Radiolabeled Inositol Phosphates Research Articles

The Pathway for the Production of Inositol Hexakisphosphate in Human Cells

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

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

Scintillation proximity assay of inositol phosphates in cell extracts: High-throughput measurement of G-protein-coupled receptor activation

The phosphatidylinositol turnover assay is used widely to measure activation, and inhibition, of G q-linked G-protein-coupled receptors. Cells expressing the receptor of interest are labeled by feeding with tritiated myo-inositol. The label is incorporated into cellular phosphatidylinositol 4,5-bisphosphate, which, upon agonist binding to the receptor, is hydrolyzed by phospholipase C to inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol. In the presence of Li +, dephosphorylation of IP 3 to inositol is blocked, and the mass of soluble inositol phosphates is a quantitative readout of receptor activation. Current protocols for this assay all involve an anion-exchange chromatography step to separate radiolabeled inositol phosphates from radiolabeled inositol, making the assay cumbersome and difficult to automate. We now describe a scintillation proximity assay to measure soluble inositol phosphate mass in cell extracts, thus obviating the need for the standard chromatography step. The method uses positively charged yttrium silicate beads that bind inositol phosphates, but not inositol. We have used this assay to measure activation of recombinant and endogenous muscarinic acetylcholine receptors and activation of recombinant neuropeptide FF2 receptor coupled to IP 3 production by coexpression of a chimeric G protein. Further, we demonstrate the use and functional validity of this assay in a semiautomated, 384-well format, by characterizing the muscarinic receptor antagonists pirenzepine and atropine.

Comparative studies on prostanoid receptors in human non-pigmented ciliary epithelial and mouse fibroblast cell lines

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1–2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca 2+] i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1–2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E 2, butaprost and 11-deoxy-prostaglandin E 1. The stimulation of cyclic AMP production by prostaglandin E 2 was partially inhibited by the EP 4 receptor antagonist AH23848. Prostaglandin E 2 and 11-deoxy-prostaglandin E 1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1–2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F 2α, 17-phenyl-trinor-prostaglandin E 2 or 11-deoxy-prostaglandin E 1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E 1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F 2α and 17-phenyl-trinor-prostaglandin F 2α increased [Ca 2+] i in a dose-dependent manner; in ODMC1–2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E 2 increased [Ca 2+] i. The present studies suggest that in ODMC1–2 cells, EP 2 and EP 4 receptors but neither EP 1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP 2 and FP receptors, while EP 1 receptors appear to be absent in this cell line.

Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta. Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M coeluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [3H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.

Low CO2 stimulates inositol phosphate turnover and increased inositol 1,4,5-trisphosphate levels in piglet cerebral microvascular smooth muscle cells.

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.

Intracellular Ca 2+ transition assessed differentially among neocortical layers in brain slices during Ischemia-like condition

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1–2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca2+]i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1–2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E2, butaprost and 11-deoxy-prostaglandin E1. The stimulation of cyclic AMP production by prostaglandin E2 was partially inhibited by the EP4 receptor antagonist AH23848. Prostaglandin E2 and 11-deoxy-prostaglandin E1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1–2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F2α, 17-phenyl-trinor-prostaglandin E2 or 11-deoxy-prostaglandin E1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F2α and 17-phenyl-trinor-prostaglandin F2α increased [Ca2+]i in a dose-dependent manner; in ODMC1–2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E2 increased [Ca2+]i. The present studies suggest that in ODMC1–2 cells, EP2 and EP4 receptors but neither EP1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP2 and FP receptors, while EP1 receptors appear to be absent in this cell line.

Activation of phosphoinositide metabolism by parathyroid hormone in growth plate chondrocytes

Parathyroid hormone (PTH) is one of the most potent stimulators of growth plate chondrocyte mitogenesis that has been reported. However, study of the second messenger signaling mechanisms involved in the transduction of the hormone's effects on these cells is incomplete. Our data indicate that in addition to stimulating cyclic adenosine-3′5′-monophosphate metabolism, PTH also activates the phosphoinositide cascade, the pathway responsible for the generation of inositol-1,4,5-trisphosphate dependent Ca 2+ signals. Our conclusion that PTH activates the phosphoinositide cascade is based on data that demonstrate: (1) the Ca 2+ transients evoked by the hormone are dependent on intracellular Ca 2+ stores; (2) the hormone stimulates the release of radiolabeled inositol phosphates from GPC plasma membranes; and (3) the hormone stimulates a greater than 8-fold increase in cytosolic inositol-1,4,5-trisphosphate pool size.

G-proteins coupled to phosphoinositide hydrolysis in the cochlear and vestibular sensory epithelia of the rat are insensitive to cholera and pertussis toxins

In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoinositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P 2y agonists. Such receptor-mediated activation of PLC is expected to be coupled through guanine nucleotide-binding proteins (G-proteins). Although several classes of G-proteins have been identified in the inner ear, nothing is known about the type of G-proteins associated with the phosphoinositide second messenger system in CSE and VSE. Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs). Ten mM NaF plus 10 μM AlCl 3 increased basal InsPs accumulation 2-fold in both CSE and VSE of the rat. Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-γ-S) in saponin-permeabilized tissues. Furthermore, release of InsPs stimulated by both carbamylcholine (CCh) and adenosine 5'-O-[3-thiotriphosphate](ATP-γ-S) was significantly inhibited by 100 μM guanosine 5'-O-[2-thiodiphosphate](GDP-β-S). These results strongly suggest the involvement of G-proteins in the receptor-PLC coupling in CSE and VSE. ADP-ribosylation in membrane fractions of CSE and VSE in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicated the existence of G s- and G i-type G-proteins. However, neither CTX nor PTX affected basal or agonist-stimulated release of InsPs. These observations suggest that muscarinic and P 2y purinergic receptors are coupled to PLC via CTX- and PTX-insensitive G-proteins in CSE and VSE.

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: differential effects of myo-inositol.

To investigate the effects of increasing concentrations of myo-inositol (inositol) on receptor stimulated [3H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [3H]inositol (1 to 30 microM). Carbachol stimulated formation of [3H]inositol trisphosphate (InsP3) and [3H]inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 microM inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 microM), [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 microM inositol, carbachol stimulated [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 microM inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [3H]Ins(1,3,4,5)P4 formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P4 mass formation by seven fold. Furthermore, Li+ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P4 mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P4. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li+, increasing the inositol concentration greatly increases the stimulated component of [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P4 formation is not reflected as an increase in mass formation.

Elevation of inositol tetrakisphosphate parallels inhibition of Ca(2+)-dependent Cl- secretion in T84 cells.

Carbachol and histamine both stimulate calcium-dependent chloride secretion in the colonic epithelial cell line, T84. However, pretreatment of cell monolayers with carbachol blocks subsequent chloride secretion induced by thapsigargin but not the calcium elevation stimulated by this agent, whereas histamine pretreatment blocks neither thapsigargin-induced chloride secretion nor calcium elevation. To examine whether inositol phosphate metabolism might account for this difference, we measured levels of radiolabeled inositol phosphates: Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins-(1,3,4,6)P4, Ins(3,4,5,6)P4, InsP5, and InsP6 after cell stimulation. Although both carbachol and histamine increase Ins (1,4,5)P3 at 5 s, there is a greater and more persistent increase in the levels of Ins(1,3,4)P3 and InsP4 at later time points after carbachol than histamine, which corresponded to the suppression of the chloride secretory response.

Rapid and sensitive anion-exchange high-performance liquid chromatographic determination of radiolabeled inositol phosphates and inositol trisphosphate isomers in cellular systems

A rapid and sensitive high-performance liquid chromatographic method for the determination of multiple inositol phosphates and inositol trisphosphate isomers was developed. The separation of inositol phosphates was optimized by controlling the ionic strength with stepped gradient programs and the pH of mobile phase. Six inositol phosphates were determined within 22 min or the six compounds plus an inositol trisphosphate isomer within 24 min using a single anion-exchange column containing the quaternary ammonium functional group. This technique was successfully applied to the determination of inositol phosphatide turnover by AIF 4 − stimulation in a small amount (5 · 10 5-1 · 10 6 cells) of cultured retinal capillary pericytes. Because of its efficiency, accuracy and applicability to the separation of inositol phosphates from biological samples, this method may be useful in signal transduction studies in cellular systems.

Phospholipase C responses in cells from spontaneously hypertensive rats.

We tested the hypothesis that increased systemic vascular resistance in spontaneously hypertensive rats may be secondary to enhanced phospholipase C activity in response to vasoconstrictor stimuli. Activation of phospholipase C by angiotensin II (Ang II), thromboxane A2, arginine vasopressin, and endothelin-1 was compared in cultured glomerular mesangial cells and mesenteric vascular smooth muscle cells taken from 13- to 14-week-old hypertensive and normotensive Wistar-Kyoto rats (blood pressure, 185 +/- 1 versus 135 +/- 2 mm Hg). Phospholipase C was assessed by measuring cytosolic free calcium and by the accumulation of radiolabeled inositol phosphates. Basal cytosolic calcium did not differ between mesangial cells taken from both strains but was greater in smooth muscle cells from hypertensive rats (210.1 +/- 8.2 versus 149.2 +/- 4.7 nM). The responsiveness of cytosolic calcium and inositol phosphate accumulation to Ang II was significantly enhanced in mesangial cells from hypertensive rats (10(-7) M Ang II: peak increase of calcium, 1,266 +/- 181 versus 603 +/- 93 nM; percent increment of inositol phosphates at 1 minute, 266 +/- 26 versus 98 +/- 10%). Vascular smooth muscle cells from hypertensive rats, when compared with normotensive rats, showed a similar augmentation of Ang II-stimulated intracellular calcium and inositol phosphates. Thromboxane A2-induced enhancement of intracellular calcium and inositol phosphate accumulation in vascular smooth muscle cells was also greater in hypertensive animals. However, the responses to vasopressin and endothelin in mesangial or vascular smooth muscle cells did not differ between the normotensive and hypertensive animals. There was no significant difference in Ang II receptor number and affinity between hypertensive- and normotensive-derived mesangial cells. We conclude that genetically increased blood pressure in rats may be secondary to enhanced post-receptor signaling in glomerular mesangial cells activated by Ang II and to enhanced signaling in vascular smooth muscle cells stimulated by either Ang II or thromboxane A2.

Rapid and selective isolation of radiolabelled inositol phosphates from cancer cells using solid-phase extraction

A method is described for rapid and selective determination of radiolabelled inositol phosphates in cancer cells using solid-phase extraction with Bond Elut strong anion-exchange minicolumns. The inositol phosphates IP1, IP2 and IP3 are selectively eluted with 0.05, 0.3 and 0.8 M ammonium formate—0.1 M formic acid, respectively. Cancer cells are extracted with 10% perchloric acid which is then neutralised prior to loading samples on to the minicolumns. Recovery is 54.1, 66.6 and 61.3% for IP1, IP2 and IP3 with between-day coefficients of variation of 7.6, 6.8 and 1.9%, respectively. When the method was applied to cancer cells high-performance liquid chromatographic analyses confirmed both the identity of the IP1, IP2 and IP3 fractions and showed that there was no detectable cross contamination of these inositol phosphates with each other.

Changes in inositol lipids and phosphates after stimulation of the MAS-transfected NG115-401L-C3 cell line by mitogenic and non-mitogenic stimuli

A neuronal cell line (NG115-401L-C3) was stimulated by mitogenic (angiotensin) and non-mitogenic (bradykinin) peptides and examined for the time course of changes in the levels of radiolabelled inositol phosphates and phospholipids. Both peptides stimulated the time-dependent production of Ins(1,4,5)P3 and related metabolites. Bradykinin caused a much larger increase in Ins(1,4,5)P3 than did angiotensin. However, both peptides stimulated similar rises in the levels of Ins(1,3,4)P3 and InsP4. Bradykinin, but not angiotensin, caused a rapid (within 2 s) fall in the levels of PtdIns(4,5)P2 and PtdIns(4)P. Serum pretreatment of the cells caused a 2-3-fold potentiation of both the responses to bradykinin and angiotensin. Although significant levels of PtdIns(3)P were detected in resting cells, neither mitogenic (angiotensin, insulin-like growth factor I, transforming growth factor beta) nor non-mitogenic (bradykinin, nerve growth factor, interleukin-1) receptor activation changed its levels, arguing against regulation of either PtdIns 3-kinase or PtdIns(3)P phosphatase. We conclude that, as judged by the levels of its product. PtdIns(3)P, the enzyme PtdIns 3-kinase is not activated. This questions the significance of this activity in the receptor-mediated initiation of DNA synthesis.

High-performance liquid chromatographic analysis of radiolabeled inositol phosphates

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 preelude 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 modifled 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.

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.

Dexamethasone does not interfere with hormone-sensitive PI hydrolysis.

Serum, but not epidermal growth factor (EGF), stimulated the release of radiolabeled inositol phosphates from human embryo palate mesenchyme (HEPM) cells prelabeled with [3H]-myoinositol. Pretreatment of cells with 10(-6) M dexamethasone (DEX) for 48 h had no effect on the release of inositol phosphates in response to serum. Furthermore, although treatment of the glucocorticoid-sensitive A/J strain of mouse embryo palate mesenchyme (MEPM) cells with 10(-6) M DEX inhibited their proliferation by 40%, it had no effect on the activity of phospholipase(s) C. However, DEX did enhance the incorporation of [3H]-myoinositol into membrane lipids. We interpret these data to mean that 1) serum factors enhance metabolism of inositol lipids in HEPM cells, 2) DEX does not interfere with the primary events by which agonists utilize metabolism of inositol lipids as a mechanism for transmembrane signaling, and 3) DEX may affect synthesis of phosphoinositides, as reported by Grove et al. (Biochem. Biophys. Res. Commun. 110:200-207, 1983; J. Craniofac. Genet. Dev. Biol. Suppl. 2:285-292, 1986).

Effects of Nucleotides on the Activity of Phospholipase C in Rabbit Thymus Lymphocytes: Differences in assays using endogenous [3H]inositol-prelabeled membranes or exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate

Abstract The influence of nucleotides and pyrophosphate on phospholipase C from rabbit thymocytes was investigated by using two different methods for the determination of phospholipase C activity. In a first approach the release of radiolabeled inositol phosphates from [3H]inositol-labeled membranes was examined. By a second type of experiment the cleavage of exogenously added radiolabeled phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) was measured. Using internally labeled membranes only guanosine 5'-O-(thiotriphosphate) exhibited a stimulatory effect on the phospholipase C suggesting the involvement of a G-protein. When exogenous [3H]PtdIns-4,5-P2 was used as substrate, cleavage of PtdIns-4,5-P2 was stimulated by all nucleotides investigated; in addition pyrophosphate showed a stimulatory effect. From these data we conclude that the increased cleavage of exogenous PtdIns-4,5-P2 induced by GTP analogues is not conclusive in terms of the involvement of a G-protein. Rather than induced by a G-protein this activation may be caused by an increased substrate accessibility. Our experiments with endogenous substrate clearly established the regulatory role of G-proteins for membrane-bound phospholipase C.

Phospholipase C activation by prostaglandins and thromboxane A2 in cultured mesangial cells.

Phospholipase C activation by prostaglandins (PG) and thromboxane A2 (TxA2) was studied in cultured rat and human glomerular mesangial cells, measuring accumulation of radiolabeled inositol phosphates and cytosolic free calcium ([Ca2+]i) with the fluorescent intracellular probe fura-2. Prostaglandin F2 alpha (PGF2 alpha) and TxA2 were found to be the major eicosanoids active on this signaling pathway in rat and human cells, respectively, whereas other PG had lesser or no effects. PGF2 alpha and TxA2 rapidly induced accumulation of inositol trisphosphate accompanied by a simultaneous transient rise of [Ca2+]i, followed by sustained elevation or, in human cells, by a distinct second increase of [Ca2+]i within 45 s. A minor initial accumulation of inositol monophosphate was followed by marked elevation greater than 5 min after the early responses. Responses to different eicosanoids were mediated by separate receptors, functionally characterized using receptor antagonists or heterologous desensitization during sequential applications. Protein kinase C activation by serum and phorbol esters potently inhibited inositol phosphate accumulation and/or [Ca2+]i transients, indicating a pathway for a negative feedback on PG-evoked intracellular signals. We conclude that receptor-mediated phospholipase C activation underlies the biological effects of certain eicosanoids on the glomerular mesangium.