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PubMed1990

High-performance liquid chromatographic separation of inositol phosphate isomers employing a reversed-phase column and a micellar mobile phase.

Surfactants have been employed in high-performance liquid chromatography (HPLC) for the separation of ionic and non-ionic compounds. We have developed a method employing a reversed-phase column and a mobile phase containing a surfactant, hexadecyltrimethylammonium hydroxide (HDTMA+OH-), for the separation of several inositol phosphate positional isomers. Various parameters were studied for their effect on the chromatographic capacity factor (k'). They included the concentration of HDTMA+OH-, the pH of the bulk micellar suspension and the addition of inorganic salts to the mobile phase. Resolution of the inositol monophosphates was controlled by a mixed mechanism, where the predominant elements were electrostatic forces and the formation of micelles. The elution of the inositol polyphosphate isomers was obtained by increasing the amount of a non-polar solvent, in agreement with an ion-pairing process. This method represents an alternative to ion-exchange HPLC. If offers a practical advantage when detection of radiolabeled samples by in-line radioactive flow detectors is required, because low-quenching solvents with good miscibility with scintillant fluids are employed. The analysis of various chromatographic conditions, the system reproducibility and its application to the analysis of biological samples are described.

Chromatography, High Pressure Liquid↗
PubMed1997

A role for tyrosine phosphorylation in generation of inositol phosphates and prostacyclin production in endothelial cells.

We have examined the effects of the protein tyrosine phosphatase inhibitor pervanadate on activation of signal transduction in human umbilical vein endothelial cells. Endothelial cells responded to pervanadate treatment by increasing tyrosine phosphorylation of cellular proteins, including phospholipase C (PLC) gamma 1, generating inositol phosphates (IPs), releasing arachidonic acid, and producing prostacyclin (prostaglandin [PG] I2). The dose and time responses for these events were similar. Tyrosine phosphorylation and formation of IPs in response to pervanadate were reduced by both staurosporine and genistein. Short-term incubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which inhibits thrombin-induced IP generation, did not affect the IP response to pervanadate. To investigate the possible involvement of tyrosine phosphorylation in thrombin or histamine-induced IP generation and PGI2 production, we examined the effects of costimulation with pervanadate and either thrombin or histamine. These responses proved to be different. While the tyrosine phosphorylation of PLC gamma 1 was enhanced after cotreatment with thrombin and pervanadate compared with pervanadate alone, costimulation with pervanadate and histamine resulted in no more tyrosine phosphorylation of PLC gamma 1 than after pervanadate alone. Similarly, while cotreatment with pervanadate and thrombin caused synergistic increase in IP generation, costimulation with pervanadate and histamine resulted in an additive response. However, PGI2 responses to costimulation of pervanadate with either thrombin or histamine were both synergistic. Furthermore, stimulation with histamine, thrombin, or pervanadate all caused tyrosine phosphorylation of a mitogen-activated protein kinase (ERK1/p44). The results suggest that a tyrosine phosphorylation-dependent mechanism has a role in the phosphoinositide signal transduction pathway of human endothelial cells. Moreover, thrombin- but not histamine-induced generation of IPs appears to be partly caused by tyrosine phosphorylation of PLC gamma 1.

Arachidonic Acid↗
PubMed1991

Bombesin and bradykinin increase inositol phosphates and cytosolic free Ca2+, and stimulate DNA synthesis in human endometrial stromal cells.

The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells. In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca2+ and in labelled inositol phosphates when pre-incubated with [3H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca2+ represented a mobilization of Ca2+ from intracellular stores and the second phase of the response depended on Ca2+ influx from the extracellular medium. [3H]Thymidine was added to human cultured endometrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2 alpha, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [3H]thymidine into DNA in quiescent cells. In conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C.

Bombesin↗
PubMed2005

Dynamics of inositol phosphate pools (tris-, tetrakis- and pentakisphosphate) in relation to the rate of phytate synthesis during seed development in common bean (Phaseolus vulgaris).

Four cultivars of Phaseolus vulgaris were grown in a greenhouse and each flower was Labeled with date of anthesis. Seeds were collected at six different stages of development and inositol phosphates (InsPs) were analyzed by ion-pair reversed-phase HPLC. Phytate accumulation was similar in all cultivars, and the specific rate of phytate synthesis (Rs) peaked at about 22 days after flowering (DAF). Variations in the concentrations of the InsP3 and InsP4 pools matched changes in Rs in cultivars Una and Aruã. These results suggest mass-action effects. Thus, the rates of conversion of InsP3 to InsP5 appeared to be at least partly dependent on substrate concentration. Proportional increases in size of all InsP pools up to 21 DAF are also consistent with Little regulation in this part of the pathway. However, this did not appear to be the case in cv. Diamante Negro or with the conversion of InsP5 to InsP6 in all cultivars, where concentrations of the InsP precursor pools peaked earlier or even dropped as Rs peaked, suggesting activation of enzyme activity. Therefore, the evidence is consistent with a control point regulating this metabolic route upstream of InsP3 and possibly in the conversion of InsP5 to InsP6.

Flowers↗
PubMed1991

Changes in inositol phosphate metabolism are associated with terminal differentiation and neoplasia in mouse keratinocytes.

Cultured murine keratinocytes respond to specific Ca2+ levels in medium (Ca0) by expressing markers of terminal differentiation. A Ca0 of 0.05 mM selects for a basal cell phenotype, whereas spinous cell characteristics occur in 0.12 mM Ca2+ and cornified envelopes develop in 1.0 mM Ca2+. An increase in inositol phosphate (InsP) metabolism is associated with higher Ca2+ in the medium. The magnitude of Ca(2+)-stimulated InsP turnover is Ca0-dependent, whereas Ca0 of 0.05, 0.12 or 1.4 mM resulted in a graded, sustained (greater than 24 h) increase in InsPs. Diacylglycerol (DAG) levels similarly increased in a graded manner. The major inositol trisphosphate (InsP3) to accumulate was Ins-1,3,4-P3 while Ins-1,4,5-P3 increased transiently. Neoplastic keratinocyte cell lines, 308 and SP-1, which produce benign tumors and have a mutated c-rasHa gene, do not express markers of differentiation in response to Ca2+. Basal InsP and DAG are 2- and 5-fold higher respectively in the neoplastic cells relative to normal keratinocytes. However, the metabolic profiles of InsPs were similar in normal and neoplastic cells. In neoplastic cells, InsP metabolism was stimulated even further following a Ca2+ increase, and this was graded to the Ca0. The unusual, sustained Ca(2+)-graded InsP response in normal cells is consistent with the turnover of InsP contributing to the signals controlling expression of markers of differentiation. Very high InsP turnover and DAG levels, as in neoplastic cells, may be inhibitory to marker expression.

Animals↗
PubMed1990

Inositol phosphate metabolism and signal transduction.

Activation of a variety of cell surface receptors results in a biphasic increase in the cytoplasmic Ca2+ concentration, due to the release or mobilization of intracellular Ca2+ stores and to the entry of Ca2+ from the extracellular space. Stimulation of these same receptors also results in the hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with the concomitant formation of (1,4,5)inositol trisphosphate [(1,4,5)IP3] and diacylglycerol. It is well established that phosphatidylinositol 4,5-bisphosphate hydrolysis is responsible for the changes in Ca2+ homeostasis. There is strong evidence that (1,4,5)IP3 stimulates Ca2+ release from intracellular stores. The Ca2(+)-releasing actions of (1,4,5)IP3 are terminated by its metabolism through two distinct pathways: (1,4,5)IP3 is dephosphorylated by a 5-phosphatase to (1,4)IP2; alternatively, (1,4,5)IP3 is phosphorylated to (1,3,4,5)IP4 by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known. A recent proposal that (1,4,5)IP3, by emptying an intracellular Ca2+ pool, secondarily elicits Ca2+ entry will be considered. This review summarizes recent studies of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations.

Calcium↗
PubMed1991

Potentiation by a sodium channel activator of effects of lithium ion on cyclic AMP, cyclic GMP and inositol phosphates.

The effects of the lithium ion (Li+) on receptor-mediated synthesis of second messengers were determined, when cellular sodium channels were quiescent or excited, using the murine neuroblastoma clone (N1E-115). In this clone, lithium inhibited the receptor-mediated synthesis of cyclic AMP and cyclic GMP and it also increased the accumulation of inositol phosphates by a receptor-mediated process. When veratridine (20 microM) excited the sodium channel, the effects of lithium were potentiated. However, tetrodotoxin, a sodium channel blocker, completely prevented this potentiation. These results suggest that when neurons are depolarizing actively and intraneuronal levels of lithium increase by entry through the sodium channel, lithium has a more potent intracellular effect. As a result, lithium would have more potent and selective effects in those pathologically-active neurons underlying manic-depressive disorder.

Animals↗
PubMed1996

Insect adipokinetic hormone stimulates inositol phosphate metabolism: roles for both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in signal transduction?

Adipokinetic hormones (AKHs) control the mobilization of energy reserves from the insect fat body as fuels for flight activity. As a part of our investigations on AKH signal transduction, we demonstrate in this study that the inositol lipid cycle may be involved in the action of AKH-I on fat body of the migratory locust. We show that [3H]inositol is incorporated into fat body phosphoinositides in vitro, whose hydrolysis leads to the formation of the following inositol phosphates (InsPs): Ins(1 and/or 3)P, Ins(4)P, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P3, Ins(1,3,4)P3, Ins(1,4,5)P3 and Ins(1,3,4,5)P4. AKH stimulates the formation of these isomers, eliciting an increase in radioactivity of total InsPs already after 1 min. Mass measurements show that Ins(1,4,5)P3 levels are substantially enhanced by AKH, which is indicative of hormonal activation of phospholipase C. In cell-free tissue preparations, Ins(1,4,5)P3 is metabolized through dephosphorylation as well as further phosphorylation. Ins(1,3,4,5)P4 is dephosphorylated primarily to Ins(1,3,4)P3, although the ability for its reconversion to Ins(1,4,5)P3 suggests that in vivo Ins(1,3,4,5)P4 may function as a rapidly mobilizable pool for Ins(1,4,5)P3 generation. Metabolic pathways for the conversion of InsPs to inositol in the locust fat body are proposed.

Animals↗
PubMed1998

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera.

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell-surface Ca2+-sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca2+, 5.5 mM Mg2+, or 10 microM Gd3+ significantly increased 45Ca efflux (p < 0.01). InsP accumulation also increased dramatically when adding these cations to human embryonic kidney (HEK) 293 cells stably transfected with wild-type bovine parathyroid CaR cDNA. Raising the extracellular [Ca2+] ([Ca2+]o) from 0.1 to > 1.4 mM in oocytes and to > 1.0 mM in HEK 293 cells stimulated significant increments in 45Ca efflux and InsP accumulation, respectively (p < 0.05). In contrast, Ca2+ and Mg2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA. In HEK 293 cells stably expressing CaR cDNA, there were significant reductions in cAMP content when adding high Ca2+, Mg2+, Gd3+, or the CaR modulator NPS R-467. Three region-specific anti-CaR peptide antisera immunoblotted bands of approximately 140 and 155 kDa in membranes from CaR-transfected HEK 293 cells and bovine parathyroid tissue. Immunocytochemistry demonstrated strong cell-surface staining in CaR-transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These results indicate that the activation of the recombinant CaR by extracellular Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+]o.

Amino Acid Sequence↗
PubMed1990

Inositol phosphate formation and its relationship to calcium signaling.

The activation of a variety of cell surface receptors results in a biphasic increase in the cytoplasmic Ca2+ concentration due to the release or mobilization of Ca2+ from intracellular stores and to the entry of Ca2+ from the extracellular space. It is well established that phosphatidylinositol 4,5-bisphosphate hydrolysis is responsible for the changes in Ca2+ homeostasis. Stimulation of Ca2(+)-mobilizing receptors also results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with the concomitant formation of inositol (1,4,5) trisphosphate [1,4,5)IP3) and diacylglycerol. Analogous to the adenylyl cyclase signaling system, receptor-mediated stimulation of phospholipase C also appears to be mediated by one or more intermediary guanine nucleotide-dependent regulatory proteins. There is strong evidence that (1,4,5)IP3 stimulates Ca2+ release from intracellular stores. The Ca2(+)-releasing actions of (1,4,5)IP3 are terminated by its metabolism through two distinct pathways. (1,4,5)IP3 is dephosphorylated by a 5-phosphatase to inositol (1,4) bisphosphate; alternatively, (1,4,5)IP3 can be phosphorylated to inositol (1,3,4,5) tetrakisphosphate by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known. A recent proposal that (1,4,5)IP3 secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool will be considered. This review summarizes our current understanding of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations.

Animals↗
PubMed1987

Estrogen increases adrenergic- but not cholinergic-mediated production of inositol phosphates in rabbit uterus.

alpha 1-Adrenergic and muscarinic cholinergic stimuli activate uterine contraction. Estrogen increases adrenergic but not cholinergic sensitivity of rabbit myometrium independent of its effects on adrenoceptor concentration. Since both alpha 1-adrenergic and muscarinic receptors are coupled to phosphatidylinositol hydrolysis, we tested the hypothesis that estrogen increases adrenergic- but not cholinergic-mediated inositol triphosphate production. We found that maximal production of inositol phosphates stimulated by norepinephrine was increased approximately 3-fold following estrogen treatment. Cholinergic-stimulated production was not increased by estrogen treatment. These results demonstrate that the effect of estrogen to enhance uterine adrenergic sensitivity is associated with an increased post-receptor response. The nature of the selectivity of estrogen for adrenergic versus cholinergic response remains obscure, but the results suggest the presence of parallel pathways for receptor activation of a common post-receptor response.

Animals↗
PubMed1999

PKCbetaI mediates the inhibition of P2Y receptor-induced inositol phosphate formation in endothelial cells.

1. Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC)-linked P2Y1 and P2Y2 receptors, for them 2-methylthio-ATP (2MeSATP) and UTP are respective agonists. Here, we have investigated the particular protein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2 receptor-evoked inositol phosphate (IP) formation by phorbol 12-myristate 13-acetate (PMA). 2. Although short-term (20 min) pretreatment of cells with PMA attenuated 2MeSATP- and UTP-induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potentiated 2MeSATP and UTP responses. The IP formation stimulated by NaF was unaltered by PMA pretreatment. 3. Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform betaI, epsilon and mu, but not lambda, from the cytosol to the membrane fraction. 4. Pretreatment of the selective PKC inhibitor Ro 31-8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventional PKCalpha, beta and gamma) and LY 379196 (a selective PKCbeta inhibitor) also dose-dependently inhibited the PMA-mediated desensitization. 5. Transfection of PKCbeta-specific antisense oligonucleotide reduced PKCbetaI protein level and inhibited PMA-mediated PI reduction. 6. RT - PCR analysis showed that PMA treatment for 4 - 24 h up-regulated P2Y1 and P2Y2 receptors at the mRNA levels. 7. These results suggest that PKCbetaI may exert a negative feedback regulation on endothelial P2Y1 and P2Y2 receptor-mediated PI turnover. The down-regulation of PKCbetaI and enhanced P2Y receptor expression together might contribute to the late PI enhancing effect of PMA.

Adenosine Triphosphate↗
PubMed1992

Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number.

After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Aluminum fluoride stimulates inositol phosphate metabolism and inhibits expression of differentiation markers in mouse keratinocytes.

Mouse keratinocytes are induced to differentiate in vitro by elevating the level of extracellular calcium from 0.05 mM, where keratinocytes express a basal cell phenotype, to greater than 0.10 mM, where they express the differentiated phenotype. This process has been associated with a rapid, sustained increase in inositol phosphate (InsP) turnover, which precedes the expression of differentiation-specific proteins. In 0.05 mM Ca2+ medium, aluminum and fluoride salts (AIF4-), which combine to activate nonspecifically heterotrimeric guanine nucleotide-binding (G) proteins, cause a concentration-dependent increase in InsP metabolism in keratinocytes, and generate elevated intracellular diacylglycerol levels. This is associated with an inhibition of cell growth. Treatment with both AIF4- and Ca2+ greater than 0.10 mM resulted in an additive increase in InsP turnover, implying the presence of at least two responsive InsP pools. AIF4- inhibited the expression of differentiation markers induced by Ca2+ greater than 0.10 mM and altered the morphology of keratinocytes from squamous to dendritic, which was reversible upon withdrawal of AIF4-. Neoplastic keratinocytes, in which basal levels of InsP metabolism are higher than in normal cells, do not differentiate in response to Ca2+. Neoplastic keratinocytes responded to AIF-4 treatment with an even greater rise in InsP metabolism. AIF-4 also inhibited cell growth and reversibly altered morphology in neoplastic keratinocytes. These data suggest that InsP metabolism in keratinocytes is at least partially regulated by a G protein mechanism. Furthermore, an increase in InsP metabolism is not sufficient to stimulate differentiation and may be inhibitory to differentiation if exceeding limited increases. However, these observations cannot exclude the possibility that other AIF-4 stimulated pathways involving G or non-G proteins can also influence keratinocyte biology.

Aluminum↗
PubMed1992

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

Calcium↗
PubMed1987

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes.

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.

Calcium↗
PubMed1996

Mutation of an aspartate at position 63 in the human platelet-activating factor receptor augments binding affinity but abolishes G-protein-coupling and inositol phosphate production.

Platelet-activating factor is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor (GPCR) family. In the present report, we demonstrate that the highly conserved aspartate 63 is critical in G protein coupling of the PAF receptor: substitution of an asparagine for the aspartate 63 (D63N) abolished inositol phosphate production following agonist stimulation; moreover, binding isotherms of the D63N mutant were monophasic and unaffected by GTPgammaS. We also demonstrate that aspartate 63 is not involved in direct interaction with the agonist: the D63N mutant displayed a higher intrinsic affinity for PAF than the uncoupled WT receptor. Sodium decreased specific (3)H-PAF and antagonist (3)H-WEB2086 binding to the PAF receptor, but the aspartate 63 residue was not involved in this regulation, contrary to cognate aspartate residues in other GPCRs. Our data suggest that aspartate 63 in the PAF receptor may be involved in the structural requirement for G protein coupling to the receptor and may contribute to receptor affinity for the ligand.

Amino Acid Sequence↗