An Optimized Fixation and Extraction Technique for High Resolution of Inositol Phosphate Signals in Rodent Brain

Members of lower and higher inositol phosphates distinctly participate in signal transduction (1). Relatively little is known regarding possible biological functions of inositol phosphates in functionally different areas of the intact brain. A detailed study on the regional distribution of biologically important inositol phosphates may help elucidate their physiological functions in different brain regions in the regional tissue context. We now show a novel technique which allows fixation and subsequent dissection of whole rat brains into small volume elements for mapping of the whole range of inositol phosphates from Ins(1,4,5)P3 to InsP6. The method has been successfully applied to investigate regional differences of a broader spectrum of inositol phosphates in microdissected brain tissue and to construct 3D-maps of these signaling compounds. The technique can be particularly well employed to investigate regional changes in the spectrum of higher inositol phosphates and phosphoinositides upon neuronal stimulation induced by motor activity or drug treatment.     

Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.     

A multi-well filtration assay for quantitation of inositol phosphates in biological samples

The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming conventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. The response of recombinant HEK-293 cells expressing human LHRH receptor cDNA to LHRH agonists was used as a model system to develop the assay conditions. Cell lysates containing labeled IPs were applied in 96-well plates fitted with filtration discs containing regenerated Dowex AGI-X8 resin. Specifically bound inositol phosphates were eluted with 1 M ammonium formate in 0.1 M formic acid directly into a fresh 96-well plate and an aliquot of the eluate from each well is transferred into a 96-well plate and counted. The results were comparable to those obtained with the conventional column method and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste.     

Properties of phospholipase C in isolated olfactory cilia from the channel catfish (Ictalurus punctatus)

1. Cilia were isolated from the olfactory epithelium of the channel catfish (Ictalurus punctatus) with improved yield. The isolated preparations were enriched in cilia as indicated by electron microscopy, tubulin immunoblotting and identification of a ciliary-specific glycoprotein. 2. The isolated cilia preparations exhibited phospholipase C (EC 3.1.4.11) activity. The enzyme was maximally active at pH 6.7. 3. Analysis of inositol phosphates resulting from the hydrolysis of exogenous radiolabeled phosphatidylinositol-4,5-bisphosphate in isolated cilia, indicated that inositol triphosphate was the major (90%) inositol phosphate produced. 4. Three molecular forms of the enzyme, Mr greater than or equal to 100,000, 82,000 and 60,000 were resolved by gel filtration chromatography from a cytosolic fraction from the olfactory epithelium.     

Activation of phospholiphase C by different effectors in rat placental cells

The present communication documents the accumulation of inositol phosphates in rat placental cells by fluoride as well as by vanadate. These findings suggest the existence of the phosphoinositide pathway and its modulation by a G-protein. A concomitant action of fluoride on phosphoinositide breakdown was also observed. As is often the case in intact cells from different organs, protein kinase C exerts a feeback regulatory control on this signalling system. Gonadotropin-releasing hormone (GnRH) also stimulated the accumulation of inositol phosphates in cultured cells but no effect could be detected in freshly isolated cells. Therefore, the phosphoinositide pathway seems to be involved in the mechanism of action of GnRH in rat placental cells.     

The separation of [32P]inositol phosphates by ion-pair chromatography: optimization of the method and biological applications

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [3H]inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.     

Isolation of soluble metabolites of the phosphatidylinositol cycle from Samanea saman

An improved protocol for the separation of inositol phosphates by high performance liquid chromatography was used to resolve inositol phosphates from pulvini (motor organs) of the legume, Samanea saman. The pulvini contained inositol phosphate, inositol bisphosphate, and inositol trisphosphate isomers which co-migrated with those of mammalian red blood cells, and one or more other inositol metabolites which, to our knowledge, have not been previously noted in preparations of inositol phosphates. The finding of inositol phosphates in Samanea which comigrate with mammalian inositol phosphates supports the possibility that the phosphatidylinositol cycle may function in signal transduction in plants as well as in animals.Abbreviations HPLC high performance liquid chromatography - PI phosphatidylinositol     

Denervation Supersensitivity of the Rat Pineal to Norepinephrine-Stimulated [3H]Inositide Turnover Revealed by Lithium and a Convenient Procedure

A convenient procedure for the assay of myo-[2-3H(N)]inositol ([3H]inositol) metabolites in cells or small amounts of tissue was developed. The procedure is a composite of modifications of published methods. After preincubation with [3H]inositol, rat pineal glands were disrupted in an acidified organic solvent mixture. Lipids were separated from the hydrophilic products and precursor using Sephadex G-25 columns and further analyzed by TLC. Hydrophilic products were further analyzed by anion-exchange column chromatography using Dowex AG1-X8 (formate form). In the presence of lithium, increases in inositol phosphates consequent to stimulation of the glands by norepinephrine were apparent within 10 min. The response in denervated glands was considerably greater than in intact pineals.     

Angiotensin II and bradykinin stimulate phosphoinositide breakdown in intact rat kidney glomeruli but not in proximal tubules: glomerular response modulated by phorbol ester

We have investigated the effect of angiotensin II, bradykinin, insulin and insulin-like growth factor I on phosphoinositide turnover in intact rat glomeruli and tubules. Angiotensin II produced a dose-dependent increase in inositol monophosphate formation with an IC50 of 10(-7)M, when added to isolated rat glomeruli. Angiotensin II-stimulated inositol phosphates formation was inhibited by the angiotensin receptor antagonist [Sar-Leu8]angiotensin II, indicating that the above response was mediated through activation of an angiotensin receptor in intact glomeruli. Besides angiotensin, in intact glomeruli, only bradykinin stimulated a phosphoinositide response, while in intact proximal tubules, none of the agonists tested produced an activation of the inositol phosphate formation. Angiotensin II- and bradykinin-stimulated inositol phosphate accumulation in intact glomeruli was inhibited by phorbol myristate acetate, an activator of protein kinase C.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Methods for the analysis of inositol phosphates

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.     

Isolation and assay of red-cell inositol polyphosphates

An inositol pentaphosphate has been found to be present in high concentration in red cells of a number of species of birds examined and is thought to be a major control of the oxygen affinity of hemoglobin. to facilitate further studies on the distribution, function, and metabolism of inositol polyphosphates in red cells of birds and other vertebrates, improved methods were developed for their isolation by ion-exchange column chromatography, for their hydrolysis to free inositol from the isolated phosphates, and for colorimetric analysis of the free inositol. Examples are given of the inositol phosphates found in red cells of a duck, an ostrich, the Pseudemys turtle, a fish (Arapaima gigas), commercial inositol hexaphosphate, corn seeds, and corn seedlings.     

Differences in inositol phosphate production in rat tail artery and thoracic aorta

Pharmacomechanical coupling of vascular smooth muscle is believed to be mediated by inositol trisphosphate (IP3). Numerous studies have demonstrated an increase in inositol phosphates following tissue stimulation using either intact aortic strips or cultured cells from aorta. However, little information is available concerning inositol phosphates in vascular tissue other than in the large conduit vessel, the aorta. This present study was designed to examine the role of inositol phosphate metabolism following adrenergic stimulation of the muscular rat tail artery as compared to the aorta. Segments of thoracic aorta and tail artery from male Sprague Dawley rats were labeled with [3H]inositol and stimulated with norepinephrine. The norepinephrine concentration that resulted in a half-maximal stimulation of inositol phosphates was approximately 10(-6) M in both the aorta and tail artery. Although the sensitivity of the two vessels to norepinephrine stimulation were similar, the stimulated levels of IP, IP2, and IP3 were from 1 to 2 orders of magnitude greater in the tail artery than in aorta. IP production in aorta and tail artery was a linear function of time (from 0 to 30 min). Significant levels of IP3 (the 1,4,5-IP3 isomer as determined by HPLC) could only be detected in the tail artery and appeared to be produced optimally after 5 min of stimulation. The several order of magnitude increase in adrenergic stimulated inositol phosphate production in the tail artery was not due to either an increased magnitude of [3H]inositol incorporated into PI, PIP, and PIP2 or to a greater percentage of smooth muscle cells per unit tissue of the rat tail artery. We believe the results of this study demonstrate that the increased inositol phosphate metabolism in the vascular smooth muscle cells of the tail artery is an intrinsic property of the cell. Moreover, due to the significant levels of all inositol phosphates produced in the tail artery, this muscular artery may be a better model, as compared to the aorta, for future studies investigating pharmacomechanical coupling of vascular smooth muscle.     

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.     

Metabolism of the biologically active inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by ovarian follicles of Xenopus laevis.

The metabolism of biologically active inositol phosphates in developed ovarian follicles from Xenopus laevis was investigated. Techniques used were microinjection of tracer into the intact oocyte coupled by gap junctions to follicle cells, as well as addition of tracer to homogenates of ovarian follicles and to homogenates of oocytes stripped of outer follicle-cell layers. Metabolism was similar to that previously described for other types of cell and tissue, with several unusual features. Homogenates of ovarian follicles were shown to contain an apparent 3'-phosphomonoesterase capable of converting [3H]Ins(1,3,4,5)P4 predominantly into a substance with h.p.l.c. elution characteristics of Ins(1,4,5)P3. In intact ovarian follicles, little Ins(1,4,5)P3 was formed but the esterase was activated by the phorbol ester activator of protein kinase C, PMA (phorbol 12-myristate 13-acetate; 60 nM), as well as by acetylcholine (200 microM). In follicle homogenates, this enzyme also appeared to be active in converting [3H]Ins(1,3,4)P3 into a substance eluting as Ins(1,4)P2. The apparent 3'-phosphomonoesterase activity was not inhibited by intracellular (or higher) levels of Mg2+. Although PMA activated this enzyme in intact oocytes relative to 5'-phosphomonoesterase activation, it did not enhance overall metabolism, in contrast with reports on other tissues. Compared with the processing of inositol phosphates injected into the intact follicle, homogenization in simulated intracellular medium appeared to alter the activity and/or accessibility of several enzymes. The metabolism of inositol phosphates appears to occur predominantly in the follicle cells surrounding the oocyte, as collagenase treatment followed by defolliculation greatly diminished the rates of metabolism of several inositol phosphates. The presence in Xenopus ovarian follicles of a 3'-phosphomonoesterase activated by protein kinase C in addition to the well-known 3'-kinase suggests that, by forming a reversible interconversion between Ins(1,4,5)P3 and Ins(1,3,4,5)P4, this tissue may have the potential to prolong stimulatory signals on binding of appropriate agonists to receptors.     

A sensitive method for the quantification of the mass of inositol phosphates using gas chromatography-mass spectrometry

A sensitive method to directly measure the mass of inositol phosphates from biologic samples is described. The procedure uses ammonium sulfate gradient elution anion exchange column chromatography to isolate inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate. The isolated fractions are dephosphorylated and subsequently desalted by a novel approach using solid barium hydroxide in a 1:1 stoichiometric ratio to the amount of ammonium sulfate present in the dephosphorylated sample. The myo-inositol derived from each inositol phosphate species was quantified by stable isotope dilution gas chromatography-mass spectrometry of the hexakis(trimethylsilyl) derivative using hexadeutero-myo-inositol as the internal standard. The applicability and sensitivity of this method are illustrated by measuring the mass of individual inositol phosphates in isolated adult canine cardiac myocytes.     

Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate–fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.     

Brain factor induced formation of inositol phosphates in tick salivary glands

A factor from tick brain increases inositol phosphates in isolated, whole tick salivary glands. The factor is sensitive to trypsin and heat (5 min, 100°C) suggesting that it may be a neuropeptide or protein. The salivary glands undergo growth and differentiation accompanied by considerable proliferation of plasma membranes during tick feeding. Salivary glands from ticks in later stages of feeding produce higher levels of inositol phosphates than glands from ticks in early stages of feeding. Cyclic AMP modulates the formation of inositol phosphates suggesting interaction of salivary gland function by the transducing system that employs cyclic AMP as a “second messenger” and that which employs inositol phosphates.     

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.