Concentrations Of Inositol Phosphates Research Articles

Amino acid and mineral digestibility, bone ash, and plasma inositol is increased by including microbial phytase in diets for growing pigs

BackgroundThe effect of microbial phytase on amino acid and energy digestibility is not consistent in pigs, which may be related to the phytase dosage or the adaptation length to the diet. Therefore, an experiment was conducted to test the hypotheses that increasing dietary phytase after an 18-day adaptation period: 1) increases nutrient and energy digestibility; 2) increases plasma P, plasma inositol, and bone ash of young pigs; and 3) demonstrates that maximum phytate degradation requires more phytase than maximum P digestibility.ResultsData indicated that increasing inclusion of phytase [0, 250, 500, 1,000, 2,000, and 4,000 phytase units (FTU)/kg feed] in corn-soybean meal-based diets increased apparent ileal digestibility (AID) of Trp (quadratic; P < 0.05), and of Lys and Thr (linear; P < 0.05), and tended to increase AID of Met (linear; P < 0.10). Increasing dietary phytase also increased AID and apparent total tract digestibility (ATTD) of Ca and P (quadratic; P < 0.05) and increased ATTD of K and Na (linear; P < 0.05), but phytase did not influence the ATTD of Mg or gross energy. Concentrations of plasma P and bone ash increased (quadratic; P < 0.05), and plasma inositol also increased (linear; P < 0.05) with increasing inclusion of phytase. Reduced concentrations of inositol phosphate (IP)6 and IP5 (quadratic; P < 0.05), reduced IP4 and IP3 (linear; P < 0.05), but increased inositol concentrations (linear; P < 0.05) were observed in ileal digesta as dietary phytase increased. The ATTD of P was maximized if at least 1,200 FTU/kg were used, whereas more than 4,000 FTU/kg were needed to maximize inositol release.ConclusionsIncreasing dietary levels of phytase after an 18-day adaptation period increased phytate and IP ester degradation and inositol release in the small intestine. Consequently, increasing dietary phytase resulted in improved digestibility of Ca, P, K, Na, and the first 4 limiting amino acids, and in increased concentrations of bone ash and plasma P and inositol. In a corn-soybean meal diet, maximum inositol release requires approximately 3,200 FTU/kg more phytase than that required for maximum P digestibility.

Phytase dose-dependent response of kidney inositol phosphate levels in poultry.

Phytases, enzymes that degrade phytate present in feedstuffs, are widely added to the diets of monogastric animals. Many studies have correlated phytase addition with improved animal productivity and a subset of these have sought to correlate animal performance with phytase-mediated generation of inositol phosphates in different parts of the gastro-intestinal tract or with release of inositol or of phosphate, the absorbable products of phytate degradation. Remarkably, the effect of dietary phytase on tissue inositol phosphates has not been studied. The objective of this study was to determine effect of phytase supplementation on liver and kidney myo-inositol and myo-inositol phosphates in broiler chickens. For this, methods were developed to measure inositol phosphates in chicken tissues. The study comprised wheat/soy-based diets containing one of three levels of phytase (0, 500 and 6,000 FTU/kg of modified E. coli 6-phytase). Diets were provided to broilers for 21 D and on day 21 digesta were collected from the gizzard and ileum. Liver and kidney tissue were harvested. Myo-inositol and inositol phosphates were measured in diet, digesta, liver and kidney. Gizzard and ileal content inositol was increased progressively, and total inositol phosphates reduced progressively, by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D-and/or L-Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5, differed from those (D-and/or L-Ins(1,2,3,4)P4, D-and/or L-Ins(1,2,5,6)P4, Ins(1,2,3,4,6)P5, D-and/or L-Ins(1,2,3,4,5)P5 and D-and/or L-Ins(1,2,4,5,6)P5) generated from phytate (InsP6) degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively by phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rate and that such effects are tissue specific, though the consequences for physiology of such changes have yet to be elucidated.

Apparent digestibility of energy and nutrients and efficiency of microbial phytase is influenced by body weight of pigs.

An experiment was conducted to test the hypothesis that regardless of pig body weight (BW), increasing dietary phytase results in increased phytate degradation and improved digestibility of minerals, amino acids (AA), and gross energy (GE). Eighteen pigs were equipped with a T-cannula in the distal ileum and allotted to a triplicated 6 × 3 Youden square design with six diets and three collection periods of 7 d, for a total of nine replicate pigs per diet. This design was repeated four times to simulate four production phases, and there was a 7-d resting period before each collection phase started (BW at start of collections: 29.3, 53.6, 85.1, and 114.4 kg for phases 1, 2, 3, and 4, respectively). Six corn-soybean meal diets were formulated by including 0, 250, 500, 1,000, 2,000, or 4,000 phytase units/kg feed (FTU). The six diets were used throughout the experiment. Samples of feces and ileal digesta were collected in each period. Results indicated that regardless of pig BW, increasing inclusion of phytase increased (quadratic; P < 0.05) apparent ileal digestibility (AID) of crude protein (CP) and most AA, increased apparent total tract digestibility (ATTD) of Ca, P, K, Mg (linear and quadratic; P < 0.05), and Na (linear; P < 0.05), but decreased (linear and quadratic; P < 0.05) AID and ATTD of GE. In all phases, ileal concentrations of inositol phosphate (IP) 6, IP5, IP4, and IP3 decreased (linear and quadratic; P < 0.05), whereas ileal inositol increased (linear and quadratic; P < 0.05) with increasing dietary phytase. However, as pig BW increased, AID of GE, CP, and AA increased (linear, P < 0.05), and the AID of a few AA (Met, Phe, Thr, Trp, Ala, Asp, Gly, and Ser) also increased quadratically (P < 0.05). The ATTD of GE, K, and Mg increased (linear and quadratic; P < 0.05), but ATTD of Ca and Na (linear; P < 0.05) and of P (linear and quadratic; P < 0.05) decreased as pig BW increased. Ileal IP6 and IP3 (linear and quadratic; P < 0.05) and ileal IP5 and IP4 (linear; P < 0.05) increased, whereas ileal inositol decreased (linear; P < 0.05) as pig BW increased. In conclusion, regardless of pig BW, increasing dietary phytase increased phytate degradation and inositol release in the small intestine, and consequently increased mineral and AA digestibility. Older pigs have reduced Ca, P, and Na digestibility, but increased K, Mg, AA, and GE digestibility compared with younger pigs. The efficiency of dietary phytase to degrade phytate appears to decrease as pigs get older.

Effect of low-temperature drying on the nitrogenous compounds and inositol phosphates in broiler chickens and cecectomized laying hen excreta.

We investigated how the chemical composition of broiler chicken and cecectomized laying hen excreta is affected by drying in a forced-air drying chamber at low temperatures. Excreta that was immediately frozen after voiding provided the reference values. The excreta were dried in drying chambers for 4hr, 6hr, and 12hr at 23°C or 33°C in the broiler experiment and 19°C or 29°C in the cecectomized laying hen experiment. The total N and inositol phosphate concentrations in the excreta of broiler chickens and cecectomized laying hens were not influenced (p>.050), except for one inositol tetrakisphosphate isomer (p=.026) in broilers. Compared to fresh excreta, drying did not affect the ammonia concentrations in the cecectomized laying hen experiment (p>.050), but the ammonia concentration was lower when dried for 12hr at 33°C in the broiler experiment (p=.002). Amino acid concentrations in cecectomized laying hen excreta decreased until 4hr of drying and then increased at both drying temperatures (p<.001). The results indicate that the applicability of drying poultry excreta at low temperatures in forced-air drying chambers to determine the chemical compound concentrations is trait-dependent. Future studies are necessary to investigate whether these results are also dependent upon the amount of excreta stored in the drying chambers.

Identification of lower-order inositol phosphates (IP&amp;lt;sub&amp;gt;5&amp;lt;/sub&amp;gt; and IP&amp;lt;sub&amp;gt;4&amp;lt;/sub&amp;gt;) in soil extracts as determined by hypobromite oxidation and solution &amp;lt;sup&amp;gt;31&amp;lt;/sup&amp;gt;P NMR spectroscopy

Abstract. Inositol phosphates (IPs) are a major pool of identifiable organic phosphorus (P) in soil. However, insight into their distribution and cycling in soil remains limited, particularly of lower-order IP (IP5 and IP4). This is because the quantification of lower-order IP typically requires a series of chemical extractions, including hypobromite oxidation to isolate IP, followed by chromatographic separation. Here, for the first time, we identify the chemical nature of organic P in four soil extracts following hypobromite oxidation using solution 31P NMR spectroscopy and transverse relaxation (T2) experiments. Soil samples analysed include A horizons from a Ferralsol (Colombia), a Cambisol and a Gleysol from Switzerland, and a Cambisol from Germany. Solution 31P nuclear magnetic resonance (NMR) spectra of the phosphomonoester region in soil extracts following hypobromite oxidation revealed an increase in the number of sharp signals (up to 70) and an on average 2-fold decrease in the concentration of the broad signal compared to the untreated soil extracts. We identified the presence of four stereoisomers of IP6, four stereoisomers of IP5, and scyllo-IP4. We also identified for the first time two isomers of myo-IP5 in soil extracts: myo-(1,2,4,5,6)-IP5 and myo-(1,3,4,5,6)-IP5. Concentrations of total IP ranged from 1.4 to 159.3 mg P per kg soil across all soils, of which between 9 % and 50 % were comprised of lower-order IP. Furthermore, we found that the T2 times, which are considered to be inversely related to the tumbling of a molecule in solution and hence its molecular size, were significantly shorter for the underlying broad signal compared to for the sharp signals (IP6) in soil extracts following hypobromite oxidation. In summary, we demonstrate the presence of a plethora of organic P compounds in soil extracts, largely attributed to IPs of various orders, and provide new insight into the chemical stability of complex forms of organic P associated with soil organic matter.

Phytate and phosphorus utilization by broiler chickens and laying hens fed maize-based diets

Maize grain is primarily used as an energy source for poultry and other animals. Maize has relatively high phytate-P content and very low intrinsic phytase activity. Given that feed phosphates are produced from finite rock phosphate resources, a reduction in the use of feed phosphates in maize-based diets by increasing the utilization of plant P sources by animals is necessary to make poultry meat and egg production more sustainable. The utilization of P by poultry is affected by two intrinsic characteristics of maize: the concentration of inositol phosphates and the activity of the intrinsic phytase of the grain in the digestive tract. The objective of this review is to present data on the variation that exists in composition of maize relevant for P use and to address factors that influence P utilization in maize-based diets of poultry. Broiler chickens and laying hens have the potential to degrade phytate in the gastrointestinal tract, but this is depressed by high dietary Ca and P concentrations. Published values of phytate degradation in broilers are overall higher than those in laying hens. Differences also exist between broiler chickens and growing turkeys and Pekin ducks. The exogenous supplementation of microbial phytases and the introduction of transgenic high phytase maize in poultry diets are efficient not only for the improvement of phytate-P digestibility, production performance, egg quality and bone mineralization, but also for the reduction of P excreta to control environmental impact.

Influence of pH and redox on mobilization of inositol hexakisphosphate from oligotrophic lake sediment

It has been suggested that inositol hexakisphosphate (IP6) contributes to the release of phosphorus (P) from lake sediments, but a mechanistic understanding remains elusive. We investigated the potential mobilization and mineralization of myo- and scyllo-IP6 from the sediment of an oligotrophic Danish lake known to contain high concentrations of inositol phosphates. Solution 31P NMR spectroscopy was used to determine changes in myo- and scyllo-IP6 in laboratory microcosms incubated under either oxic or anoxic conditions. In addition, we incubated sediment slurries adjusted to pH between 4.9 and 6.6, with and without addition of myo-IP6, and induced redox changes by adding starch and sulfate. We observed no significant changes in myo- or scyllo-IP6 after 1 year of incubation under anaerobic conditions. A sequential extraction procedure revealed that one half of the added myo-IP6 was recovered in the humic acid fraction (acid-insoluble organic matter) and the other half in the fulvic acid fraction (acid-soluble organic matter). Reduction in redox potential by starch addition did not mobilize myo-IP6, but myo-IP6 bound to humic acids was released to the pore water when the pH was increased to ≥ 5.8. This pH-induced mobilization of IP6 occurred in parallel with increases in dissolved iron and organic matter, suggesting the release of IP6 bound to humic acids through metal bridges. We conclude that myo-IP6 mobilization from this oligotrophic lake sediment is driven by changes in pH rather than by changes in the redox potential.

Effects of a high level of phytase on broiler performance, bone ash, phosphorus utilization, and phytate dephosphorylation to inositol.

An experiment was conducted to evaluate how the addition of microbial phytase influenced growth performance, bone mineralization, tissue P content, apparent digestibility and retention, and inositol phosphate (IP) concentrations in broilers fed diets with varying mineral matrices from 2 to 23 d of age. At 2 d of age, chicks were randomly allotted to receive 1 of 6 experimental diets arranged as a 3 × 2 factorial of mineral matrix [control diet with 1.0% Ca and 0.5% non-phytate phosphorus (NPP); mineral matrix 1 with 0.84% Ca and 0.35% NPP; and mineral matrix 2 with 0.77% Ca and 0.29% NPP] and phytase supplementation (0 or 1,500 FTU/kg). Feed intake was influenced (quadratic, P = 0.012) by the mineral matrix, but no interaction or main effect of phytase were observed. Phytase increased (P = 0.011) BW gain regardless of the mineral matrix applied. Feed efficiency was not influenced (P >0.05) by mineral matrix, phytase, or their interaction. Phytase increased bone ash content differentially across matrices (interaction, P <0.01), and tibia P content was lowest in birds fed matrix 2 and highest in the control (linear, P <0.05). Concentrations of P in muscle, spleen, and liver were not affected by treatment. An interactive effect (P <0.01) was observed for apparent ileal digestibility (AID) of P, where phytase increased AID in matrix 1. An interactive effect (P <0.01) was observed for apparent retention of P and Ca, where phytase reduced P and Ca retention in the control diet. A main effect (P <0.01) of mineral matrix was observed for AID of Ca, with birds fed matrix 1 having the lowest AID of Ca compared with control and matrix 2 treatments. Phytase influenced (P <0.05) IP concentrations differently across matrices. Overall, phytase and the mineral matrix, either as main effects or in an interactive manner, influenced growth performance, apparent nutrient digestibility and retention, bone and IP concentration responses in broiler chicks.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.

Suppression of Thyrotropin Receptor-G Protein-Phospholipase C Coupling by Activation of Protein Kinase C in Thyroid Carcinoma Cells*

In human thyroid follicular cells TSH exerts its action on growth and function at least via two distinct pathways, the adenylate cyclase cascade and the phospholipase Cbeta (PLCbeta)-mediated inositol phosphate generation. We investigated the effect of TSH on activation of phosphoinositide hydrolysis and inositol phosphate generation by PLCbeta in HTh74 thyroid carcinoma cells that express functional TSH receptors and in HTC-TSHr thyroid carcinoma cells that are devoid of endogenous TSH receptors but express recombinant human TSH receptors. In both cell lines, TSH up to concentrations of 300 mU/ml failed to stimulate myo-inositol 1,4,5-trisphosphate and myo-inositol-tetrakisphosphate generation, but led to a decrease in these compounds within 1 min of stimulation. However, ATP and bradykinin increased concentrations of inositol phosphates in both thyroid carcinoma cell lines. In contrast, in differentiated FRTL5 thyroid cell line and CHO-TSHr cell line expressing recombinant human TSH receptors, TSH elicited a significant increase in myo-inositol 1,4,5-trisphosphate and its metabolic derivatives. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of protein kinase C, a TSH concentration of 20 mU/ml enhanced generation of inositol phosphates in these cells. From our data we conclude that in HTC-TSHr and HTh74 thyroid carcinoma cells, the coupling within the TSH receptor-Gq protein-PLCbeta signaling pathway is impaired compared to that in nontransformed cells. It is conceivable that this is at least in part dependent on the level of protein kinase C activation in these cells.

Inositol Phosphate Content of Selected Dry Beans, Peas, and Lentils, Raw and Cooked

Fourteen varieties of dry legumes available in local supermarkets were analyzed for inositol phosphate profile in the raw state and after cooking. Inositol tris-, tetrakis-, pentakis-, and hexakisphosphate (IP3–IP6) were determined by a HPLC procedure. The concentration of inositol phosphates in raw dry legumes did not differ significantly among three different brands. Phytic acid (IP6) concentration (per kg dry basis) ranged from 6.0 mmol in chickpeas to 14.2 mmol in black beans. Phytic acid was the predominate inositol phosphate of the total inositol phosphates determined in raw dry legumes, ranging from 77% in chickpeas and pigeon peas to 88% in black beans. The remainder of the inositol phosphates in raw, dry legumes were IP4and IP5, except lentils contained, in addition, a detectable amount of IP3. The cooked legumes all contained detectable amounts of IP3, increased amounts of both IP4and IP5, but lower amounts of IP6in comparison to the raw dry legume (differences between uncooked and cooked were statistically significant,P< 0.05). The concentration of IP6in the cooked legumes averaged 83%, ranging from 68% in red kidney beans to 86% in chickpeas, of the concentrations in the raw dry legumes. Phytic acid remained the predominate inositol phosphate in cooked legumes, averaging 68% of the total and ranging from 60% in pigeon peas to 78% in yellow split peas. Quick soak vs overnight soak of beans before cooking resulted in no difference in inositol phosphate concentration or profile of cooked beans. Although the total inositol phosphates did not differ significantly between raw and cooked dry legumes (P> 0.1), the IP6and IP5+ IP6represented a smaller percentage of the total in the cooked legumes, suggesting that cooking decreases the potential adverse impact of inositol phosphates on mineral utilization when legumes are included in the diet.

Inositol Phosphate, Calcium, Magnesium, and Zinc Contents of Selected Breakfast Cereals

The concentrations in selected breakfast cereals of inositol tris-, tetra-, penta-, and hexakisphosphate (IP3, IP4, IP5, IP6) were determined by HPLC methodology. In addition, the calcium, magnesium, and zinc concentrations were determined by atomic absorption spectrometry. All cereals examined contained each of the inositol phosphate (IP) species. Wheat and oat cereals contained an overall average of 5 and 8 μmol/g, respectively, of IP6, representing 46 and 65% of total IP. The mean IP6 content of bran or bran added cereals was 13 μmol/g, 65% of the total IP content. Corn, rice, and most multigrain-based cereals contained lower concentrations of IPs. Whole grain or bran cereals to which no source of Ca or Zn was added averaged 10.9 μmol (0.44mg)/g of Ca, 0.48 μmol (0.032 mg)/g of Zn, and 57.1 μmol (1.4 mg)/g of Mg. Corn and rice cereals contained lower amounts of Ca, Zn, and Mg. Calculated indices of bioavailability, such as phytate/Zn and phytate/Ca molar ratios are most meaningful if based on values for IP6 or IP6 + IP5 rather than total IPs.

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

Inducers of the heat shock response stimulate phospholipase C and phospholipase A2 activity in mammalian cells.

Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat-shocked cells of phosphoinositide-specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shock, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response.

Flow-related responses of intracellular inositol phosphate levels in cultured aortic endothelial cells.

In vitro and in vivo evidence indicates that hemodynamic wall shear stress evokes a diversity of biological responses in vascular endothelial cells, ranging from cell shape changes to alterations in low density lipoprotein receptor expression. The signal transduction mechanisms by which the level of fluid mechanical shear stress is recognized by the endothelial cell and translated into these diverse biological responses remain to be elucidated. The present study focuses on the association between the onset of elevated shear stress and activation of the phosphoinositide signal transduction pathway, as measured by the intracellular release of inositol phosphates, in cultured bovine aortic endothelial cells (BAECs). BAECs were seeded, grown to confluence on large polyester sheets, and preincubated with 0.3 microCi/ml [3H]inositol for 24 hours before insertion in parallel-plate flow chambers for exposure to high shear stress (HS) at 30 dynes/cm2 or low shear stress (LS) at < 0.5 dyne/cm2 for periods ranging from 15 seconds to 24 hours. The induction of HS was associated with an early, transient but significant increase (142%, HS/LS x 100%) in inositol trisphosphate (IP3) measured at 15 seconds of shear stress exposure followed by a major peak in IP3 (189%) observed at 5 minutes after HS onset. After these initial increases, IP3 levels returned to near resting levels within 30 minutes of continued HS exposure and then continued to decline to significantly lower (75%) levels relative to LS-treated cells within 4 hours and remained lower throughout the remainder of the 24-hour HS exposure. LS-treated cells exhibited no significant changes in inositol phosphate levels throughout the 24-hour exposure periods. Exposure of BAECs to shear stress of 60 dynes/cm2 resulted in an approximately fourfold increase in IP3 levels (396%) measured at 5 minutes, almost double the levels measured in cells exposed to 30 dynes/cm2 for 5 minutes. Pretreatment of BAECs for 30 minutes with 5 mM neomycin, an inhibitor of phosphoinositide metabolism, before HS exposure inhibited both the early increases in inositol phosphates and subsequent cell elongation and alignment observed in untreated BAECs simultaneously exposed to HS without inhibiting protein synthesis. These results indicate that the exposure of cultured BAECs to elevated wall shear stress is associated with an early biphasic IP3 increase followed by a resetting of intracellular inositol phosphate concentrations to levels below that observed in static cultured BAECs. Furthermore, neomycin inhibition of this IP3 response to shear stress is associated with an inhibition of one of the major endothelial biological responses to shear stress, i.e., cell shape change and orientation.(ABSTRACT TRUNCATED AT 400 WORDS)

Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangment of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA / AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.

Salmonella typhimuriuminduces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangment of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA / AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.

Bradykinin stimulates DNA synthesis in competent Balb/c 3T3 cells and enhances inositol phosphate formation induced by platelet- derived growth factor

Both platelet-derived growth factor (PDGF) and bradykinin were found to induce a growth response in Balb/c 3T3 cells. However, whereas PDGF brought about a five-fold increase in the incorporation of [ 3H]thymidine into DNA, the response to bradykinin was never more than 50%. When bradykinin was present simultaneously with sub-optimal concentrations of PDGF the response was about 15% greater than with PDGF alone. In contrast, if the cells were made competent by a 5 hr preincubation with PDGF which was then washed away, subsequent addition of bradykinin induced a more than two-fold increase in incorporation of [ 3H]thymidine into DNA compared with competent cells subsequently incubated with serum-free medium alone. Bradykinin also acted synergistically with insulin when the two agents were added simultaneously to competent cells. PDGF induced marked increases in the concentration of inositol phosphates at 30 min after stimulation, but by this time point any effect of bradykinin had disappeared. However, the simultaneous presence of PDGF and bradykinin induced increases at 30 min that were 50–100% greater than with PDGF alone. It is concluded that the pathways by which PDGF and bradykinin initiate a growth response in BALB/c 3T3 cells only partly overlap. Their actions on the synthesis of inositol phosphates exhibit distinctive temporal characteristics, but can be co-operative at 30 min and at earlier time intervals. This effect was found to be time-dependent, and developed over the first 5 min.

Decapitation-induced changes in inositol phosphates in rat brain

Decapitation resulted in a time-dependent production of inositol phosphates in rat brain. This production was analyzed by measuring both the radioactivity and the concentrations of inositol phosphates generated from [3H]inositol-labeled phospholipids. Both measurements produced the same time-dependent changes, including a rapid decrease in inositol 1,4,5-trisphosphate within 1.5 min, a 6-fold increase in inositol 1,4-bisphosphate to a maximum at 1.5 min, a 5-fold rise in inositol 4-monophosphate to a maximum at 2.5 min, and little change in inositol 1-monophosphate. The temporal changes in the mass and radioactivity of these compounds, together with the decrease in labeling of phosphatidylinositol 4,5-bisphosphates, support the idea that the inositol phosphates originate from the hydrolysis of phosphatidylinositol 4,5-bisphosphates and not from either the direct hydrolysis of phosphatidylinositol 4-phosphates or phosphatidylinositols.