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Helicobacter pylori is a notorious human pathogen and the appearance of antibiotic resistance of this bacterium has posed a serious threat to human health. Lipopolysaccharide (LPS) is a key virulence factor and plays important roles in pathogenesis of H. pylori infection. Sedoheptulose 7-phosphate isomerase (GmhA), as an enzyme participating in the first step of heptose biosynthesis, is indispensable for the formation of inner core oligosaccharide of LPS. In this study, we cloned one putative gmhA ortholog, hp0857, from H. pylori 26695 and overexpressed it in Eschericha coli. Based on the results of molecular weight determination, the recombinant HP0857 is likely a homodimer. Analysis of enzymatic kinetic properties of this protein confirmed that hp0857 is indeed encoded a phosphoheptose isomerase which can utilize sedoheptulose 7-phosphate as the substrate in the ADP-L-glycero-D-manno-heptose (ADP- L,D-Hep) biosynthesis pathway. We also generated an HP0857 knockout mutant and explored its phenotypic changes. This mutant exhibited a decreased growth rate and displayed a "deep rough" type of LPS structure. In addition, it also had a slight decrease in its motility and was more susceptible to hydrophobic antibiotic novobiocin and detergents Triton X-100 and SDS. Furthermore, the adhesive capacity of the HP0857 knockout mutant to AGS cells was reduced significantly, and most of the infected cells didn't show a classic hummingbird phenotype. However, complementation of the HP0857 knockout mutation restored most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for inner core biosynthesis of H. pylori LPS and is a potential target for developing new antimicrobial agents against H. pylori infection.

The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme. To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E. coli and yeast transketolase (TK). Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity.

The kdsA gene from the hyperthermophilic bacterium Aquifex aeolicus was cloned into a vector for expression in Escherichia coli and the kdsA gene product, 3-deoxy-d-manno-octulosonic acid 8-phosphate synthase (KdsA), was overexpressed under optimized growth conditions. The thermophilic KdsA was purified using an efficient purification procedure including a heat-treatment step. Purified KdsA was shown to catalyze the formation of 3-deoxy-d-manno-octulosonic acid 8-phosphate from phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A 5-P) as determined from (1)H NMR analysis of the product. Analytical gel filtration analysis indicated the native enzyme to be oligomeric. KdsA was extremely thermostable, exhibiting maximal activity at 95 degrees C and with half-lives of 1.5 h (90 degrees C), 8.1 h (80 degrees C), and 30.3 h (70 degrees C). KdsA appeared to follow Michaelis-Menton kinetics with K(A5-P)(m) = 8 - 74 microM, K(PEP)(m) = 43-28 microM, and k(cat) = 0.4-2.0 s(-1) between 60 and 90 degrees C.

To clarify roles of intermediate metabolites of the glycolytic pathway and the polyol pathway in nonenzymatic glycation under physiological conditions, we incubated bovine serum albumin with intermediates of both pathways in the micromolar range as well as with 20 mmol/l glucose, and observed the formation of advanced glycation end products (AGEs). We found that triose phosphates, glyceraldehyde, and a novel polyol pathway-related metabolite, fructose 3-phosphate along with its breakdown product, 3-deoxyglucosone were extremely potent glycating agents that at nearly physiological concentrations on incubation with albumin produced substantial amounts of AGEs as early as 24 hours, while 20 mmol/l glucose afforded trace amounts of AGEs after two week incubation. The results along with the previous evidence of the increased level of intermediates in diabetic states may suggest that the intermediate metabolites rather than glucose contribute to enhanced glycation in diabetic tissues, inspite of the much lower concentrations compared with glucose.

Sugar phosphates are required to maintain active rates of translation in gel-filtered rabbit reticulocyte lysates. They may stimulate polypeptide chain initiation by acting as NADPH generators or by a direct interaction with initiation factor(s). We now provide evidence for the allosteric activation of the purified guanine nucleotide exchange factor (eIF-2B) by sugar phosphates and inositol phosphates. In the presence of microM fructose 1,6-bisphosphate, the rate of eIF-2B-catalyzed GDP/GTP exchange is increased approximately 2-fold. The half-maximal concentration for stimulation of eIF-2B activity (SC50) is 57 microM. The binding of GTP to isolated eIF-2B is stimulated 1.5-fold, whereas GTP-binding to ALP-treated eIF-2B is not affected by sugar phosphates. Inositol 1,4-bisphosphate, like fructose 1,6-bisphosphate, stimulates 2-3-fold the activity of the isolated eIF-2B (SC50, 140 microM).

Rabbit muscle aldolase was found to be inactivated in a slow, reversible manner by D-erythrulose 1-phosphate. This compound combined rapidly and reversibly with the enzyme to form an initial complex, which then only slowly (ki = 0.28 min-1) converted to a kinetically more stable form. This stable enzyme-ligand form was inactive toward the normal substrate of aldolase, fructose 1,6-bisphosphate. The inactive enzyme-ligand complex, however, could be decomposed (kr = 0.0041 min-1) to yield active enzyme once again by incubation in a solution devoid of D-erythrulose 1-phosphate.

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when e is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)33+ in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B–A conformation transition takes place at more than 60% ethanol solution.

For the detailed analysis of energy metabolism, a HPLC method is described allowing the single-run separation and quantification of most metabolites from glycolysis and the Krebs cycle including the high energy phosphates. With a detection limit in the picomolar range this method is even applicable when only small sample sizes of tissue are obtained.

The steady-state and dynamic behavior of a partial glycolytic reaction sequence are investigated in cell-free extracts of yeast. Pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase cooperate to a multienzyme system centered around the 6-phosphofructokinase (6-PFK) and fructose 1,6-bisphosphatase (FBPase) cycle. The reaction system operates under thermodynamically open conditions maintained by a continuous supply of substrates, i.e., glucose 6-phosphate (Glc6P), ATP and phosphoenolpyruvate (PPrv) in a flow-through reaction chamber. Appropriate conditions lead to the occurence of (two) coexisting and markedly different time-independent states in the metabolite concentrations and fluxes. For particular experimental conditions, changes in the influx adenylic energy charge, [AEC]IN, may cause transitions between these alternative steady states which are either reversible as it occurs in classical hysteresis phenomena, or, more importantly, irreversible (irreversible transitions, IT) where the system is not able to switch back to its previous state even when the perturbation is reverted. The emergence of these irreversible transitions do not result from artificial or non-realistic experimental constraints, but are a potential intrinsic property of any non-linear dynamic system exhibiting bi- or multistability. These one-way transitions may well have important biological implications with respect to switching, adaptation and memory phenomena.

The capacity to modify the incorporation of [2-3H]myo-inositol into inositides and inositol phosphates was different for three psychotropic cationic amphiphilic drugs. Chlorpromazine, desmethylimipramine and propranolol were able to increase the labeling of inositol-containing lipids, but only chlorpromazine dramatically increased the incorporation into inositol phosphate, -bisphosphate and -trisphosphate. The increase was 10- to 50-fold in 60 min as compared with controls. This effect is not due to stimulation of lipid labeling, because in chase experiments radioactivity in inositol phosphates increased to a greater extent than in their parent lipids. It is possible that the alteration of phosphoinositide catabolism is related to the neuroleptic activity of the drug.

Several inositol trisphosphate isomers and inositol tetrakisphosphate activate a rat brain phosphoprotein phosphatase, using phosphohistone as well as phosphorylase kinase as substrate. Inositol mono- and bisphosphate have no effect. The protein phosphatase may correspond to type-1 since it is associated with the particulate fraction and is inhibited by heparin. Evidence is presented for the target of inositol phosphate being the catalytic subunit of the protein phosphatase. A parallelism is observed between the ability of the several inositol trisphosphates to activate the protein phosphatase and reported data indicating their ability to release calcium in permeabilized cells.

Clonal neurohybridoma NCB-20 cells expressed muscarinic cholinergic receptors coupled to phospholipase C. Addition of carbachol in the presence of Li+ to cells prelabeled with 3H-inositol increased 3H-inositol-l-phosphate (3H-IP1) accumulation by more than 4-fold with an EC50 of about 50 microM. This carbachol-induced response was blocked by atropine and pirenzepine with a Ki of 0.5 and 25 nM, respectively. The EC50 of Li+ for the carbachol-induced phosphoinositide turnover was 17 +/- 1.2 mM compared with a value of 1.8 +/- 0.2 mM in brain slices, suggesting the presence of an unusual type of inositol-l-phosphatase in NCB-20 cells. Carbachol-induced IP1 accumulation in these cells was potently and noncompetitively inhibited by the biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA), while the biologically inactive phorbol, 4 beta-phorbol, failed to affect this phosphoinositide breakdown. The basal IP1 accumulation was also significantly attenuated by PDB and PMA but not by 4 beta-phorbol.

In analyzing the 31P NMR spectra of extracts of mammalian tissues and cells we have identified inositol pentakis- and hexakis-phosphates in essentially all of the samples examined. These compounds were present at concentrations of at least 5-15 microM. While the sources and functions of these compounds in mammalian cells are not clear, they may play an important role in phosphoinositol metabolism. For example, one obvious possibility is that these compounds may be sources of or sinks for the Ca++ mobilizing inositol tris- and tetrakis-phosphates.

Angiotensin II stimulates the formation of several inositol polyphosphates in cultured bovine adrenal glomerulosa cells prelabelled with [3H] inositol. Analysis by high performance anion exchange chromatography of the inositol-phosphate compounds revealed the existence of two additional inositol tetrakisphosphate (InsP4) isomers in proximity to Ins-1,3,4,5-P4, the known phosphorylation product of Ins-1,4,5-trisphosphate and precursor of Ins-1,3,4-trisphosphate. Both of these new compounds showed a slow increase after stimulation with angiotensin II. The structure of one of these new InsP4 isomers, which is a phosphorylation product of Ins-1,3,4-P3, was deduced by its resistance to periodate oxidation to be Ins-1,3,4,6-P4. The existence of multiple cycles of phosphorylation-dephosphorylation reactions for the processing of Ins-1,4,5-P4 may represent a new aspect of the inositol-lipid related signalling mechanism in agonist-activated target cells.

Semi-synthetic inositol 1,2-cyclic 4,5-trisphosphate is 1/16th as potent as inositol 1,4,5-trisphosphate in releasing Ca2+ from intracellular stores in permeabilized mouse pancreatic acinar cells. Competitive displacement studies in mouse pancreatic microsomes show that the affinity of inositol 1,2-cyclic 4,5-trisphosphate is 1/20th of that of inositol 1,4,5-trisphosphate at the latter's receptor, indicating that the lower potency of inositol 1,2-cyclic 4,5-trisphosphate in releasing Ca2+ can be accounted for by a weaker affinity at the receptor. These results suggest that inositol 1,2-cyclic 4,5-trisphosphate is unlikely to play any significant role in Ca2+ mobilization, at least in mouse pancreatic acinar cells.

The initial signal for thyroid cell proliferation is unknown. This is the first report to show that epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium ([Ca2+]i) in the thyroid gland. In cultured porcine thyroid cells, 10 nM EGF produces a breakdown of phosphatidylinositol and stimulates inositol phosphate production. Ten nM EGF increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the EGF-induced [Ca2+]i response occurs immediately, reaches a maximum within several seconds, and then slowly declines. EGF stimulates production of inositol phosphates, which seem to increase [Ca2+]i. Inositol phosphate production and an increase in [Ca2+]i after EGF-stimulation may function as an initial signal for thyroid cell proliferation.

EGF is a low molecular weight polypeptide hormone which acts as a regulator of cell growth and differentiation. The A-431 cell line has been used frequently to examine receptor-mediated biochemical effects of EGF, since this cell line has an increased (20-50 fold) level of EGF receptors. We have utilized A-431 cells to examine the influence of EGF on formation of an intracellular second messenger, inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3), and other inositol phosphates. The results show that EGF induces rapid formation of Ins-1,4,5-P3 as well as Ins-1,3,4-P3 and Ins-1,3,4,5-P4. There is a concurrent decrease in the level of the lipid precursor for Ins-1,4,5-P3, phosphatidylinositol 4,5-biphosphate (PIP2). Furthermore, we have examined five other cell lines that overexpress the EGF receptor and find that EGF treatment induces formation of inositol polyphosphates in those cell lines also.

The effects of histamine on [3H]inositol phosphate ([3H]IP) accumulation was examined in the presence of lithium in [3H]inositol-prelabelled human umbilical vein endothelial cells. Histamine stimulated total [3H]IP formation in a dose-dependent manner with a half-maximal value (EC50) of around 1-2 X 10(-6) M. Mepyramine, but not cimetidine, completely abolished the histamine response indicating that activation of phosphoinositide hydrolysis is mediated via H1-receptors. These data are the first to suggest that activation of inositol lipid hydrolysis is the underlying transmembrane signalling mechanism histamine H1-receptors employ in mediating various endothelial cell functions.

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.

Summary Stimulation of mouse peritoneal macrophages by Interleukin-one (IL-1) provoked rapid increases in the levels of inositol mono, bis, tris and tetrakisphosphates (IP 1 , IP 2 , IP 3 and IP 4 respectively). IP 3 was by far the major metabolite formed and time course studies revealed that IP 2 and IP 3 were formed more rapidly than IP 1 and IP 4 in response to IL-1 stimulation. The IP 2 and IP 3 levels peaked at five seconds while there was a time lag of five seconds in the IP 4 response and the IP 1 levels increased relatively steadily over the time course of the experiments. Levels of IP 2 , IP 3 and IP 4 all returned almost to control levels by 60s. The rapid formation of the inositol phosphate metabolites was concomitant with a decrease (84%) in the levels of phosphatidyl-inositol 4,5-bisphosphate (PIP 2 ) in the macrophages. These results suggest that the mechanism of IL-1 receptor activation is by the rapid hydrolysis of phosphoinositides and generation of the second messenger IP 3 .

The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoyl lysophosphatidic acid involves an initial mobilization of intracellular Ca2+ with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C.

IP3-induced Ca2+ release from the endoplasmic reticulum (ER) of islets is believed to be a key intracellular event in glucose-induced insulin secretion. Calmodulin was shown to increase ATP-dependent Ca2+ steady-state and inhibit by 57.2% IP3-induced Ca2+ mobilization from the ER. Conversely, the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7), induced Ca2+ release from the ER. The combination of W-7 (100 microM) and IP3 (10 microM), resulted in a greater release of Ca2+ from the ER than either W-7 or IP3 alone. W-7 was shown not to affect the structural integrity of the ER. Our results suggest that IP3-induced Ca2+ release from the ER is regulated by a calmodulin-dependent process.

A non-hydrolysable GTP analogue enhanced the formation of [3H]inositol polyphosphates in permeabilized adrenal glomerulosa cells. Pertussis toxin, which ADP-ribosylated Ni, failed to influence angiotensin-induced formation of 3H-labelled inositol phosphates and the incorporation of [32P]phosphate into phosphatidyl inositol and phosphatidic acid. These results show that Ni, is prescot and a G-protein activates phospholipase C also in glomerulosa cells, however, it is not Ni which couples angiotensin receptors to the enzyme.

The effects of heparin on the release of intracellular Ca2+, assessed by tension development in saponin-permeabilized rabbit main pulmonary artery, were determined. Heparin inhibited (IC50 = 5 micrograms/ml) inositol 1,4,5-trisphosphate (InsP3)-induced, but not caffeine-induced, Ca2+ release. The initial (InsP3-dependent) component of GTP gamma S-induced Ca2+-release was also inhibited by heparin, but the InsP3-independent component was resistant to both heparin and procaine. These results support the existence of a G protein activated mechanism of Ca2+ release that is not mediated by InsP3 or by Ca2+-induced Ca2+ release.

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF 2α , PGE 2 and the thromboxane A 2 /endoperoxide analogue U-46619 rapidly increased cytosolic free Ca 2+ , measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca 2+ , indicating release from internal stores. The thromboxane A 2 -receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI 2 analogue Iloprost had no effect on cytosolic Ca 2+ . PGF 2α , PGE 2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.

The release of IP3 and the incorporation of arachidonic acid into phospholipids was measured in a stimulated murine alloantigen-specific, non-cytolytic T cell clone. While Concanavalin A provoked a sharp increase in IP3, Interleukin 2 had no effect on the production of IP3. An increased reacylation of phospholipids with arachidonic acid was seen within the first 4 hours after addition of Concanavalin A, while an effect upon Interleukin 2 was only observed after 8 hours of incubation with Interleukin 2. A similar retarded response to Interleukin 2 was observed in proliferation experiments. These retarded cell responses may be due to changed properties of IL 2 receptors induced by IL 2.

Summary Angiotensin II stimulates rapid formation of inositol-1,4,5-triphosphate (Ins-1,4,5-P 3 ) in bovine adrenal glomerulosa cells. In addition to being rapidly metabolized to lower inositol phosphates, Ins-1,4,5-P 3 is converted to Ins-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P 4 ) and Ins-1,3,4-P 3 which is in turn phosphorylated to a further Ins-P 4 isomer, namely Ins-1,3,4,6-P 4 . In bovine adrenocortical cytosol [ 3 H]Ins-1,3,4,5-P 4 and [ 3 H]Ins-1,3,4-P 3 were converted to Ins-1,3,4,6-P 4 and inositol pentakisphosphate (Ins-P 5 ) in a metabolic sequence suggesting that unlike Ins-1,3,4,5-P 4 , Ins-1,3,4,6-P 4 is a direct precursor of Ins-P 5 . Consistent with this assumption, [ 3 H]Ins-1,3,4,6-P 4 was converted to Ins-P 5 in electropermeabilized adrenal glomerulosa cells. These findings demonstrate that Ins-1,3,4,6-P 4 is an intermediate link between InsP 3 metabolism and the higher inositol phosphates detected in several tissues.

In mouse neuroblastoma × Chinese hamster brain clonal cell line NCB-20, bradykinin (BK) receptor stimulation causes phosphoinositide hydrolysis and release of inositol phosphates. Maximum stimulation (4-fold) of [2- 3 H]inositol trisphosphate (IP 3 ) release in the absence of Li + from NCB-20's prelabelled for 20–24 hours with [2- 3 H]myo-inositol (15 μCi/confluent 60 mm dish) occurred after 5–10 seconds of bradykinin exposure, with an EC 50 of approximately 100 nM. Inositol bisphosphate (IP 2 ) and inositol monophosphate (IP 1 ) also showed increases (2.9-fold and 1.5 fold, respectively), with peaks at 15–20 seconds and 50 seconds, respectively. Under these same conditions, D-Ala 2 -D-Leu 5 enkephalin (DADLE) (10 μM), an opiate agonist with 2 nM affinity, gave no stimulation of IP 3 release. Furthermore, it did not block BK-initiated release, both when applied simultaneously with BK and when cells were preincubated with DADLE for 100 minutes to lower cyclic AMP levels. These results show that (1) pain-inducing BK has a major acute stimulatory effect on receptor-phospholipase C-coupled IP 3 release, (2) the opioid peptide DADLE has no such effect and (3), DADLE does not block the IP 3 release induced by BK.

The effects of supramaximal concentrations of substance P and the cholinergic agonist carbachol on the accumulation of inositol trisphosphate and the elevation of the intracellular free calcium concentration were compared in rat parotid acinar cells. Substance P was fully as effective as carbachol at initial times, but there was a rapid loss of the substance P responses while the effects of carbachol were well maintained. The loss of the substance P responses represented desensitization rather than degradation of the peptide since further additions of substance P were without effect. Desensitization to substance P did not involve long-term loss of substance P receptors as it was fully reversible in less than twenty minutes, the minimum time to extensively wash previously desensitized cells.

Bradykinin produces increases in cytosolic calcium in MDCK cells. We have extracted and separated Inositol 1,4,5 trisphosphate by HPLC and after-acid hydrolysis and conversion to the hexatrifluoro-acetyl derivative quantitated by negative ion chemical ionization mass spectrometry the mass of inositol trisphosphate in MDCK cells. Bradykinin causes an increase in the mass of Inositol trisphosphate from basal levels of 152 pmoles/mg cell protein to 537 pmoles/mg cell protein by 10 secs of stimulation. We conclude that bradykinin stimulates PLC hydrolysis of PIP2 with rapid release of IP3 in sufficient amount to account for the increase in cytosolic Ca++.

Summary Analysis of inositol phosphate formation in chemoattractant-stimulated human polymorphonuclear leukocytes demonstrated the production of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,4-bisphosphate and another inositol bisphosphate isomer not detected in unstimulated cells. Studies in cell sonicates provided evidence that the previously unidentified inositol bisphosphate isomer is produced via the degradation of inositol 1,3,4-trisphosphate. This unidentified inositol bisphosphate peak was purified by high pressure liquid chromatography, and base hydrolyzed to form a mixture of inositol monophosphate isomers. Based on these studies, the unidentified peak was identified as inositol 3,4-bisphosphate. Identification of this isomer defines a new metabolic product derived from the initial inositol 1,4,5-trisphosphate formation, and also suggests another substrate for the inositol 1-phosphatase.

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca 2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca 2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10 −2 M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca 2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca 2+ is dose-dependent (maximal at 10 −4 M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca 2+ are of the P 2 -purinoceptor subtype. The physiological functions of these receptors are not presently known.

Summary Carbamazepine is used to treat manic-depressive disorder, and is also an anticonvulsant. Rats were injected with this drug 90 min prior to this experiment, when mild inhibition of convulsions took place. Intraventricular injections of 14 uCi [ 3 H]myoinositol were made 20–24 hrs prior to the experiment. Ninety min after intraperitoneal injection of carbamazepine or vehicle, rats were given electroconvulsive shock or sham procedure and sacrificed 30 sec later. Incorporation of radiolabel into inositol lipids and inositol phosphates was analyzed in cerebral cortex and hippocampus. Carbamazepine's effects on the brain inositol lipid cycle, studied here for the first time, showed 1) enhanced labeling in the polyphosphoinositides (carbamazepine-ECS groups showed increases of about 40% in PIP 2 ); 2) decreased [H]IP 1 levels; and 3) inhibition of ECS-induced [ 3 H]-IP 3 accumulation.

Summary The net content of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was measured in bradykinin (BK)-stimulated NIH3T3 fibroblasts and neuroblastomaglioma hybrid cells (NG108-15). BK-mediated production of Ins(1,4,5)P3 was not affected by replacing the medium with Ca2+-free medium, but addition of EGTA(1mM) to Ca2+-free medium markedly prevented production of Ins(1,4,5)P3. Although perussis toxin (PT) treatment caused ADP-ribosylation in both N1H3T3 cells and NG108-15 cells, the BK-induced Ins(1,4,5)P3 formation was considerably reduced in the former cells but not in the latter cells, suggesting that PT-sensitive and PT-insensitive GTP-binding proteins are involved in phosphoinositide phospholipase C (PI-PLC) activation in fibroblasts and neuroblastoma cells, respectively. In NG108-15 cells down-regulated in protein kinase C (PKC) by long-term exposure to phorbol 12-myristate 13-acetate (PMA), BK-stimulated Ins(1,4,5)P3 accumulation was significantly enhanced compared to control cells.

When isolated rat hearts were perfused with medium containing 3-0-methyl-D-glucose (3MG) a compound with the chromatographic behavior expected for 3MG-6-phosphate was formed. This assignment is supported by the action of alkaline phosphatase and by production of an identical peak on incubation of 3MG with yeast hexokinase in a buffer containing adenosine triphosphate and Mg ++ ions. The clearance of labeled 3MG from isolated perfused rat hearts was biphasic and eventually total. Since sugar phosphates do not cross cellular membranes, this behavior is consistent with both formation and dephosphorylation of 3MG-6-phosphate, with dephosphorylation rate-limiting the slow phase of the clearance. Phosphatase activity was also indicated by a decrease in 3MG-6-phosphate when hearts were not chilled and homogenized immediately after perfusion. The common assumption that 3MG is not metabolized after transport into cells is shown by these experiments to be erroneous.

Inositol (1,4,5) triphosphate (IP 3 ) was observed to induce release of sequestered Ca 2+ from crude human platelet membranes. This activity was also shown to be present in purified membranes enriched in Ca 2+ -ATPase activity. Maximal Ca 2+ release occurred at 8 μM IP 3 and half maximal activity was at 0.4 μM. Release was quite rapid and was complete by 40 s. Released Ca 2+ was pumped back into the membrane vesicles and the rate of this reuptake was increased by the presence of phosphate. These results demonstrate that internal platelet membranes that possess an active Ca 2+ -pump will release sequestered Ca 2+ in the presence of the second messenger IP 3 . IP 3 did not induce release of Ca 2+ from skeletal muscle sarcoplasmic reticulum when ATP was present.

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.

Neoplastic rat liver epithelial (261B) cells made permeable by electroporation released 0.2-0.3 μM Ca 2+ from intracellular stores in response to 0.5 μM Ins(1,4,5)P 3 stimulation. This Ca 2+ release response was found to be inhibited by heparin in a dose-dependent manner (K i of 15 μg/ml). Two other glycosaminoglycans, chondroitin sulfate and hyaluronic acid, showed no inhibitory effect at doses as high as 0.2 mg/ml. Passive Ca 2+ release, and sequestration of Ca 2+ into intracellular storage sites by the action of Ca 2+ -ATPase were unaffected by heparin treatment. We conclude that the inhibitory action of heparin treatment on Ca 2+ mobilization in permeabilized 261B cells is mediated through its interaction at the Ins(1,4,5)P 3 receptor binding site.

Low concentrations of 6-phosphogluconate activated phosphofructokinase from rat liver. 6-phosphogluconate (0.2mM) lowered the concentration of fructose-6-phosphate required to achieve one-half the maximum velocity (S0 · 5) to a value within the physiological range of fructose-6-phosphate concentrations, but had no effect on the maximum velocity. 6-phosphogluconate also changed the kinetics of the enzyme from sigmoidal to hyperbolic. The effect of 6-phosphogluconate on phosphofructokinase was abolished by heating the enzyme.

Inositol (1,4,5)P3 (IP3) receptors in rat cerebellar membranes have been used to develop a radioreceptor assay for endogenous IP3. The assay is sensitive, detecting as little as 1 pmole IP3 in a 0.5 ml volume. The receptor is highly selective for the 1,4,5-isomer of IP3 so that assays can employ crude tissue extracts with no purification. Chromatographic analysis and selective enzymatic hydrolysis of IP3 show that in crude tissue extracts only authentic IP3 is detected. A hundred or more samples can be assayed in a day for endogenous IP3.

Pretreatment of bovine aortic endothelial cells with pertussis toxin inhibited partially the accumulation of inositol phosphates in response to ATP, whereas cholera toxin had no effect. Both pertussis and cholera toxins enhanced the stimulatory effect of ATP on prostacyclin release from the same cells. This action of cholera toxin was mimicked neither by an increase of cyclic AMP nor by the dissociated subunits of the toxin. Cholera and pertussis toxins, as well as aluminum fluoride, also potentiated the release of prostacyclin induced by ionophore A23187. These results suggest that a pertussis toxin-sensitive GTP-binding protein is involved in the coupling between P2-purinergic receptors and phospholipase C. In addition, another GTP-binding protein would play a crucial role at a further step in the control of PGI2 biosynthesis., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The facile formation of glycosylamines suggests that a base liberated by depurination might react at the free C1 position of the sugar phosphate from which it had been hydrolyzed, effectively repurinating the site. Model experiments testing this hypothesis demonstrate that such a reaction does take place. The primary product of a reaction between 2-aminopyrimidine (a model for guanine) and ribose-5-phosphate is characterized by enzymatic and chemical degradation, and UV spectra. It is shown to be a novel nucleoside with the base attached via its exocyclic amino group to the C1 of the ribose-5-phosphate.

Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 microM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that by mobilizing intracellular Ca2+, inositol trisphosphate may be the link between hormonal stimuli and smooth muscle contraction.

The effect of inositol 1,4,5-trisphosphate (IP 3 ) on Ca 2+ release in the transformed murine mast cells, mastocytoma P-815 cells permeabilized with digitonin was studied. Ca 2+ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca 2+ concentration was lowered to a new steady-state level. The subsequent addition of IP 3 caused a rapid Ca 2+ release, which was followed by a slow re-uptake of Ca 2+ . Fifty percent of the sequestered Ca 2+ was released by 10 μM IP 3 . Maximal Ca 2+ release occurred at 10 μM and half maximal activity was at 1.3 μM. These results indicate that IP 3 may function as a messenger of intracellular Ca 2+ mobilization in mastocytoma cells.

The concentrations of glycolytic intermediates, including 2,3-diphosphoglycerate, were similar in rat reticulocytes and erythrocytes. There were striking differences, however, in the content and kind of water-soluble nucleotides. Reticulocytes contained much higher concentrations of ATP, GTP, UTP and CTP and had nucleotides not detected in the mature cell including UDP-acetylhexosamine, guanosine diphosphomannose and an unidentified cytidine compound. A large fraction of the total GTP found in the reticulocyte was in the form of a 1:1 complex of ferric iron with GTP.

Platelet activation is often associated with an increase in the cytosolic free Ca concentration that is due in part to Ca release from the dense tubular system. The present studies examine whether the diacylglycerol formed by phosphoinositide hydrolysis during platelet activation contributes to this process. The effect of diacylglycerol on the dense tubular system was tested using platelets that were permeabilized with saponin and then allowed to accumulate 45Ca. A synthetic diacylglycerol, 1-oleoyl-2-acetoyl glycerol (OAG), released up to 70% of the ionophore A23187-releasable 45Ca, a fraction identical to that discharged by inositol 1,4,5-triphosphate (IP3) under the same conditions. 45Ca release was half-maximal at 40 μM OAG and 1 μM IP3. The response to OAG was not inhibited by aspirin and could not be reproduced by the addition of a phorbol ester, which suggests that it involves neither arachidonic acid metabolism nor protein kinase C activation. The time course of OAG-induced 45Ca release, which was slower than IP3-induced 45Ca release, corresponded to the time course of conversion of the OAG to 1-oleoyl-2-acetoyl phosphatidic acid (OAG-PA). When either OAG-PA or lysophosphatidic acid was added to the saponin-treated platelets, the extent of 45Ca release was similar to that observed with OAG, but both the OAG-PA and the lysophosphatidic acid were 5 to 10 times more potent than OAG on a molar basis. These data suggest: (1) that the Ca release caused by diacylglycerol is actually due to formation of phosphatidic acid and/or lysophosphatidic acid, (2) that these molecules are not acting as simple Ca ionophores and (3) that diacylglycerol metabolites may augment the changes in Ca homeostasis caused by IP3 during platelet activation.

Summary Calf-liver rough microsomes incorporated N-acetyl[3H] glucosamine from UDP-[3H]GlcNAc or [14C]mannose from GDP-[14C]Man into products soluble in chloroform-methanol (2:1). The [3H] GlcNAclabeled material had the hydrolytic and chromatographic properties of a polyisoprenyl N-acetylglucosaminyl pyrophosphate whereas the 14C-mannolipid exhibited the characteristics of a polyisoprenyl mannosyl phosphate. Tunicamycin inhibited the production of the [3H]G1cNAc-lipid but did not affect the synthesis of the 14C-mannolipid. This inhibition accounts for the effect of the antibiotic upon glycoprotein formation in vivo since polyprenyl sugar phosphates are intermediates in the biosynthesis of certain glycoproteins.

Application of acetylcholine (ACh) to C62B glioma cells results in a rapid release of inositol phosphates. Since this response is transient, we evaluated the possible role of protein kinase C (PKC) in its desensitization. Pretreatment with 100nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited ACh-induced accumulation of [ 3 H]inositol mono-, bis-, and trisphosphates. However, interpretation of this result as proof of PKC involvement was complicated by the failure of 1,2-dioctanoylglycerol, 1,2-didecanoylglycerol, or 1-oleoyl-2-acetylglycerol pretreatments to mimic the phorbol ester effect. Further evidence against PKC involvement was obtained using the PKC inhibitor sphingosine; PDBu inhibition of inositol phosphate formation was not reversed by sphingosine pretreatments at concentrations which blocked ACh-stimulated PKC activation of inositol trisphosphate phosphatase activity. These results suggest that there may be phorbol effects not mediated by PKC.

31P-Magnetic resonance spectroscopy has been used to assess the changes in the levels of water-soluble phosphate pools in T47-D breast carcinoma cells induced by the antimitochondrial drugs, gossypol and 6-aminonicotinamide. A decrease in the NTP/Pi ratio occurred after treatment with gossypol. No change in the NTP/Pi ratio occurred on treatment with 6-aminonicotinamide; however, a substantial accumulation of 6-phosphogluconate was observed. Pretreatment of T47-D cells with gossypol prevented the accumulation of 6-phosphogluconate. This facile and non-invasive approach suggests that the oxidative part of the pentose-phosphate shuttle is an important source of reducing equivalents in T47-D cells. This pathway may prove to be a useful target for site-directed drug attack in carcinoma cell lines that require large quantities of NADP for the synthesis of fatty acids and steroids.

2-p-toluidino-naphthalene-6-sulfonate (TNS) is a sensitive fluorescent reporter group for the detection of the events at the reaction centres of the ribulose biphosphate carboxylase/oxygenase from spinach. The formation of binary complexes of the carboxylase with substrates and effectors is associated with significant changes (ΔF) of the fluorescence emission of the enzyme-TNS-complex. This indicates substrate and effector induced conformational changes of the enzyme. From the concentration dependence of ΔF the following dissociation constants for ribulose biphosphate (RuBP) and Mg2+ were determined: K(RuBP) = 0,5 μM and K(Mg2+) = 1 mM. Sugar phosphates, e.g. 6-phosphogluconate, which show regulatory effects in the carboxylation and oxygenation of RuBP, function antagonistically to RuBP, presumably by competition with RuBP for its allosteric binding site.