PHAS-I as a link between mitogen-activated protein kinase and translation initiation.

PHAS-I is a heat-stable protein (relative molecular mass approximately 12,400) found in many tissues. It is rapidly phosphorylated in rat adipocytes incubated with insulin or growth factors. Nonphosphorylated PHAS-I bound to initiation factor 4E (eIF-4E) and inhibited protein synthesis. Serine-64 in PHAS-I was rapidly phosphorylated by mitogen-activated (MAP) kinase, the major insulin-stimulated PHAS-I kinase in adipocyte extracts. Results obtained with antibodies, immobilized PHAS-I, and a messenger RNA cap affinity resin indicated that PHAS-I did not bind eIF-4E when serine-64 was phosphorylated. Thus, PHAS-I may be a key mediator of the stimulation of protein synthesis by the diverse group of agents and stimuli that activate MAP kinase.

Modulation of the protein kinase activity of mTOR.

mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope.

Effects of focal cerebral ischemia on expression and activity of inositol 1,4,5-trisphosphate 3-kinase in rat cortex.

Results from this study clearly indicate that Ins(1,4,5)P3 3-kinase is a target enzyme of cerebral ischemia insult. This enzyme is responsible for removal of Ins(1,4,5)P3 which, in turn, plays an important role in the maintenance of intracellular Ca2+ homeostasis. Not only did a time-dependent decrease in enzyme activity occur due to the focal cerebral ischemic insult, but there was also a second phase for the decline in enzyme activity around 6 h after the insult. Examination of the mRNA for the 3-kinase in frozen brain sections suggested an increase in message at a time (around 8 h) prior to development of tissue infarct. Since the initial decline in enzyme activity during ligation correlated well with the time for development of an infarct, assay of this enzyme could be used as a biochemical marker of cerebral ischemic insult.

Metabolism of inositol 1,4,5-trisphosphate in mouse brain due to decapitation ischemic insult: effects of acute lithium administration and temporal relationship to diacylglycerols, free fatty acids and energy metabolites.

Previous studies have shown that global cerebral ischemia induced by decapitation leads to the stimulated hydrolysis of poly-phosphoinositides. In this study, the decapitation model was used to further examine the temporal events related to metabolism of Ins(1,4,5)P3 and the release of diacylglycerols (DGs) and free fatty acids (FFAs) in the mouse brain. Since lithium administration is known to inhibit inositol monophosphatase activity in brain, the effects of acute lithium injection on Ins(1,4,5)P3 metabolism were also examined. Cerebral ischemia induced by decapitation of C57 Bl/6J mice resulted in transient increases of Ins(1,4,5)P3, Ins(1,4)P2 and Ins(4)P which peaked at 35, 65 and 125 s, respectively. The level of Ins(1)P, however, was not altered. Mice administered lithium by intraperitoneal injection (8 meq/kg for 4 h) gave rise to a 40- and 4-fold increase in levels of Ins(1)P, Ins(4)P, respectively, a 20% increase in levels of Ins(1,4)P2 but no apparent changes in the levels of Ins(1,4,5)P3. Decapitation also induced an increase in the levels of DGs and FFAs. Unlike the transient appearance of Ins(1,4,5)P3, however, DG levels increased steadily for 2 min and then reached a plateau whereas the FFAs showed a lag time of 35 s prior to a biphasic increase. During the initial 2 min after decapitation, there was a preferential increase in the DG species containing 18:0 and 20:4. Lithium administration did not alter the decapitation-induced release of DG and FFA. As expected, decapitation gave rise to a rapid decrease in the levels of phosphocreatine and ATP and the decline in ATP was marked by a transient appearance of ADP and a concomitant increase in AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

The cholinergic receptor-linked phosphoinositide metabolism in mouse cerebrum and cerebellum in vivo.

The cholinergic receptor-linked poly-phosphoinositide hydrolysis was studied in mouse cerebrum and cerebellum after prelabeling the brain with [3H]inositol. I.p. injection of Li (8 meq/kg) to C57Bl/6J mice for 4 h resulted in 14- and five-fold increases in [3H]inositol-labeled inositol monophosphate (IP1) in cerebrum and cerebellum, respectively. The labeled inositol bisphosphate (IP2) was also increased 83 and 19% in cerebrum and cerebellum, respectively. Prior injection of atropine (100 mg/kg) resulted in inhibition of Li-induced increases in labeled IP1 by 74 and 56% in cerebrum and cerebellum, respectively. Administration of pilocarpine (20 mg/kg) to the Li-treated mice for 30 min resulted in further increases in labeled IP1 and IP2 and a concomitant decrease in labeled inositol in cerebrum but not in cerebellum. Mass measurements of IP1 and IP2 isomers by HPLC revealed that inositol 1-monophosphate (Ins(1)P), inositol 4-monophosphate (Ins(4)P) and inositol 1,4-bisphosphate (Ins(1,4)P2) were all increased by pilocarpine administration in the Li-treated mouse cerebrum. The effects of pilocarpine administration in mouse cerebrum (increases in IP1 and IP2) could be completely inhibited by preinjection of atropine. Atropine injection also decreased the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Surprisingly, a decrease in Ins(1,4,5)P3 level was also found in non-Li-treated mice after pilocarpine administration (30 mg/kg, 10-40 min). Except for the increase (20%) in [32P]-labeled PIP in the cerebrum, Li or Li together with pilocarpine administration did not alter the levels of [3H]inositol or [32P]phosphate-labeled phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of the stereoselectivity of L-glutamate receptors by synthetic 4(R)- and 4(S)-substituted L-glutamate analogues.

R- and S-stereoisomers of 4-substituted L-glutamate analogues are used to study the stereoselectivity of L-glutamate receptors. It is found that 4(R)-substituted analogues are more potent than their 4(S)-isomers in interacting with L-glutamate receptors both at porcine brain synaptic junctions and on drosophila muscles. This demonstrates that the ligand recognition site of L-glutamate receptors has chiral selectivity discriminating L-glutamate analogues with bulky 4(R)- and 4(S)-substituent groups.

PHAS proteins as mediators of the actions of insulin, growth factors and cAMP on protein synthesis and cell proliferation.

PHAS-I and PHAS-II are members of a newly discovered family of proteins that regulate translation initiation. PHAS-I is expressed in a wide variety of cell types, but it is highest in adipocytes, where protein synthesis is markedly increased by insulin. PHAS-II is highest in liver and kidney, where very little PHAS-I is found. PHAS proteins bind to eIF-4E, the mRNA cap-binding protein, and inhibit translation of capped mRNA in vitro and in cells. In rat adipocytes PHAS-I is phosphorylated in at least five sites, all of which conform to the consensus, (Ser/Thr)-Pro. Both PHAS proteins are phosphorylated in response to insulin or growth factors, such as EGF, PDGF and IGF-1. Phosphorylation in the appropriate site(s) promotes dissociation of PHAS/eIF-4E complexes. This allows eIF-4E to bind to eIF-4G (p220), thereby increasing the amount of the eIF-4F complex and the rate of translation initiation. Increasing cAMP promotes PHAS-I dephosphorylation and increases binding to eIF-4E. Unlike PHAS-I, PHAS-II is readily phosphorylated by PKA in vitro, suggesting that regulation of the two proteins differs. However, increasing cAMP in cells also promotes dephosphorylation of PHAS-II. Thus, PHAS proteins appear to be key mediators not only of the stimulatory effects of insulin and growth factors on protein synthesis, but also of the inhibitory effects of cAMP. Moreover, by controlling eIF-4E PHAS proteins may be involved in the control of cell proliferation, as increasing eIF-4E is mitogenic and can even cause malignant transformation of cells. MAP kinase readily phosphorylates both PHAS-I and PHAS-II in vitro, but inhibiting activation of MAP kinase does not attenuate the effects of insulin on increasing phosphorylation of the PHAS proteins in adipocytes or skeletal muscle. MAP kinase phosphorylates neither PHAS-I nor PHAS-II at a significant rate when the proteins are bound to eIF-4E. Therefore, the role of MAP kinase in promoting the dissociation of PHAS/eIF-4E complexes is not clear. Of several protein kinases tested, only casein kinase-II phosphorylated PHAS-I when it was bound eIF-4E. Indeed, the bound form of PHAS-I was phosphorylated more rapidly than the free form. However, it is unlikely that casein kinase II regulates either PHAS protein, as the major site (Ser111) in PHAS-I phosphorylated by casein kinase II in vitro is not phosphorylated in adipocytes, and PHAS-II is not a substrate for casein kinase-II. Pharmacological and genetic evidence indicates that the mTOR/p70S6K pathway is involved in the control of PHAS-I and -II. Thus, PHAS proteins may be mediators of the effects of this pathway on protein synthesis and cell proliferation.

Synergistic effect of trichostatin A and scriptaid on the development of cloned rabbit embryos.

The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits.

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage.

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.

Dual bronchodilatory and pulmonary anti-inflammatory activity of RO5024118, a novel agonist at vasoactive intestinal peptide VPAC2 receptors.

BACKGROUND AND PURPOSE: Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC(1) and VPAC(2). RO5024118 is a selective VPAC(2) receptor agonist derived via chemical modification of an earlier VPAC(2) agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118. EXPERIMENTAL APPROACH: Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model. KEY RESULTS: RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting ß-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting ß-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8(+) cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Activation of ribosomal protein S6 kinases does not increase glycogen synthesis or glucose transport in rat adipocytes.

Rat adipocytes were incubated with insulin or epidermal growth factor (EGF) before the mitogen-activated protein (MAP) kinases, ERK-1 and ERK-2, and the ribosomal protein S6 kinases, Rsk-2 and p70S6K, were resolved by ion exchange chromatography and identified by immunoblotting. EGF was more effective than insulin in increasing the activity of two kinases that reacted with Rsk-2 antibody (2- and 2.5-fold with EGF versus 1.6- and 1.2-fold with insulin). EGF was also more effective than insulin in increasing the activity of ERK-1 (5-fold versus 2-fold) and ERK-2 (2.5-fold versus 1.5 fold). The activity of p70S6K was increased by approximately the same extent by EGF and insulin (1.7-fold versus 2-fold). Rapamycin blocked activation of p70S6K by insulin, but it did not attenuate the effect (2-fold) of insulin on increasing the glycogen synthase activity ratio (+/-glucose-6-P). Insulin increased glucose incorporation into glycogen and 2-deoxyglucose uptake by approximately 5-fold, whereas EGF and phorbol 12-myristate were without effect. Thus, activation of MAP kinases and ribosomal protein S6 kinases appears insufficient to activate glycogen synthase or glucose transport, the two key components in the stimulation of glycogen synthesis by insulin.

Control of PHAS-I phosphorylation in 3T3-L1 adipocytes: effects of inhibiting protein phosphatases and the p70S6K signalling pathway.

PHAS-I is a recently discovered regulator of translation initiation. Non-phosphorylated PHAS-I binds and inhibits eukaryotic initiation factor-4E, the mRNA cap-binding protein that mediates a rate-limiting step in translation initiation. When PHAS-I is phosphorylated in response to insulin, the PHAS-I/eukaryotic initiation factor-4E complex dissociates. The present study was conducted to investigate mechanisms involved in the control of PHAS-I. Phosphorylation of PHAS-I was monitored by immunoblotting after subjecting extracts to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This was possible because phosphorylation markedly decreases the electrophoretic mobility of PHAS-I. Incubating 3T3-L1 adipocytes with rapamycin and wortmannin inhibited insulin-stimulated phosphorylation of PHAS-I at concentrations similar to those that inhibited activation of p70S6K. Both agents increased the amount of PHAS-I that co-purified with eukaryotic initiation factor-4E when extracts were fractionated using a cap affinity resin, indicating that PHAS-I binding to the initiation factor was increased. Incubating adipocytes with the protein phosphatase inhibitors, calyculin A and okadaic acid, increased PHAS-I phosphorylation and opposed the effects of rapamycin on decreasing PHAS-I phosphorylation. However, neither okadaic acid nor calyculin A abolished the effects of rapamycin on PHAS-I. These results suggest that the phosphorylation of PHAS-I in response to insulin occurs via the p70S6K signalling pathway. By regulating eukaryotic initiation factor-4E, PHAS-I may have important roles in the control of both protein synthesis and mitogenesis.

Control of the translational regulators PHAS-I and PHAS-II by insulin and cAMP in 3T3-L1 adipocytes.

The eukaryotic initiation factor 4E (eIF-4E)-binding proteins PHAS-I and PHAS-II were found to have overlapping but different patterns of expression in tissues. Both PHAS proteins were expressed in 3T3-L1 adipocytes, in which insulin stimulated their phosphorylation, promoted dissociation of PHAS.eIF-4E complexes, and decreased the ability of both to bind exogenous eIF-4E. The effects of insulin were attenuated by rapamycin and wortmannin, two agents that block activation of p70(S6K). Unlike PHAS-I, PHAS-II was readily phosphorylated by cAMP-dependent protein kinase in vitro; however, the effects of insulin on both PHAS proteins were attenuated by agents that increase intracellular cAMP, by cAMP derivatives, and by phosphodiesterase inhibitors. These agents also markedly inhibited the activation of p70(S6K). In summary, our results indicate that PHAS-I and -II are controlled by the mammalian target of rapamycin and p70(S6K) signaling pathway and that in 3T3-L1 adipocytes this pathway is inhibited by increased cAMP.

Effects of acute ethanol administration on polyphosphoinositide turnover and levels of inositol 1,4,5-trisphosphate in mouse cerebrum and cerebellum.

Although ethanol is known for its central depressant action, its effect on the polyphosphoinositide (poly-PI) signal transduction activity in brain has not been examined in detail. In this study, C57Bl/6J mice were injected intracerebrally with [3H]inositol, and poly-PI turnover in brain was assessed by determining the levels of labeled inositol monophosphates (IP1) accumulated after intraperitoneal injection of LiCl (6 meq/kg body weight) 4 hr before killing. Using this experimental protocol, acute ethanol administration (by gavage) resulted in time- and dose-dependent decreases in the levels of labeled IP1 in both cerebrum and cerebellum as compared with controls. The ethanol-induced decrease in labeled IP1 correlated well with the decrease in levels of inositol 1,4,5-triphosphate (as measured by the radioreceptor assay) and the increase in blood ethanol concentration. Despite a 4-fold higher accumulation of labeled IP1 in the cerebrum compared with the cerebellum, there were no major differences in the steady-state levels of inositol 1,4,5-triphosphate (based on tissue weight) in either brain region. Intraperitoneal injection of atropine (50 mg/kg) (a muscarinic cholinergic receptor antagonist) to the lithium-treated mice resulted in a 34% decrease in labeled IP1 as compared with controls. This result suggests that a substantial proportion of the signals transduced were due to activation of the muscarinic cholinergic receptor. Administration of ethanol (5 g/kg) to the atropine-treated mice resulted in a further decrease in labeled IP1 and longer sleep time as compared with those given ethanol alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of PHAS-I by insulin in 3T3-L1 adipocytes. Synthesis, degradation, and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-independent pathway.

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-I eukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H6]PHAS-I when the [H6]PHAS-I.eIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-I.eIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70S6K. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes.

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.

Increased inositol 1,4,5-trisphosphate accumulation correlates with an up-regulation of bradykinin receptors in Alzheimer's disease.

An alteration in signal transduction systems in Alzheimer's disease (AD) would likely be of pathophysiological significance, because these processes control normal brain functions. Previously, a diminished beta-adrenergic-mediated cyclic AMP response was found in cultured fibroblasts from AD patients. Because cross-talk between the phosphoinositide and cyclic AMP pathways exists, the phosphoinositide cascade was studied under conditions that were similar to those for studying the cyclic AMP response. Cells from AD patients and age-matched controls responded to bradykinin (BK) and released inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in a time- and dose-dependent manner. The level of Ins(1,4,5)P3 increased rapidly and transiently in response to BK, peaked at 5 s, but still remained 116-132% above the basal level by 30 s. Although the temporal patterns were similar in both groups, the Ins(1,4,5)P3 concentrations in AD fibroblasts were 73 and 89% above levels in the age-matched controls at 5 and 10 s, respectively. Prostaglandin E1 also increased Ins(1,4,5)P3 formation, but this response was not different between the two groups. Although KD (affinity) values for the BK receptor were similar in both control and AD cells, the number of BK receptors (Bmax) was significantly elevated in AD fibroblasts (186.8 +/- 0.8 fmol/mg of protein) as compared with control fibroblasts (57.2 +/- 15.3 fmol/mg of protein). These results indicate that the elevated Ins(1,4,5)P3 production in response to BK in AD fibroblasts is positively correlated with an increase in the receptor numbers.

Signal transduction pathways coupled to a P2U receptor in neuroblastoma x glioma (NG108-15) cells.

Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma x glioma hybrid cell line (NG108-15 cells). The addition of > or = 1 microM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations > or = 500 microM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis of [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP gamma S, 2-methylthio ATP, beta, gamma-imidoATP or 3'-O-(4-benzoyl)benzoylATP, but not CTP, AMP, beta, gamma-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]P(i) or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D.