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The effect of luteinising hormone (LH) on bovine cervical tissues from three phases of the estrous cycle was studied. It was found that in the luteal phase cervix contained an LH receptor mRNA transcript and the 93 kDa LH receptor protein. Incubation of cervical minces from luteal phase with LH significantly increased (P < 0.05) the intracellular concentrations of LH receptor protein, cAMP, inositol phosphate (IP) and cyclooxygenase as well as the production of prostaglandin (PG) E2 (but not PGF2alpha). In contrast in the pre-oestrus/oestrus or post-ovulatory cervix, the signal for the transcript for LH receptor and the LH receptor protein was significantly lower than luteal cervix (P < 0.05) and the tissue did not respond to LH. We conclude that bovine cervix at luteal phase has high levels of LH receptor mRNA and receptor protein associated with the protein coupled receptor family that mediates cAMP and IP signalling responses. The receptors are coupled to PG synthase and generate cervical PGE2.
Phagocytosis of C3bi- or IgG-opsonized yeast particles in human neutrophils was found to be associated with an increased formation of inositol phosphates and diacylglycerol. Pertussis toxin only marginally affected phagocytosis of IgG- and C3bi-opsonized particles and the associated formation of second messengers. Forskolin, which induced a threefold rise of cellular cAMP, however, markedly inhibited both C3bi- and IgG-mediated phagocytosis as well as the particle-induced formation of inositol phosphates and diacylglycerol. These observations are in contrast to what was found to occur with chemotactic factors and indicate that chemotactic and phagocytic signaling can be regulated independently in human neutrophils. Since C3bi-mediated phagocytosis has been shown to occur at vanishingly low cytosolic free calcium levels, calcium-depleted cells were used to study the importance of the inositol cycle for the engulfment of C3bi-opsonized particles. Despite a total lack of receptor-induced formation of inositol phosphates, a significantly increased accumulation of diacylglycerol accompanied the ingestion of C3bi-opsonized particles. These data show that the engulfment of C3bi-opsonized particles can occur independently of both a calcium transient and an increased inositol phosphate production. However, the observed accumulation of diacylglycerol, not derived from phosphoinositides, suggests that this second messenger play a role in the control of the engulfment process.
In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO2 tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [3H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO2 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3 protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO2 20 mm Hg versus control pH 7.40, PCO2 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P3. aCSFs of control pH 7.40, and PCO2s of 70, 36, or 25 mm Hg were studied both by [3H]-myoinositol (HPLC) and PBA to further determine the importance of CO2 tension, in the presence of fixed pH, on Ins(1,4,5)P3 production. When PCO2 was constant, inositol phosphate turnover (as measured by [3H]-Ins[1,4,5]P3 accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [3H]-Ins(1,4,5)P3 accumulation at pHs of 7.40 and 7.80, constant PCO2 of 36 mm Hg, were 2.9 +/- 0.7 and 4.1 +/- 0.8 cpm/micrograms protein, respectively. Ins(1,4,5)P3 levels for pH of 7.40 or 7.80 and constant PCO2 of 36 mm Hg, were 25.4 +/- 1.8 and 38 +/- 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P3 levels. For pH of 7.40, PCO2 36 mm Hg and pH 7.60, PCO2 20 mm Hg, Ins(1,4,5)P3 levels were 37.6 +/- 16 and 64.1 +/- 25 pmol/well, respectively. Decreasing CO2 tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P3 levels. The present data demonstrate that decreased CO2 tension stimulates an increase in Ins(1,4,5)P3 production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO2 increases the level of Ins(1,4,5)P3 even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO2 without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.
Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)
Separation of inositol phosphates by low-pressure anion-exchange chromatography yields unsatisfactory results, while previously described anion-exchange HPLC methods require such extensive processing times that they preclude efficient sample analysis. Using a low-capacity Vydac nucleotide anion-exchange column, we have developed a method which allows complete separation of myo-inositol, inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in approximately 10 min followed by a 5-min column regeneration time. This method provided exceptional reproducibility and quantitative recovery of each inositol phosphate. One column was used for over 300 separations with no loss in performance or alteration in elution pattern. A modified procedure with a 14-min gradient was developed to separate the 1,3,4- and 1,4,5-isomers of inositol trisphosphate. These separation procedures were used to characterize the kinetics of degradation of inositol phosphates by lysates of erythrocytes and neutrophils. We conclude that these procedures are applicable for rapid and quantitative analysis of radiolabeled inositol phosphates in cellular extracts.
Interest in the inositol phospholipids was stimulated by the simultaneous discoveries that the products of hydrolysis of these lipids could serve as messengers to activate to synergistic signaling pathways in hormonally responsive cells, namely, inositol 1,4,5-trisphosphate which causes the release of Ca2+ from intracellular stores and diacylglycerol which promotes the activation of protein kinase C. At the same time, Berridge and co-workers introduced relatively simple approaches to study the inositol phospholipid cycle. These included the use of [3H]inositol to label the inositol metabolites, all of which are confined to this cycle, and of Li+ to decrease the rate of degradation of the inositol phosphates. Water-soluble inositol phosphates and chloroform-soluble inositol phospholipids could then be separated by solvent partition and the inositol phosphates further separated by use of an anion-exchange resin. However, the subsequent application of high-performance liquid chromatography as a separation technique indicated the existence of many isomers of the inositol phosphates formed by different pathways of dephosphorylation and phosphorylation. Mapping of these metabolic pathways may be substantially complete, but novel pathways may still be discovered. We review both old and new methods of analysis of the inositol phosphates for the measurement of mass and radioactivity. Although the complexity of the cycle sometimes demands the use of sophisticated methods of separation and rigorous identification, older and inexpensive methods may still be useful for some purposes.
We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP(4) 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P(4) with a catalytic efficiency (V(max)/K(m)) 43-fold greater than that of Ins(1,3,4,5)P(4) and 2-fold greater than that of Ins(1,4,5)P(3). The apparent V(max) was 114 nmol of Ins(1,3,4,5,6)P(5) formed/min/mg of protein, and the apparent K(m) was 0.3 microm Ins(1,3,4,6)P(4). The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P(5) or InsP(6). Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP(4) 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P(4), and the reaction of the human protein with Ins(1,3,4,5)P(4) is insufficient to effect rescue or synthesis of InsP(5) and InsP(6). Therefore the major activity of human InsP(4) 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P(5) in yeast versus humans are different.
The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.
Inositol phospholipids and their hydrolysis products have been recognized to play important roles in an intracellular signal transduction system. Since their contents in cells are quite low, synthetic methods for their production are required. Chemical synthesis of various inositol phosphates and inositol phospholipids, including natural phosphatidylinositol 3,4,5-trisphosphate, have been efficiently accomplished. For this purpose, several new reactions and reagents were developed.
The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5 microM Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1 microM FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.
Inositol phosphate release and metabolism were studied in right atrial appendages obtained from 18 patients undergoing coronary artery bypass surgery and/or mitral valve replacement. [3H]Inositol-labeled human atria contained inositol(1,4. 5)trisphosphate, inositol(1,4)bisphosphate and the 1- (or 3) and 4-isomers of inositol monophosphate. Addition of norepinephrine (100 mumol/l) activated the release of inositol phosphates, as indicated by increased [3H]inositol label in all of these inositol phosphates. However, the phosphorylation product of inositol (1.4.5)trisphosphate, inositol-(1,3,4,5)tetrakisphosphate, and its metabolic products were not detected, either in control or stimulated atria. Similar inositol phosphate profiles were observed in rat right atria. Furthermore, both human and rat atria contained high concentrations of inositol(1,4,5)trisphosphate, which were not observed to increase with norepinephrine stimulation. The inositol phosphate responses to norepinephrine in rat and human cardiac tissue appear to be similar, except for the generally lower activity observed in human tissue. Thus, the rat provides a suitable model for the study of cardiac phosphatidylinositol turnover.
Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[3H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 microM, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)P5. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50-85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal-transduction mechanisms is unlikely. In homogenates prepared from myo-[3H]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InsP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 microM were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1.
scyllo-Inositol is the all equatorial stereoisomer of myo-inositol. All possible 12 regioisomers of scyllo-inositol phosphate were synthesized for the first time via a scyllo-inositol benzoate intermediate, which was derived from a myo-inositol derivative. The stereoinversion of myo-inositol into scyllo-inositol was accomplished by Mitsunobu reaction of the vicinal cis-diol. The requisite intermediates, scyllo-inositol benzoates were obtained by benzoyl migration or random benzoylation, and phosphorylated to give scyllo-IPn.
The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.
Inositol phosphate release in intact heart in response to norepinephrine involves primarily release of inositol(1,4)bisphosphate (Ins(1,4)P2) rather than inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) but Ins(1,4,5)P3 release predominates under conditions of post-ischemic reperfusion. In the current study, effects of myocardial ischemia on inositol phosphate responses were examined. Global myocardial ischemia in rat ventricle caused a reduction in the content of [3H]Ins(1,4)P3 (70-90%) and [3H]Ins(1,4,5)P3 (46%) and altered the pattern of norepinephrine stimulation such that increases in [3H]Ins(1,4,5)P3 were observed. Simulated ischemia in isolated right atria or isolated ventricular myocytes (P alpha 2 16-20 mmHg. pH 6.7. KCl 10 mM) produced similar changes. Reduction in O2 in the absence of other changes reduced the content of [3H]Ins (1,4)P2 (79%) in right atria whereas hypoxia and reduced pH were required to alter the [3H]Ins(1,4,5)P3 response. Progressive reduction in atrial ATP content using metabolic inhibitors caused a parallel decrease in [3H]Ins(1,4,5)P3 content (r = 0.96) without affecting [3H]Ins(1,4)P2 or the isomers of InsP1, showing that levels of Ins(1,4)P2 and Ins(1,4,5)P3 are regulated differently in the heart. These findings show that effects of ischemia on inositol phosphates in heart are complex and multifactorial, with Ins(1,4)P2 being affected under more moderately ischemic conditions than required for alterations in Ins(1,4,5)P3. These studies also demonstrate that ischemia produces similar effects on the release and metabolism of inositol phosphates in heart regardless of the ischemic model or the myocardial preparation used.
A systematic effort was made to elucidate the mode of recognition at the inositol 1,4,5-trisphosphate-specific receptor. Eleven D-myo-inositol phosphates were synthesized and tested for Ca(2+)-mobilizing and receptor-binding activities, which included Ins(1,3,4,5,6)P5, Ins(1,2,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(3,4,5,6)P4, Ins(1,3,4)P4, Ins(1,4,5)P3, Ins(1,5,6)P3, Ins(1,4)P2, and Ins(4,5)P2. Of these, Ins(1,4,5)P3, Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(1,4,5,6)P4, and Ins(4,5)P2 were able to elicit Ca2+ release from rat brain microsomes. Binding experiments suggest that the ability of these polyphosphates to effect Ca2+ mobilization arises from interactions with the Ins(1,4,5)P3-specific receptor. Accordingly, a model accounting for the ligand recognition is proposed. The Ins(1,4,5)P3-binding site is presumably composed of two domains. The anchoring domain binds the 4,5-bisphosphate 6-hydroxy motif. Disruption of this structural feature abolishes the agonist activity. The auxiliary domain exerts long-range interactions with the 1-phosphate, thus enhancing the binding affinity. The stereochemical requirement for this electrostatic interaction is, however, less stringent. Evidence suggests that Ca(2+)-mobilizing inositol phosphates are able to effect productive binding by assuming conformations displaying or mimicking these essential structural features.
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.