random order. Blood samples were taken 14 days later for stable isotope measurements. RESULTS: The mean fractional iron absorption from the fortified oat beverage without vitamin C (5.65 ± 0.54%) was significantly lower than that from the beverage with vitamin C (7.14 ± 0.90%; p < 0.05). CONCLUSION: Fortified oat beverages may offer a convenient and effective mechanism to improve the iron status of children. The addition of vitamin C improved iron absorption by an additional 1.5%.

An integrated approach to the degradation of phytates in the corn wet milling process.

An integrated process was developed to hydrolyze the phytates in light steep water (LSW) and to simultaneously isolate inorganic phosphate (Pi) and myo-inositol products. The proposed integrated process will be helpful in resolving the environmental and nutritional concerns in the use of corn gluten feed (CGF) in the animal diets. This process comprised of partial and total hydrolysis of LSW and intermediate anion exchange separation technique. The phytates in LSW were initially degraded to negatively charged myo-inositol phosphates (InsP(2)-InsP(5)). The optimized experimental parameters for the partial hydrolysis of LSW were determined to be 2 h hydrolysis with 1FTU Aspergillus niger/g substrate at 35 degrees C. The negatively charged species of the partially hydrolyzed substrate were separated on a strong base anion exchange resin. The negatively charged species, retained by the resin, were eluded with 1M NaCl solution and were subjected to complete hydrolysis with the Escherichia coli, A. niger derived phytases and their respective combinations. The maximum amount of myo-inositol released from the anion exchange column was 3.73+/-0.03 mg/NaCl elution which was detected after 48 h reactions catalyzed by 100 FTU E. coli, 150 FTU E. coli, and 150 FTU the combination of A. niger and E. coli. The time course of Pi released showed a similar trend to that of myo-inositol and the released Pi reached a maximum amount of 3.30+/-0.05 mg/g NaCl elution after 48 h incubation at the enzyme loadings for which the maximum concentration of myo-inositol were reached.

Antioxidants in thermally treated buckwheat groats.

The seeds of buckwheat (Fagopyrum esculentum Moench L.) were dehulled and then, following milling, extruded on a counter rotating, twin-screw extruder with the different barrel temperature profiles: 120, 160, and 200 degrees C. After extrusion cooking process, the following compounds were analyzed: free and conjugated phenolic acids, total polyphenols (TPC), tocopherols (T) and tocotrienols (T3), inositol phosphates (IP), reduced glutathione (GSH), and melatonin (MLT). The antioxidant capacity and superoxide dismutase-like activity (SOD-like activity) were determined in the groats and extrudates. Extrusion caused a significant decrease in all the compounds tested, except for phenolic acids. The content of IP decreased by 13%, that of GSH by 42%, and that of T + T3 by 62%. A three-fold lower level of MLT and TPC was noted whereas the SOD-like activity disappeared when compared to the nonextruded material. A two-fold higher content of phenolic acids (free and released from ester bonds) was observed. In spite of the clear decrease in the investigated antioxidants, the extruded dehulled buckwheat seeds contained still significant content of bioactive compounds, which resulted in as little as an average 10% decrease of the antioxidant capacity.

Phosphoinositides, inositol phosphates, and phospholipase C in embryonic stem cells.

The stimulation of inositol phospholipid metabolism via phospholipase C (PLC) is an important signal transduction pathway in a wide variety of cell types. Activation of the pathway is associated with many aspects of cellular activity, including cell growth and differentiation. Activation of hormone-sensitive PLC results in the rapid breakdown of polyphosphoinositides to generate two second messengers: inositol trisphosphate and diacylglycerol. The water-soluble inositol trisphosphate is involved in the release of intracellular calcium from internal stores, whereas the lipophilic diacylglycerol is involved in protein kinase C activation. Inositol supplementation is essential for the in vitro growth of rabbit blastocysts, and studies have shown that the components of the signaling system are present in mouse and cattle embryos and in mouse embryonic stem (ES) cells. In ES cells, the signaling system appears to be constitutively active and essential for normal ES cell proliferation. Here, we describe in detail the materials required and some of techniques involved in studying the phosphoinositide signaling system in mouse ES cells. Furthermore, we describe methods of analyzing the effects of modulating the PtdIns signaling system on ES cell proliferation and the induction of apoptosis.

A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation.

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

Buckwheat concentrate reduces serum glucose in streptozotocin-diabetic rats.

The antihyperglycemic effects of chemically synthesized d-chiro-inositol (d-CI), a component of an insulin mediator, have been demonstrated in rats. Buckwheat contains relatively high levels of d-CI: thus, it has been proposed as a source of d-CI for reducing serum glucose concentrations in diabetics. The present study evaluates the effects of a buckwheat concentrate, containing d-CI, on hyperglycemia and glucose tolerance in streptozotocin (STZ) rats. In fed STZ rats, both doses of the buckwheat concentrate (containing 10 and 20 mg of d-CI/kg of body weight) were effective for lowering serum glucose concentrations by 12-19% at 90 and 120 min after administration. Findings from this study demonstrate that a buckwheat concentrate is an effective source of d-CI for lowering serum glucose concentrations in rats and therefore may be useful in the treatment of diabetes.

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation.

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.

Inositol phosphates in the environment.

The inositol phosphates are a group of organic phosphorus compounds found widely in the natural environment, but that represent the greatest gap in our understanding of the global phosphorus cycle. They exist as inositols in various states of phosphorylation (bound to between one and six phosphate groups) and isomeric forms (e.g. myo, D-chiro, scyllo, neo), although myo-inositol hexakisphosphate is by far the most prevalent form in nature. In terrestrial environments, inositol phosphates are principally derived from plants and accumulate in soils to become the dominant class of organic phosphorus compounds. Inositol phosphates are also present in large amounts in aquatic environments, where they may contribute to eutrophication. Despite the prevalence of inositol phosphates in the environment, their cycling, mobility and bioavailability are poorly understood. This is largely related to analytical difficulties associated with the extraction, separation and detection of inositol phosphates in environmental samples. This review summarizes the current knowledge of inositol phosphates in the environment and the analytical techniques currently available for their detection in environmental samples. Recent advances in technology, such as the development of suitable chromatographic and capillary electrophoresis separation techniques, should help to elucidate some of the more pertinent questions regarding inositol phosphates in the natural environment.

Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.

Anti-nutritional constituents of six underutilized legumes grown in Nigeria.

Six underutilized legume seeds grown in Nigeria namely, red and white lima beans, brown and cream pigeon pea, African yam bean and jackbean were analysed for different anti-nutritional factors Sojasapogenol B was identified as the predominant sapogenol in lima beans and jackbeans by capillary gas chromatography. The content of total inositol phosphates and individual inositol phosphates (IP6, IP5, IP4 and IP3) were analysed by ion-pair HPLC, being in the range of other legumes. Trace quantities of lupanine were identified as the alkaloid in jackbean. alpha-Galactosides were present in all the legume seeds, stachyose being the predominant galactoside in lima beans, African yam bean and jackbean, and verbascose in pigeon pea. The haemagglutinating activity was estimated as a measure of the lectin content of the samples. African yam bean was found to have the highest heamagglutinating activity. Tannins were found to be in low quantities. The presence of these anti-nutrients in relation to the nutritional value of the legume is discussed.

Brain biochemistry in Williams syndrome: evidence for a role of the cerebellum in cognition?

OBJECTIVE: To determine what biochemical changes may occur in the brain in Williams syndrome (WS) and whether these changes may be related to the cognitive deficits. BACKGROUND: WS is a rare, congenital disorder with a characteristic physical, linguistic, and behavioral phenotype with known cognitive deficits. METHODS: We obtained 31P magnetic resonance spectra (MRS) from a region consisting of mostly frontal and parietal lobe of 14 patients with WS (age, 8 to 37 years) and 48 similarly-aged controls. 1H MRS (27 cm3) localized to the left cerebellum obtained from the WS cohort were compared with those from 16 chronological age- and sex-matched normal controls. A battery of cognitive tests were administered to all subjects undergoing 1H MRS. RESULTS: WS brains exhibited significant biochemical abnormalities. All 31P MRS ratios containing the phosphomonoester (PME) peak were significantly altered in WS, suggesting that PME is significantly decreased. Ratios of choline-containing compounds and creatine-containing compounds to N-acetylaspartate (Cho/NA and Cre/NA) were significantly elevated in the cerebellum in WS cf. controls, whereas the ratio of Cho/Cre was not altered. This suggests a decrease in the neuronal marker N-acetylaspartate in the cerebellum. Significant correlations were found between the cerebellar ratios Cho/NA and Cre/NA and the ability of all subjects at various neuropsychological tests, including Verbal and Performance IQ, British Picture Vocabulary Scale, Ravens Progressive Matrices, and Inspection Time. CONCLUSIONS: The correlations can be interpreted in two ways: 1) Our sampling of cerebellar biochemistry reflects a measure of "global" cerebral biochemistry and is unrelated to cerebellar function, or 2) The relations indicate that cerebellar neuronal integrity is a requirement (on a developmental time scale or in real-time) for ability on a variety of cognitive tests.

Effect of natural fermentation on the content of inositol phosphates in lentils.

The effects of natural fermentation upon phytic acid and less phosphorylated inositol phosphates of Lens culinaris var vulgaris cultivar Magda-20 were investigated. Seven fermentation runs were made following a 2(2) complete factorial design with three replicated centre points to study the effect of different conditions of temperature (28, 35 and 42 degrees C) and broth concentration (79, 150 and 221 g/l). Samples were taken for each of them at daily intervals (0, 24, 48, 72 and 96 h). The pH value declined sharply in the first 24 h of fermentation, becoming stabilized from this time. The relation between lactic acid and titratable acidity presented important differences between the different fermentations, ranging from 30-80%. Phytic acid (IP6), inositol pentakis (IP5), tetrakis (IP4) and tris-(IP3) phosphates were quantitatively determined. The content of total inositol phosphates showed a maximum reduction of 63% at 72 h under the fermentation conditions of 42 degrees C and 79 g/l.

Pharmacological evaluation of N-methyl-actinodaphnine, a new vascular alpha-adrenoceptor antagonist, isolated from Illigera luzonensis.

The pharmacological activity of N-methyl-actinodaphnine, isolated from Illigera luzonensis, was determined by functional and binding experiments with peripheral tissues. In a functional study, N-methyl-actinodaphnine was a simple competitive antagonist of contractions elicited by phenylephrine (pA2 = 7.11) in rat thoracic aorta; it also competitively antagonised the clonidine-induced inhibition of the twitch response of rat vas deferens (pA2 = 5.01). In addition, [3H]inositol monophosphate formation caused by noradrenaline (3 microM) in rat isolated thoracic aorta was concentration dependently inhibited by N-methyl-actinodaphnine (1 and 10 microM); however, it had no effect on cyclic AMP and cyclic GMP contents. Additionally, N-methyl-actinodaphnine had extremely low affinity for thromboxane receptors, prostaglandin receptors, Ca2+ channels, muscarinic receptors, histamine receptors, beta-adrenoceptors, neurokinin and leukotriene receptors in vitro. However, N-methyl-actinodaphnine also possessed 5-hydroxytryptamine (5-HT) receptor blocking activity. Its potency for blocking 5-HT receptors was about 14 times less than that for blocking alpha 1-adrenoceptors. In binding experiments, N-methyl-actinodaphnine displaced biphasically the binding of 0.2 nM [3H]prazosin to cultured A10 cells. The selectivity for alpha 1-adrenoceptor subtypes was also investigated in rat vas deferens and spleens. The contractile response in rat vas deferens to noradrenaline was competitively inhibited by N-methyl-actinodaphnine with a pA2 value of 6.58; N-methyl-actinodaphnine also competitively antagonized the phenylephrine-induced contraction in rat spleen with a pA2 value of 7.38. These results indicate that N-methyl-actinodaphnine is a selective alpha 1-adrenoceptor antagonist. Furthermore, it is more selective for the alpha 1B- than for the alpha 1A-adrenoceptor subtype.

Dietary 1,25-dihydroxycholecalciferol supplementation increases natural phytate phosphorus utilization in chickens.

These studies were conducted to determine if supplementation of a corn-soybean meal diet with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] would increase the utilization of natural phytate phosphorus by broiler chickens. Two experiments were conducted to evaluate the effect of dietary 1,25-(OH)2D3 in the presence and absence of supplemental phytase and at several dietary levels of inorganic phosphorus supplementation. The criteria measured in these studies were weight gain, gain:feed ratio, bone ash, rickets due to phosphorus deficiency, plasma calcium and phosphorus and retention of calcium, phosphorus and phytate phosphorus. In the first experiment, the types and amounts of fecal inositol phosphates were determined by HPLC, and the total fecal phytate was determined by the classic FeCl3 precipitation technique. In the first experiment, the addition of 1,25-(OH)2D3 to the diet in the presence of dietary phytase resulted in greater 9-d weight and bone ash and lower incidence of rickets; the retention of total fecal phytate and phytate phosphorus was greater than in controls. The second experiment was a complete 2 x 2 x 2 factorial design [phosphorus levels x phytase x 1,25-(OH)2D3]. The addition of 1,25-(OH)2D3 alone to the diet resulted in greater 9-d weight and bone ash, lower incidence of rickets, and greater retention of total calcium and phosphorus and phytate phosphorus. The highest retention of phytate phosphorus (79.4%) was obtained when both phytase and 1,25-(OH)2D3 were present in the diet. The possible mode of action and importance of these results in many areas of nutrition and environmental science are discussed.

Activation of calcium oscillations by thapsigargin in parotid acinar cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.

High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates. Separation of myo-inositol phosphates, some common nucleotides and sugar phosphates.

A detailed study of all the major chromatographic variables affecting the retention behaviour and separation of myo-inositol phosphates in reversed-phase ion-pair chromatographic systems was carried out. The parameters studied included the eluent concentration of the pairing ion, the eluent concentration of the organic modifier and the buffer salt, the pH of the eluent, the minimum column plate count necessary for the separation of the inositol trisphosphate isomers and isocratic and gradient modes of separation. The retention behaviour of some common nucleotides and sugar phosphates was also investigated as these phosphates present chromatographic interference problems in biochemical studies based on the cellular incorporation of [32P]Pi. The separation methods developed appear to be superior to established anion-exchange separation techniques in terms of separation speed and "mildness" of the chromatographic conditions.