Phosphatidylinositide Hydrolysis Research Articles

Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization.

Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with beta-arrestin 1 but continues to exhibit association with adaptin 2 alpha and beta subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca(2+) mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to beta-arrestin 1 but exhibit decreased binding to adaptin 2alpha and beta. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides: ENDOGENOUS 5-HT2C RECEPTORS IN CHOROID PLEXUS EPITHELIAL CELLS

To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.

Gestational exposure to methylmercury alters neurotrophin- and carbachol-stimulated phosphatidylinositide hydrolysis in cerebral cortex of neonatal rats.

Neurotrophin-stimulated signal transduction through the Trk receptors has been implicated in the development and survival of the nervous system. Phospholipase Cgamma (PLCgamma) is an early downstream effector for the Trk receptors, and catalyzes the hydrolysis of phosphatidylinositides (PI) to inositol phosphates (IPs) and diacylglycerol. The current study demonstrated that PI hydrolysis can be used as a measure of Trk stimulation in slices from neonatal rat brain, and examined changes in the ontogeny of neurotrophin-stimulated PI hydrolysis in animals exposed to MeHg during gestation. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) stimulated PI hydrolysis in neocortical and cerebellar slices from neonatal rats in a concentration-dependent manner (30-1000 ng/ml). The neurotrophin-stimulated PI hydrolysis was completely blocked by K-252a, a compound known to inhibit Trk autophosphorylation. To examine the effects of MeHg on PI hydrolysis, Long-Evans dams were dosed p.o. on gestational days 6-15 with 0 or 2 mg/kg/day MeHg dissolved in saline. Pups were sacrificed on postnatal days (PND) 1, 4, 10, 14, and 21 and brain slices prepared from the neocortex and cerebellum. Neurotrophin-stimulated PI hydrolysis was highest on PND 1-4 and decreased with age in slices from both regions. Prior exposure to MeHg had no effect on NT-3 or BDNF-stimulated PI hydrolysis in the cerebellum; however, in the neocortex carbachol-stimulated PI hydrolysis and NT-3-stimulated PI hydrolysis were decreased on PND 1. In addition, NT-3-stimulated PI hydrolysis was increased on PND 14 compared to controls. Nerve growth factor (NGF), which had no effect in controls, increased PI hydrolysis in MeHg exposed animals. Acute exposure to 10 micro M MeHg increased basal PI hydrolysis in cortical slices and increased NT-3- and BDNF-stimulated PI hydrolysis in slices from the cerebellum. These data indicate that gestational exposure to MeHg can alter neurotrophin signaling in the neocortex at early postnatal times.

Histamine-induced calcium mobilization in single cultured cells expressing histamine H1 receptors: a relationship between its sensitivity and the density of H1 receptors.

Stimulation of histamine H1 receptors initiates the hydrolysis of phosphatidylinositides and results in the production of inositol (1, 4,5)-triphosphate and intracellular Ca2+ mobilization. Although the mechanism for signal transduction via the H1 recptor has been extensively investigated, little is known about the correlation between the sensitivity of histamine-induced Ca2+ mobilization and the density of H1 receptors in cultured cells. Cytosolic free Ca2+ concentration ([Ca2+]i) after stimulation by histamine was monitored in single CHO and rat C6-glioma cells stably expressed with H1 receptors and astrocytoma 1321N1 cells using the Ca2+-sensitive dye Indo-1 and dynamic single cell imaging techniques (ACAS 570 laser cytometer). Both of the H1 receptor-expressed CHO cells and C6-glioma cells were over 10 times more sensitive to histamine than astrocytoma 1321N1 cells in which H1 receptors were naturally present. The density of H1 receptors in the transfected cells was also more than 10-fold that of 1321N1 cells. In addition, inhibition of intracellular Ca2+-ATPase by thapsigargin elicited an increase in [Ca2+]i in H1 receptor-overexpessed cells and astrocytoma 1321N1 cells with similar sensitivity. These data suggest that the sensitivity of Ca2+ mobilization by histamine in these cells was correlatively augmented with the increase in the density of H1 receptors.

Lithium chloride does not inhibit the proliferation of L6 myoblasts by decreasing intracellular free inositol.

We conducted a series of experiments to determine whether lithium chloride (LiCl) inhibited the proliferation of L6 myoblasts by reducing the availability of intracellular free inositol. After the myoblasts were plated in DMEM + 10% fetal bovine serum (FBS) for 24 h, medium was replaced with DMEM + 10% FBS containing 0 (control), 5, 10, or 20 mM LiCl. Cell number, protein content, and [3H]thymidine incorporation into DNA were determined at 24-h intervals. Control cells exhibited a 3.8-fold increase in cell number by 96 h in culture. Although 5 mM LiCl did not affect the rate or extent of proliferation, 10 and 20 mM LiCl caused 36 and 86% decreases, respectively (P < .05), in cell number by 96 h in culture. The effects of LiCl could not be overcome by the addition of free inositol (up to 20 mM) to the medium. Lithium chloride caused 4.6- and 7.3-fold increases (P < .05) in lactate dehydrogenase activity in culture media after 96 h of exposure to 10 and 20 mM LiCl, respectively, indicating loss of viability after chronic treatment. However, the acute effects of LiCl after 24 h of treatment were reversible, as indicated by a rapid resumption of proliferation following removal of LiCl. Concentrations of 5, 10, and 20 mM LiCl caused 4.7-, 8.2-, and 9.1-fold increases (P < .05), respectively, in the accumulation of [3H]inositol within the inositol monophosphate pool. Treatment of cells with 10 and 20 mM LiCl also increased (P < .05) label recovered as inositol bisphosphate. Rather than depress phosphoinositide synthesis, the addition of 10 and 20 mM LiCl dose-dependently increased (P < . 05) the incorporation of [3H]inositol into phosphatidylinositol and phosphatidylinositol-4-phosphate. These results indicate that LiCl does not decrease proliferation of L6 myoblasts via a depletion in the intracellular free inositol pool. Instead, LiCl may block the hydrolysis of phosphatidyl inositides.

Synthesis and Kinetic Evaluation of Inhibitors of the Phosphatidylinositol-Specific Phospholipase C from Bacillus cereus.

Substrate analogues of phosphatidylinositol (1) were synthesized and evaluated as potential inhibitors of the bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. The chiral analogues of the water-soluble phospholipid substrate 5 were designed to probe the effects of varying the inositol C-2 hydroxyl group, which is generally believed to serve as the nucleophile in the first step of the hydrolysis of phosphatidylinositols by PI-PLC. In the analogues 6-9, the C-2 hydroxyl group on the inositol ring of the phosphatidylinositol derivatives was rationally altered in several ways. Inversion of the stereochemistry at C-2 of the inositol ring led to the scyllo derivative 6. The inositol C-2 hydroxy group was replaced with inversion by a fluorine to produce the scyllo-fluoro inositol 7 and with a hydrogen atom to furnish the 2-deoxy compound 8. The C-2 hydroxyl group was O-methylated to prepare the methoxy derivative 9. The natural inositol configuration at C-2 was retained in the nonhydrolyzable phosphorodithioate analogue 10. The inhibition of PI-PLC by each of these analogues was then analyzed in a continuous assay using D-myo-inositol 1-(4-nitrophenyl phosphate) (25) as a chromogenic substrate. The kinetic parameters for each of these phosphatidylinositol derivatives were determined, and each was found to be a competitive inhibitor with K(i)'s as follows: 6, 0.2 mM; 10, 0.6 mM; 8, 2.6 mM; 9, 6.6 mM; and 7, 8.8 mM. This study further establishes that the hydrolysis of phosphatidylinositol analogues by bacterial PI-PLC requires not only the presence of a C-2 hydroxyl group on the inositol ring, but the stereochemistry at this position must also correspond to the natural myo-configuration. For future inhibitor design, it is perhaps noteworthy that the best inhibitors 6 and 10 each possess a hydroxyl group at the C-2 position. Several of the inhibitors identified in this study are now being used to obtain crystallographic information for an enzyme-inhibitor complex to gain further insights regarding the mechanism of hydrolysis of phosphatidylinositides by this PI-PLC.

Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation

Signal transduction on platelet activation involves phosphoinositide- specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet- activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5′-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate- induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

Human Platelet Signaling Defect Characterized by Impaired Production of Inositol-l,4,5-Triphosphate and Phosphatic Acid and Diminished Pleckstrin Phosphorylation: Evidence for Defective Phospholipase C Activation

Signal transduction on platelet activation involves phosphoinositide-specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet-activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate-induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.

Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.

Sympathetic sprouting reverses decreases in membrane-associated activity of protein kinase C following septohippocampal denervation of the rat hippocampus

Hippocampal sympathetic ingrowth (HSI), a form of neuronal plasticity, is induced by medial septal lesions and consists of the sprouting of peripheral sympathetic fibers, arising from the superior cervical ganglion, into the dentate gyrus and CA 3 region of the hippocampus. HSI has been previously shown to alter learned and spontaneous behaviors, phosphatidyl inositide hydrolysis, and the antagonist binding kinetics of both muscarinic cholinergic receptors and phorbol ester receptors. We now report that sympathetic sprouting reverses decreases in membrane-associated activity of protein kinase C (PKC) following septohippocampal denervation of the rat hippocampus. Further, no changes were found in α, β or γ PKC isoenzymes among experimental groups, suggesting that the group A PKC isoforms do not mediate the observed changes in activity and phorbol ester binding.

Nongenomic actions of progesterone on human spermatozoa

Progesterone's (P) stimulatory actions on human spermatozoa have been known for many years. P indeed appears to be the main sperm stimulator present in women's biological fluids, particularly the follicular fluid. The nongenomic nature of the biological effects of P on human spermatozoa has been demonstrated only recently. P and 17-α-hydroxy P have been shown to increase sperm intracellular calcium, phosphatidylinositide hydrolysis, and tyrosine phosphorylation of proteins, and to induce the acrosome reaction (AR), through a rapid, nongenomic mechanism. The effect on calcium is due to influx of the ion from the extracellular medium, as it is inhibited by the calcium chelator EGTA and appears to be mediated by P-binding sites present on the sperm surface, particularly at the head level. The nature of such binding sites has not been addressed so far, but the lack of inhibition of P action by the potent antiprogestin RU486 strongly suggests a biochemical difference from the genomic ones. Evidence exists for involvement of the phospholipid platelet-activating factor (PAF), polyamines, tyrosine kinase activation, proteases, and other factors in P-mediated calcium increase and acrosome reaction. Recent studies suggest the possibility that the sperm's response to P may be functionally related to their fertilizing ability, thus opening new perspectives in the possible development of a predictive test in the assisted reproductive techniques.

Distribution of metabotropic glutamate receptor mGluR5 immunoreactivity in rat brain.

The receptor mGluR5 is a metabotropic glutamate receptor with messenger RNA abundantly present throughout cortex, hippocampus, and caudate/putamen that is also coupled to phosphatidyl inositide hydrolysis and calcium mobilization. In this study, the distribution of mGluR5 was examined in rat brain by immunocytochemistry. The antibody utilized is highly specific and does not cross react with the most closely related other metabotropic glutamate receptor, as determined by Western blot analysis of nonneuronal cells transfected with metabotropic receptor coding sequences. The receptor mGluR5 is widely expressed with the highest density in olfactory bulb, caudate/putamen, lateral septum, cortex, and hippocampus, as confirmed with both immunocytochemistry and Western blot analysis. Electron microscopic studies in hippocampus and cortex indicate that the labeling is mostly on membranes of dendritic spines and shafts. Light and electron microscopic evidence indicates that some mGluR5 immunoreactivity is located in presynaptic axon terminals, suggesting that mGluR5 may function as a presynaptic receptor.

L-2-amino-3-phosphonopropionic acid competitively antagonizes metabotropic glutamate receptors 1α and 5 in Xenopus oocytes

The aspartate analog 2-amino-3-phosphonopropionic acid (AP3) antagonizes glutamate-stimulated phosphatidyl inositide hydrolysis in brain slices, but is reportedly weak or ineffective in antagonizing the phosphatidyl inositide-coupled cloned metabotropic glutamate receptors 1α and 5. Thus we examined the pharmacological properties of AP3 on mGlu 1α and mGlu 5 receptor responses in Xenopus oocytes. DL-AP3 antagonized mGlu 1α and mGlu 5 responses, but antagonism was overcome at high glutamate concentrations consistent with competitive inhibition ( IC 50 = 2.1 nM for mGlu 1 α ). Both responses were also inhibited by (RS)-α-methyl-4-carboxyphenylglycine (MCPG). We conclude that the available antagonists cannot distinguish between the mGlu 1α receptor and mGlu 5 receptor, and that antagonism by AP3 may be obscured in the presence of high agonist concentrations or in cells with spare receptors.

Complement peptide C3a inhibits IgE-mediated triggering of rat mucosal mast cells.

The relationship between mast cells' secretory response to stimulation via their type 1 Fc epsilon receptors (Fc epsilon RI) and that provided by the C3a fragment of the complement system was investigated in the rat mucosal-type mast cell line RBL-2H3. These cells are known to be unresponsive to the so-called 'peptidergic' stimulus provided by cationic agents, such as anaphylatoxins, neuropeptides or polyamines. We now observed that C3a effectively inhibits the Fc epsilon RI clustering induced secretion of RBL-2H3 cells. This inhibition is dose-dependent and takes place at a C3a concentration range of 0.4-12.5 nM, i.e. at least three orders of magnitude lower than those where this anaphylatoxin exerts its secretory stimulus to 'serosal' mast cells. In order to identify where C3a interferes in the Fc epsilon RI coupling cascade, we have studied its effect on the cells' protein phosphorylation pattern, hydrolysis of phosphatidyl inositides, transient rise in free cytosolic Ca2+ ion concentration and Ca2+ uptake. All these processes were found to be inhibited by a similar C3a concentration range.

Ca2+-ATPase Inhibitors Induce Interleukin-2 Synthesis and T Cell Proliferation

In Jurkat cells, the three Ca2+-ATPase blockers, thapsigargin, cyclopiazonic acid, and di-teributylhydroquinone (DtBuHQ) induced both a release of Ca2+ from intracellular stores and a Ca2+ influx. In contrast to CD3 mAb, the Ca2+-ATPase inhibitors did not induce the formation of inositol trisphosphate from the hydrolysis of phosphatidylinositides. Emptying intracellular Ca2+ stores was accompanied by a decrease of phosphatidylserine (PtdSer) synthesis as previously observed in PHA- or CD3 mAb-treated Jurkat cells. In the presence of a phorbol ester able to activate protein kinase C, TPA, the three Ca2+-ATPase inhibitors induced Jurkat cells to synthesize large amounts of interleukin-2 demonstrating that early signal transduction mechanisms can be bypassed by Ca2+-ATPase inhibitors. In purified human peripheral blood T lymphocytes, the same inhibitors induced moderate if any cytosolic Ca2+ rise, in the absence of external calcium. Nevertheless analysis of PtdSer synthesis suggested that intracellular stores were efficiently depleted by DtBuHQ and cyclopiazonic acid but not by thapsigargin. In contrast, the three compounds induced similar Ca2+ influx. However, in the presence of TPA, cyclopiazonic acid and DtBuHQ induce highly purified T cells to proliferate while thapsigargin did not, suggesting that the status of internal Ca2+ store may have a decisive role in T cell activation.

Dual effect of thrombin on voltage-dependent Ca2+ channels of portal vein smooth muscle cells.

Thrombin induces a number of physiological responses in several types of cells. To determine the action of thrombin in the vein, the electrophysiological and mechanical effects of thrombin were studied in rat portal vein smooth muscle cells. Ca2+ channel currents were recorded using the whole-cell patch-clamp technique. Thrombin had both inhibitory and stimulatory effects on the Ca2+ channel current. The inhibitory effect was reversed on washout of thrombin, whereas the stimulatory effect was maintained after thrombin was removed. Thrombin (1 unit/ml) produced a reversible decrease of 27.3 +/- 3.3% (n = 12) in the current amplitude and a sustained increase of 71.2 +/- 12.9% (n = 20). The thrombin-induced inhibition of Ca2+ channel current was blocked by the thrombin inhibitor hirudin and by the protease inhibitor leupeptin. The stimulatory effect of thrombin was inhibited by hirudin, by intracellular application of guanosine 5'-O-(beta-thio)diphosphate, and by antiphophatidylinositide antibodies but not by pertussis toxin. The thrombin-induced enhancement of the Ca2+ channel current amplitude was not observed when the current was previously stimulated by phorbol 12,13-dibutyrate. This suggests that the inhibitory effect of thrombin was related to its proteolytic activity and that the stimulatory effect involved activation of a pertussis toxin-insensitive GTP-binding protein, phosphatidylinositide hydrolysis, and protein kinase C activation. Both thrombin effects occurred in the same concentration range (0.001-10 units/ml). The thrombin-induced contraction of portal vein strips was completely inhibited by isradipine, and thrombin did not produce an increase in cytosolic [Ca2+], measured by indo-1 fluorescence in cells clamped at -50 mV, sufficient to activate Ca(2+)-dependent chloride current.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of protein kinase C activation on norepinephrine-induced phosphatidylinositide hydrolysis in intact rat aorta

The purpose of this study was to investigate the role of protein kinase C in the regulation of α 1-adrenoceptor-mediated phosphatidylinositide hydrolysis in intact vascular smooth muscle. Phorbol myristate acetate (0.1 and 1 μM) and staurosporine inhibited and potentiated, respectively, norepinephrine-induced inositol phosphate formation in intact rat aorta. In contrast, 30 μM prostaglandin F 2α, which activated protein kinase C to a similar magnitude as 1 μM phorbol myristate acetate, was without effect on norepinephrine-induced inositol phosphate formation. These results suggest that protein kinase C activated in response to physiologic agonists, but not in response to phorbol esters, may be compartmentalized within the smooth muscle cell.

Choline phospholipids: signal transduction and carcinogenesis.

Phospholipids act as vital elements in transmembrane signaling. Agonist-induced hydrolysis of phosphatidylinositides has been established as a major mechanism for transmitting messages into the interior of cells via protein phosphorylation cascades, ultimately regulating gene transcription. There is a growing body of evidence that choline phospholipids (phosphatidylcholine, sphingomyelin, and their metabolites) also are important mediators and modulators of transmembrane signaling. These functions may explain how choline phospholipids influence normal physiological processes as well as a diverse group of pathological processes.

Signal transduction in human B cells initiated via Ig beta ligation.

Ig alpha and Ig beta heterodimers are non-covalently associated with Ig to compose the antigen receptor complexes on B cells. The demonstration that different sets of tyrosine kinases bind to the cytoplasmic tails of Ig alpha and Ig beta suggests that Ig alpha and Ig beta may activate distinct second messenger pathways. In this study, we examined the effects of mAbs against an exposed epitope of human Ig beta on pre-B and B cell triggering. Cross-linkage of Ig beta on B cells leads to activation of tyrosine kinases, hydrolysis of phosphatidylinositides, and elevation of intracellular Ca2+, effects qualitatively identical to those of anti-mu mAbs. Our observations thus indicate that cross-linking of Ig beta does not segregate signal transduction pathways connected with the cytoplasmic tails of Ig alpha and Ig beta. Ig alpha ligation has been reported to be more effective in triggering pre-B than B cells, whereas our results indicated that Ig beta ligation is more efficient in triggering B than pre-B cells. In addition to their activation properties, the anti-Ig beta mAbs effectively modulated B cell receptor complexes and blocked terminal differentiation of all plasma cell isotypes. The findings support the idea that anti-Ig beta could serve as a universal B cell immunosuppressant.

Interaction between phosphoinositide turnover system and cyclic AMP pathway for the secretion of pancreastatin and somatostatin from QGP-1N cells

It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12- myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin.