Scavenging Peptide Research Articles

Isolation and purification of Eleutherococcus sessiliflorus (Rupr. & Maxim.) S. Y. Hu peptides and study of their antioxidant effects and mechanisms.

Oxidative stress is a state of imbalance between oxidant and antioxidant effects in the body, which is closely associated with aging and many diseases. Therefore, the development of antioxidants has become urgent. In this study, we isolated three polypeptides, G-6-Y, P-8-R, and F-10-W, from Eleutherococcus sessiliflorus (Rupr. & Maxim.) S. Y. Hu (E. sessiliflorus), based on the antioxidant and anti-aging properties of Eleutherococcus, and screened the most powerful free radical scavenging peptide P-8-R. Ultraviolet B (UVB)-induced oxidative stress damage in the skin was established to test the efficacy of P-8-R. In cellular experiments, P-8-R not only prevented oxidative stress damage in HaCaT cells, reduced intracellular reactive oxygen species levels, and inhibited the overexpression of matrix metalloproteinases but also inhibited apoptosis via the mitochondria-dependent apoptotic pathway; in animal experiments, P-8-R was able to prevent oxidative stress damage in the skin and reduce skin collagen loss by inhibiting the overexpression of MMPs to prevent mouse skin aging. In conclusion, the present study contributes to an in-depth understanding of the active compounds of Eleutherococcus, which is of great significance for the pharmacodynamic mechanism and industrial development of Eleutherococcus, and P-8-R is likely to become a potential antioxidant and anti-aging drug or skin care cosmetic in the future.

Antioxidant activity of venison subjected to in vitro cooking and gastrointestinal digestion and isolation of its 2,2-diphenyl-1-picrylhydrazyl radical scavenging peptides.

Venison, a type of game meat, has several health benefits because it contains not only high protein and low fat but also bioactive peptides with several physiological properties, including antioxidative and angiotensin-converting enzyme inhibitory properties. The aim of the present study was to investigate the antioxidant activity of venison treated by in vitro cooking and gastrointestinal digestion. We subjected venison along with pork and beef to in vitro cooking and digestion and assessed their antioxidant activity via 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH-RS) and hydrophilic oxygen radical absorbance capacity (H-ORAC) assays. The peptide contents of all types of cooked and digested meat samples were higher than those of the untreated and cooked samples. The DPPH-RS activities and H-ORAC of digested venison, pork, and beef were increased compared with those of untreated samples. DPPH-RS activity was significantly higher in the digested venison samples than in the digested pork and beef samples. In this study, several fractions of digested venison from the chromatography exhibited DPPH-RS activity. Peptide analysis, using liquid-chromatography with tandem mass spectrometry, unveiled two peptides DIDDLELTLAK and TQTVCNFTDGALVQHQEWDGK with high DPPH-RS activities. Thus, venison is a rich source of antioxidant peptides and potentially demonstrate an antioxidation ability by digestive enzymes in vivo.

Optimization and Identification of Antioxidant Peptide from Underutilized Dunaliella salina Protein: Extraction, In Vitro Gastrointestinal Digestion, and Fractionation.

DPPH• scavenging peptides (<3kDa) from underutilized Dunaliella salina protein were obtained by the following successive treatment, i.e., ultrasound extraction, simulated in vitro gastrointestinal digestion hydrolyzation, and membrane ultrafiltration classification. The optimal condition for ultrasound-assisted extraction was an ultrasound wave with 800 W of power treating a mixture of 60 mL of 1.0 mol L−1 NaOH and 2 g algae powder for 15 min. A high correlation (r=0.8146) between DPPH• scavenging activity and yield of the intact peptides showed their antioxidant capacity. Simulated in vitro digestion assay resulted in excellent DPPH• scavenging activity of the total peptide, amounting to (86.5 ± 10.1)%, comparing with the nondigestion samples at (46.8 ± 6.5)%. After fractionation, the 500-1000 Da fraction exhibited the highest DPPH• scavenging activity (81.2 ± 4.0)%, increasing 1.5 times due to digestion. Then, the 500-1000 Da fraction was analyzed by RPLC-Q Exactive HF mass spectrometer, and 4 novel peptides, i.e., Ile-Leu-Thr-Lys-Ala-Ala-Ile-Glu-Gly-Lys, Ile-Ile-Tyr-Phe-Gln-Gly-Lys, Asn-Asp-Pro-Ser-Thr-Val-Lys, and Thr-Val-Arg-Pro-Pro-Gln-Arg, were identified. From these amino acid sequences, hydrophobic residues accounted for 56%, which indicated their high antioxidant property. The results indicated that underutilized protein of Dunaliella salina could be a potential source of antioxidative peptides through simulated in vitro gastrointestinal digestion.

Production of Whey-Derived DPP-IV Inhibitory Peptides Using an Enzymatic Membrane Reactor

Continuous processing in the production of peptides is an area of increased interest. In this study, an enzymatic membrane reactor (EMR) was developed whereby whey protein isolate was used as a substrate to prepare DPP-IV inhibitory and radical scavenging peptides via enzymatic hydrolysis. Two separate enzymes were tested: Corolase 2TS and Protamex in conventional batch processes and the EMR. Neither enzyme was considered effective at producing peptides with radical scavenging activity when measured using a DPPH assay. However, both enzymes were capable of producing DPP-IV inhibitory peptides. Corolase and Protamex both produced similar DPP-IV inhibition levels upon completion of batch experiments. In the EMR process, permeate in the Protamex run showed 33.7% lower IC50 value compared to the continuous Corolase run. Protamex was a better enzyme at producing the DPP-IV inhibitory effect. The continuous (EMR) production method showed an increased productivity over batch for both enzymes.

A scavenger peptide prevents methylglyoxal induced pain in mice

The reactive metabolite methylglyoxal (MG) has been identified as mediator of pain. Scavenging of free MG and the prevention of MG-derived post-translational modifications may provide a useful therapeutic treatment. An arginine-rich, fatty acid coupled, cyclic peptide (CycK(Myr)R4E) with high proteolytic stability and prolonged circulation was developed for the scavenging of MG. It was shown to reduce the formation of albumin-MG adducts in vitro and prevented MG-induced pain by reducing plasma MG levels through the formation of peptide-MG adducts in vivo. CycK(Myr)R4E therefore presents a promising option for the treatment of pain and other diabetic complications associated with high MG levels.

Intranasal Administration ofaPolyethylenimine-Conjugated Scavenger Peptide Reduces Amyloid-β Accumulation in a Mouse Model ofAlzheimer's Disease.

Amyloid-β (Aβ) aggregation in the brain plays a central and initiatory role in pathogenesis and/or progression of Alzheimer's disease (AD). Inhibiting Aβ aggregation is a potential strategy in the prevention of AD. A scavenger peptide, V24P(10-40), designed to decrease Aβ accumulation in the brain, was conjugated to polyethylenimine (PEI) and tested as a preventive/therapeutic strategy for AD in this study. This PEI-conjugated V24P(10-40) peptide was delivered intranasally, as nasal drops, to four-month-old APP/PS1 double transgenic mice for four or eight months. Compared with control values, peptide treatment for four months significantly reduced the amount of GdnHCl-extracted Aβ40 and Aβ42 in the mice's hippocampus and cortex. After treatment for eight months, amyloid load, as quantified by Pittsburgh compound B microPET imaging, was significantly decreased in the mice's hippocampus, cortex, amygdala, and olfactory bulb. Our data suggest that this intranasally delivered scavenger peptide is effective in decreasing Aβ accumulation in the brain of AD transgenic mice. Nasal application of peptide drops is easy to use and could be further developed to prevent and treat AD.

Association of glutathione-S-transferase with patients of type 2 diabetes mellitus with and without nephropathy

Statements of the problemHyperglycemia induced oxidative stress is implicated as a contributor to the onset and progression of type 2 diabetes mellitus (T2DM) and its complications like diabetic nephropathy (DN). Glutathione-S-transferase (GST) is primarily involved in the neutralization of reactive oxygen species (ROS) by enzymatic conjugation with the scavenger peptide glutathione (GSH). Therefore, present study was aimed to evaluate the role of GST along with oxidative stress markers and their correlation in patients with Type 2 diabetes mellitus with and without nephropathy. MethodsThis study comprised of 300 participants divided into three groups of 100 each: healthy controls (HC), T2DM without complications and DN. Plasma GST, malondialdehyde (MDA), reduced GSH levels and ferric reducing ability of plasma (FRAP) were estimated spectrophotometrically. ResultsHighest GST levels was observed in T2DM which was significantly higher (p<0.05) as compared to DN and HC. However, GSH and FRAP levels were found to be significantly lowest whereas MDA levels were significantly highest in DN as compared to T2DM and HC. GST showed a significant negative correlation with GSH, FRAP and positive correlation with MDA in both patients groups. ConclusionsHighest activity of GST in T2DM might be as a compensatory mechanism in response to oxidative stress. GST is found to have significant negative association with decreased GSH. Altered redox milieu in DN collectively conspire to increase the risk of renal damage in T2DM.

Isolation and characterisation of in vitro and cellular free radical scavenging peptides from corn peptide fractions.

Corn gluten meal, a corn processing industry by-product, is a good source for the preparation of bioactive peptides due to its special amino acid composition. In the present study, the in vitro and cellular free radical scavenging activities of corn peptide fractions (CPFs) were investigated. Results indicated that CPF1 (molecular weight less than 1 kDa) and CPF2 (molecular weight between 1 and 3 kDa) exhibited good hydroxyl radical, superoxide anion radical and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt (ABTS) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Meanwhile, the in vitro radical scavenging activity of CPF1 was slightly higher than that of CPF2. Both CPF1 and CPF2 also exhibited significant cytoprotective effects and intracellular reactive oxygen species scavenging activity in Caco-2 cells exposed to hydrogen peroxide (H2O2). The amino acid composition analysis revealed that the CPF were rich in hydrophobic amino acids, which comprised of more than 45% of total amino acids. An antioxidant peptide sequence of Tyr-Phe-Cys-Leu-Thr (YFCLT) was identified from CPF1 using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI TOF/TOF MS). The YFCLT exhibited excellent ABTS radical scavenging activity with a 50% effective concentration (EC50) value of 37.63 µM, which was much lower than that of Trolox. In conclusion, corn gluten meal might be a good source to prepare antioxidant peptides.

The Purification of Free Radical Scavenging Peptides from Naked Oats

The present study conducted enzymatic hydrolysis of naked oats glutelin by Alcalase ( FG).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result showed that the molecular weight of naked oats glutelin ranged from 14 to 97 kDa. Ion exchange chromatography was used to isolate and purify naked oats glutelin hydrolysates. The results showed that hydrolysates were purified into 4 fractions by ion exchange chromatography. Fraction D showed higher scavenging effect against hydroxyl radical (IC50 1.40 mg mL-1), and 2,2-Dipheyl-1-picrylhydrazyl (DPPH) radical (IC50 1.26 mg mL-1) than the other 3 fractions. Moreover, fraction D exhibited higher scavenging effect against hydroxyl and DPPH radical than the hydrolysates. Free radical scavenging capacity of naked oats glutelin hydrolysates was improved by isolation and purification.

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.

Purification and Characterization of a New DPPH Radical Scavenging Peptide from Shrimp Processing By-products Hydrolysate

A peptide with high antioxidant activity was isolated and identified from shrimp processing by-products hydrolysate. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity was used to evaluate the antioxidant activity of fractions. The purified antioxidant peptide was identified as Ser-Val-Ala-Met-Leu-Phe-His (804.4 Da) by electrospray ionization tandem mass spectrometry. The purified peptide at 50 μg/mL showed antioxidative activity of 65.7 ± 0.9%, which was 3.18-fold higher compared with the first step separation by ion-exchange chromatography. It is possible to produce natural antioxidative peptides from shrimp processing by-products hydrolysate. The high antioxidant activity may be due to the presence of Phe-His segment at the C-terminus of the peptide.

What’s New in Shock, December 2011?

What’s New in Shock, December 2011?

Quantitative Structure-Activity Relationship Study of Radical Scavenging Peptides Based on Orac Method by Using Different Sets of Amino Acids Descriptor

Some radical scavenging peptides by ORAC method from different hydrolysates were used for the quantitative structure-activity relationships (QSAR) research. Partial least-squares regression analysis (PLSR) was treated as the method to build the model with 17 kinds of amino acid descriptors. In order to translate the sequence to the same length, two-terminal position numbering (TTPN) was applied. Two of amino acid descriptors VSHE and VSW were selected for their excellent performance (R2, Q2, and RMSEcwith VHSE and VSW descriptor are 0.995, 0.630, 0.318 and 0.966, 0.543, 0.181 respectively). VHSE has the definite physicochemical meanings and easy to understand while VSW has good predictive ability (Rand RMSEpwith VHSE and VSW are 0.404, 2.633 and 0.635, 2.298 respectively). It is believed that the position No.2 amino acid from N-terminal (N2) have more importance than others in sequence, and most of electronic properties are negative to activity while all the steric properties are positive to activity as well as the hydrophobic properties. The suitable amino acids in sequence are as follow: G, R, K, W, Y, N, E, H, and Q are suitable for N2position which illustrated the importance of acidic amino acids in peptide sequence for radical scavenging activity.

Gβγ and the C Terminus of SNAP-25 Are Necessary for Long-Term Depression of Transmitter Release

BackgroundShort-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.Methodology/Principal FindingsThis study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca2+] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca2+]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca2+ channels, imaging of presynaptic [Ca2+] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca2+ influx, an effect not altered by infusion of Ct-SNAP-25.Conclusions/SignificanceThe C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD.

Gβγ‐activated phosphoinositide 3‐kinase γ regulates airway smooth muscle contraction by modulating calcium oscillations

Airway smooth muscle (ASM) contractile hyper‐responsiveness plays a critical role in asthma. Muscarinic receptors in ASM convey bronchoconstrictor signals by dissociating G‐proteins into active Gα and Gβγ subunits. We found that expression of a Gβγ scavenger peptide suppressed acetylcholine (ACh)‐stimulated mouse ASM cell contraction. Gβγ plays prominent roles in signal transduction through its downstream effectors including PI3Kγ. Immunohistochemistry staining and Western blot indicated expression of PI3Kγ in ASM cells of mouse trachea, lung and cultured ASM cells. Inhibitors of PI3Kγ, but not PI3Kα, β, or δ, attenuated ACh‐stimulated airway contraction of mouse lung slices. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitors still exhibited an ACh‐induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, PI3Kγ inhibitors or silence of endogenous PI3Kγ by siRNA had a small inhibitory effect on the ACh‐stimulated initial Ca2+ transient in mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. Thus, Gβγ/PI3Kγ signaling pathway controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Targeting this pathway may directly impact airway hyper‐responsiveness associated with asthma. Supported by Nebraska State LB595 and 692.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G‐protein coupled receptors (GPCRs) promotes breast cancer metastasis. G‐proteins convey GPCR signals by dissociating into Gα and Gβγ subunits. The aim of this study was to determine whether blockade of Gβγ suppresses breast cancer cell migration and invasion, critical steps of metastasis. Transwell chamber assays showed that expression of a Gβγ scavenger peptide derived from β‐adrenergic receptor kinase, attenuated migration and invasion of both breast cancer MDA‐MB‐231 and MDA‐MB‐436 cells by 50%. Migration and invasion of those cells were also blocked by M119K, an inhibitor of Gβγ, with maximum inhibition of 80% and IC50 of 1–2 μM. Activation of Gi‐protein coupled CXC chemokine receptor 4 (CXCR4) promotes breast cancer metastasis. M119K suppressed CXCR4‐dependent MDA‐MB‐231 cell migration by 75% with IC50 of 0.9 μM. Immunostaining analysis indicated that 10 μM M119K attenuated CXCR4‐stimulated, Rac‐dependent formation of lamellipodia, a key structure required for metastasis. In contrast, CXCR4‐dependent inhibition of adenylyl cyclase, a Giα‐mediated response, was not blocked by M119K but was blocked by pertussis toxin, which inactivates Giα. This is the first to directly demonstrate the role of Gβγ in breast cancer metastasis and suggests that targeting Gβγ signaling pathways may provide a novel strategy for suppressing breast cancer metastasis. Supported by Nebraska State LB595.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G protein-coupled receptors (GPCRs) promotes breast cancer metastasis. G proteins convey GPCR signals by dissociating into Galpha and Gbetagamma subunits. The aim of the present study was to determine whether blockade of Gbetagamma signaling suppresses breast cancer cell migration and invasion, which are critical components of metastasis. Conditioned media (CM) of NIH-3T3 fibroblasts are widely used as chemoattractants in in vitro cancer metastasis studies. Expression of a Gbetagamma scavenger peptide attenuated NIH-3T3 CM-induced migration and invasion of both metastatic breast cancer MDA-MB-231 and MDA-MB-436 cells by 40 to 50% without effects on cell viability. Migration and invasion of cells in response to NIH-3T3 CM were also blocked by 8-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-1-naph-thalene-carboxylic acid) (M119K), a Gbetagamma inhibitor, with maximum inhibition exceeding 80% and half-maximal inhibitory concentration (IC50) values of 1 to 2 microM. M119K also attenuated Rac-dependent formation of lamellipodia, a key structure required for metastasis. Constitutively active Rac1 rescued Gbetagamma blockade-mediated inhibition of breast cancer cell migration, whereas dominant negative Rac1 inhibited cell migration similar to Gbetagamma blockade. Furthermore, M119K suppressed Gi protein-coupled CXC chemokine receptor 4 (CXCR4)-dependent MDA-MB-231 cell migration by 80% with an IC50 value of 1 microM, whereas tyrosine kinase receptor-dependent cell migration was significantly less inhibited. However, CXCR4-dependent inhibition of adenylyl cyclase, a Gialpha-mediated response in MDA-MB-231 cells, was not blocked by M119K but was blocked by pertussis toxin, which selectively inactivates Gialpha. This report is the first to directly demonstrate the role of Gbetagamma in cancer cell migration and invasion and suggests that targeting Gbetagamma signaling pathways may provide a novel strategy for suppressing breast cancer metastasis.

Purification and Characterization of a Radical Scavenging Peptide from Rapeseed Protein Hydrolysates

AbstractWe previously reported that crude rapeseed peptides (CRPs) and peptide fractions (RP25 and RP55) prepared from aqueous enzymatic extraction of rapeseed exhibited marked antioxidant activities, among which RP55 showed the most potent effects. In the present study, RP55 was further purified using consecutive chromatographic methods for identification of antioxidant peptides. The α,α‐diphenyl‐β‐picrylhydrazyl (DPPH) radical scavenging effects of peptides were measured at each purification step to evaluate the antioxidant activities. RP55 was first fractionated by anion‐exchange chromatography, and three fractions (E1, E2, and E3) were obtained. All of them showed significantly lower scavenging activities compared to RP55, which was very probably due to the remarkable loss of tannin during the separation. Next, the active fraction E2 with higher protein content was sequentially purified by gel filtration chromatography (GFC) and reversed‐phase high performance liquid chromatography (RP‐HPLC). The purified peptide, of which the median effective dose (ED50) value for DPPH radical scavenging was 0.063 mg/mL, was identified to be Pro‐Ala‐Gly‐Pro‐Phe (487 Da) using electrospray ionization (ESI) mass spectrometry. The unique amino acid composition and sequence in the peptide might play an important role in expression of its antioxidant activity.

Membrane Electrical Activity Elicits Inositol 1,4,5-Trisphosphate-dependent Slow Ca2+ Signals through a Gβγ/Phosphatidylinositol 3-Kinase γ Pathway in Skeletal Myotubes

Tetanic electrical stimulation of myotubes evokes a ryanodine receptor-related fast calcium signal, during the stimulation, followed by a phospholipase C/inositol 1,4,5-trisphosphate-dependent slow calcium signal few seconds after stimulus end. L-type calcium channels (Cav 1.1, dihydropyridine receptors) acting as voltage sensors activate an unknown signaling pathway involved in phospholipase C activation. We demonstrated that both G protein and phosphatidylinositol 3-kinase were activated by electrical stimulation, and both the inositol 1,4,5-trisphosphate rise and slow calcium signal induced by electrical stimulation were blocked by pertussis toxin, by a Gbetagamma scavenger peptide, and by phosphatidylinositol 3-kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3-kinase gamma antibodies showed a clear location in striations within the cytoplasm, consistent with a position near the I band region of the sarcomere. The time course of phosphatidylinositol 3-kinase activation, monitored in single living cells using a pleckstrin homology domain fused to green fluorescent protein, was compatible with sequential phospholipase Cgamma1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3-kinase gamma inhibited the phosphatidylinositol 3-kinase activity as well as the slow calcium signal. We conclude that Gbetagamma/phosphatidylinositol 3-kinase gamma signaling pathway is involved in phospholipase C activation and the generation of the slow calcium signal induced by tetanic stimulation. We postulate that membrane potential fluctuations in skeletal muscle cells can activate a pertussis toxin-sensitive G protein, phosphatidylinositol 3-kinase, phospholipase C pathway toward modulation of long term, activity-dependent plastic changes.

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.