scholarly journals Ligation of the α2-macroglobulin signalling receptor on macrophages induces protein phosphorylation and an increase in cytosolic pH

1995 ◽  
Vol 309 (1) ◽  
pp. 151-158 ◽  
Author(s):  
U K Misra ◽  
G Gawdi ◽  
S V Pizzo
Keyword(s):  
Monoclonal Antibody ◽  
Growth Factors ◽  
Protein Phosphorylation ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Density Lipoprotein ◽  
Ligand Concentration ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cytosolic Ph

We have recently described an alpha 2-macroglobulin (alpha 2M) signalling receptor which is distinct from the low-density lipoprotein-related protein/alpha 2M receptor (LRP/alpha 2MR). Ligation of the macrophage signalling receptor by alpha 2M-methylamine stimulates production of several second messengers and involves a pertussis toxin-insensitive G-protein. We now report that binding of alpha 2M-methylamine, or the cloned M(r) = 20,000 receptor-binding fragment from rat alpha 1M, to macrophage alpha 2M signalling receptors induces protein phosphorylation. By use of a monoclonal antibody to phospholipase C gamma l (PLC gamma l) we were able to identify it as one target for protein phosphorylation. Phosphorylation was time and concentration dependent, being optimal at about 60 s of incubation and a 100-200 nM ligand concentration. By use of a second monoclonal antibody directed against phosphotyrosine, we were able to demonstrate that at least a portion of the label was incorporated into one or more tyrosine residues. PLC gamma l phosphorylation was then studied in membrane preparations at 4 degrees C in order to minimize serine or threonine modification. Preincubation of macrophage membranes with genistein, a tyrosine kinase inhibitor, drastically reduced phosphorylation of PLC gamma l. Receptor-associated protein, which blocks alpha 2M binding to LRP/alpha 2MR but not to the alpha 2M signalling receptor, had no effect on alpha 2M-methylamine-induced tyrosine phosphorylation of PLC gamma l. Binding of lactoferrin to LRP/alpha 2MR failed to induce phosphorylation of PLC gamma l, further supporting the hypothesis that the alpha 2M signalling receptor and LRP/alpha 2MR are distinct entities. Growth factors which induce tyrosine phosphorylation typically cause a rise in cytosolic pH. Binding of a2M-methylamine to macrophages also gradually increased the intracellular pH in a concentration-dependent manner, being optimal at a 200 nM ligand concentration. The increase in pH was amiloride sensitive. We propose that receptor-recognized forms of a2M may function like growth factors with regard to macrophage regulation.

scholarly journals The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3886
Author(s):  
Stefania Sut ◽  
Irene Ferrarese ◽  
Maria Giovanna Lupo ◽  
Nicola De Zordi ◽  
Elisa Tripicchio ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Density Lipoprotein ◽  
In Vitro Study ◽  
Volatile Constituent ◽  
Dependent Manner ◽  
Human Hepatoma Cell ◽  
Concentration Dependent Manner

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.

Sevoflurane Inhibits Guanosine 5′-[γ-thio]triphosphate–stimulated, Rho/Rho-kinase–mediated Contraction of Isolated Rat Aortic Smooth Muscle

2003 ◽  
Vol 99 (3) ◽  
pp. 646-651 ◽  
Author(s):  
Jingui Yu ◽  
Koji Ogawa ◽  
Yasuyuki Tokinaga ◽  
Yoshio Hatano
Keyword(s):  
Smooth Muscle ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Rho Kinase ◽  
Force Transducer ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Aortic Smooth Muscle ◽  
Gamma S ◽  
Kinase Pathway

Background The Rho/Rho-kinase signaling pathway plays an important role in mediating Ca2+ sensitization of vascular smooth muscle. The effect of anesthetics on Rho/Rho-kinase-mediated vasoconstriction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane on the Rho/Rho-kinase pathway by measuring guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)-stimulated contraction and translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol to the membrane in rat aortic smooth muscle. Methods GTP gamma S-induced contraction of rat aortic endothelium-denuded rings was measured using an isometric force transducer, and GTP gamma S-stimulated membrane translocation of RhoA and Rock-2 in smooth muscle cells was detected with Western blotting in the presence and absence of sevoflurane. Results GTP gamma S (10(-4) m) induced a sustained contraction, which was significantly inhibited by the Rho-kinase inhibitor, Y27632 (3 x 10(-6) m). Before treatment with GTP gamma S, RhoA and Rock-2 were detected primarily in the cytosolic fraction. GTP gamma S (10(-4) m) stimulated the translocation of RhoA and Rock-2 from the cytosol to the membrane, which was sustained for more than 60 min. Sevoflurane (1.7, 3.4, and 5.1%) concentration dependently inhibited the GTP gamma S-induced constriction of rat aortic smooth muscle with a reduction of constriction of 52-75% (P < 0.01, n = 8), and attenuated the translocation of RhoA and Rock-2 by 31-66% and 34-78%, respectively (P < 0.05-0.01, respectively; n = 4). Conclusion The current findings show that sevoflurane depresses the GTP gamma S-stimulated contraction and translocation of both Rho and Rho-kinase from the cytosol in a concentration-dependent manner, indicating that sevoflurane is able to inhibit vasoconstriction mediated by the Rho/Rho-kinase pathway in rat aortic smooth muscle.

scholarly journals Reversal of Cancer Multidrug Resistance (MDR) Mediated by ATP-Binding Cassette Transporter G2 (ABCG2) by AZ-628, a RAF Kinase Inhibitor

2020 ◽  
Vol 8 ◽  
Author(s):  
Jing-Quan Wang ◽  
Qiu-Xu Teng ◽  
Zi-Ning Lei ◽  
Ning Ji ◽  
Qingbin Cui ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kinase Inhibitor ◽  
Efflux Pump ◽  
Simulation Analysis ◽  
Chemotherapeutic Agents ◽  
Dynamics Simulation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Raf Kinase

Overexpression of ABCG2 remains a major impediment to successful cancer treatment, because ABCG2 functions as an efflux pump of chemotherapeutic agents and causes clinical multidrug resistance (MDR). Therefore, it is important to uncover effective modulators to circumvent ABCG2-mediated MDR in cancers. In this study, we reported that AZ-628, a RAF kinase inhibitor, effectively antagonizes ABCG2-mediated MDR in vitro. Our results showed that AZ-628 completely reversed ABCG2-mediated MDR at a non-toxic concentration (3 μM) without affecting ABCB1-, ABCC1-, or ABCC10 mediated MDR. Further studies revealed that the reversal mechanism was by attenuating ABCG2-mediated efflux and increasing intracellular accumulation of ABCG2 substrate drugs. Moreover, AZ-628 stimulated ABCG2-associated ATPase activity in a concentration-dependent manner. Docking and molecular dynamics simulation analysis showed that AZ-628 binds to the same site as ABCG2 substrate drugs with higher score. Taken together, our studies indicate that AZ-628 could be used in combination chemotherapy against ABCG2-mediated MDR in cancers.

Functional Characterization of BAX 855, a PEGylated Recombinant FVIII

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4359-4359
Author(s):  
Gerald Schrenk ◽  
Katalin Varadi ◽  
Herbert Gritsch ◽  
Hanspeter Rottensteiner ◽  
Hartmut J. Ehrlich ◽  
...  
Keyword(s):  
Functional Properties ◽  
Thrombin Generation ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Activated Protein C ◽  
Density Lipoprotein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Recombinant Fviii

Abstract Abstract 4359 Baxter and Nektar have developed BAX 855, a longer-acting PEGylated form of Baxter’s recombinant FVIII (ADVATE process) using stable PEG technology from Nektar. BAX 855 was functionally characterized in vitro and its features were compared with those of the unmodified parent rFVIII. The overall hemostatic potency of BAX 855 was assessed using a thrombin generation assay. Human FVIII-deficient plasma containing less than 1% of FVIII was supplemented with different concentrations of BAX 855 and unmodified rFVIII and coagulation was triggered by adding a small amount of recombinant human tissue factor complexed with phospholipid (PL) micelles to the plasma. Similar to unmodified rFVIII, BAX 855 corrected the impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner. The role of FVIII within the tenase complex was determined by measuring the kinetics of FXa generation with a FIXa-cofactor activity assay, using either untreated or thrombin activated BAX 855. Comparison of the kinetic parameters and the maximum FXa generated revealed similar characteristics between BAX 855 and unmodified rFVIII. A similar approach revealed that BAX 855 fully retained its ability to be activated and inactivated by thrombin. The susceptibility of BAX 855 to activated protein C (APC) inactivation was also similar for BAX 855 and unmodified rFVIII. The binding affinities for VWF were similar for unmodified rFVIII (KD 0.6 nM) and BAX 855 (KD 0.8 nM) and the binding capacity of BAX 855 was also only slightly reduced. In contrast, the binding capacity of BAX 855 to the low-density lipoprotein-receptor-related protein (LRP) clearance receptor was 55% less than that of the unmodified rFVIII. In summary, the functional properties of BAX 855 were fully retained, indicating that PEGylation did not have an impact on the functional properties of rFVIII. Disclosures: Schrenk: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

Autophagy and Apoptosis in the Response of Human Vascular Endothelial Cells to Oxidized Low-Density Lipoprotein

Cardiology ◽  
2015 ◽  
Vol 132 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Yanlin Zhang ◽  
Ying Xie ◽  
Shoujiang You ◽  
Qiao Han ◽  
Yongjun Cao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
P53 Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Positive Staining ◽  
Low Density ◽  
Dependent Manner ◽  
Oxidized Low Density Lipoprotein ◽  
Concentration Dependent Manner

Objectives: Oxidized low-density lipoprotein (ox-LDL) may induce autophagy, apoptosis, necrosis or proliferation of cultured endothelial cells depending on the concentration and exposure time. Our previous studies found that ox-LDL exposure for 6 h increases the autophagic level of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. The present study investigates the relationship between autophagy and apoptosis in HUVECs exposed to ox-LDL. Methods: Flow cytometry and Western blot were used to study the apoptotic and autophagic phenomena. The contribution of autophagic and apoptotic mechanisms to ox-LDL-induced upregulation of MAP1-LC3, beclin1 and p53 protein levels were assessed by pretreatment with the autophagic inhibitors 3-MA and Atg5 small interfering (si)RNA, as well as z-vad-fmk, an apoptosis inhibitor. Results: ox-LDL induced the apoptosis of HUVECs in a concentration-dependent way. The increased expression of the autophagic proteins, LC3-II and beclin1, can be reversed by 3-MA and z-vad-fmk pretreatment. 3-MA and Atg5 siRNA increased the ox-LDL-induced increases of the p53 protein level and the annexin V-positive staining, which was decreased by z-vad-fmk. Conclusion: These results suggest that overstimulation of ox-LDL can induce autophagy and apoptosis in HUVECs. Inhibition of apoptosis leads to an inhibition of autophagy induced by ox-LDL. However, inhibition of autophagy leads to an increase in the ox-LDL-induced apoptosis.

scholarly journals Identification of a Human Anti-Alpha-Toxin Monoclonal Antibody Against Staphylococcus aureus Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Fangjie Liu ◽  
Zhangchun Guan ◽  
Yu Liu ◽  
Jingjing Li ◽  
Chenghua Liu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
A549 Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Phage Display Technology ◽  
Alpha Hemolysin ◽  
Protective Strategy ◽  
Display Technology ◽  
Hla Binding

Staphylococcus aureus is a major pathogenic bacterium that causes a variety of clinical infections. The emergence of multi-drug resistant mechanisms requires novel strategies to mitigate S. aureus infection. Alpha-hemolysin (Hla) is a key virulence factor that is believed to play a significant role in the pathogenesis of S. aureus infections. In this study, we screened a naïve human Fab library for identification of monoclonal antibodies targeting Hla by phage display technology. We found that the monoclonal antibody YG1 blocked the Hla-mediated lysis of rabbit red blood cells and inhibited Hla binding to A549 cells in a concentration-dependent manner. YG1 also provided protection against acute peritoneal infection, bacteremia, and pneumonia in murine models. We further characterized its epitope using different Hla variants and found that the amino acids N209 and F210 of Hla were functionally and structurally important for YG1 binding. Overall, these results indicated that targeting Hla with YG1 could serve as a promising protective strategy against S. aureus infection.

β2 Glycoprotein-I Inhibits Factor XII Activation on Triglyceride Rich Lipoproteins: The Effect of Antibodies from Plasma of Patients with Antiphospholipid Syndrome

1996 ◽  
Vol 76 (02) ◽  
pp. 220-225 ◽  
Author(s):  
T McNally ◽  
I J Mackie ◽  
D A Isenberg ◽  
S J Machin
Keyword(s):  
Antiphospholipid Antibodies ◽  
Low Density Lipoprotein ◽  
Fetal Loss ◽  
Density Lipoprotein ◽  
Antibody Concentration ◽  
Factor Xii ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Factor Xiia

SummaryIt is now well recognised that antiphospholipid antibodies are associated with thrombosis and recurrent fetal loss. Some antiphospholipid antibodies (aPAs) have been shown to require a cofactor, β2 glyco-protein-I (β2GPI), for binding to phospholipids, and recently β2GPI has been identified as the antigenic target for some aPAs. β2GPI possesses in vitro anticoagulant properties and modulation of β2GPI function may therefore result in altered haemostatic regulation. In the present study, the influence of plasma derived aPAs and β2GPI on factor XII activation on the surface of very low density lipoprotein (VLDL) was investigated. Factor XIIa generation was dependent on lipoprotein lipase treatment of VLDL and β2GPI inhibited the factor XIIa generation in a concentration dependent manner. No consistent effects on factor XIIa generation were demonstrated with the IgG fractions from patients with aPAs. Inhibition of the β2GPI activity was demonstrated by some antibodies, and study with cardiolipin affinity purified antibody indicated that antibody concentration is critical. These results suggest that perturbation of β2GPI function may contribute to the pathogenic mechanism for thrombosis in some patients with aPAs. .

Protein-Tyrosine Phosphorylation and p72 syk Activation in Human Platelets Stimulated with Collagen Is Dependent upon Glycoprotein Ia/IIa and Actin Polymerization

1996 ◽  
Vol 75 (04) ◽  
pp. 648-654 ◽  
Author(s):  
Naoki Asazuma ◽  
Yutaka Yatomi ◽  
Yukio Ozaki ◽  
Ruomei Qi ◽  
Kenji Kuroda ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Actin Polymerization ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
Concentration Dependent Manner ◽  
Protein Tyrosine ◽  
Collagen Receptors ◽  
Fibrinogen Binding ◽  
Adp Release

SummaryIn human platelets treated with acetylsalicylic acid, collagen induced protein-tyrosine-phosphorylation of several proteins. The major 75 kDa band included cortactin and autophosphorylated p72 syk . p72 syk activity rapidly increased upon collagen stimulation, whereas p60c-src activation was below detectable levels. A combination of inhibitors to remove the effects of extracellular and intracellular Ca2+, released ADP, and fibrinogen binding to GPIIb/IIIa delayed and attenuated the major 75 kDa band. By contrast, p72 syk activation was not inhibited by these treatments. Cytochalasin D completely inhibited protein tyrosine phosphorylation and p72 syk activation. It also potently inhibited aggregation and [Ca2+]i elevation. Anti-GPMIa/IIa MoAb in a concentration-dependent manner partially attenuated protein tyrosine phosphorylation and p72 syk activation. Its inhibitory effects on intracellular Ca2+ mobilization, release of intracellular granule contents, and aggregation also were partial. No tyrosine kinase activity was coprecipitated with GPIa/IIa. These results suggest that p72 syk activation lies upstream of protein tyrosine phosphorylation, Ca2+ mobilization, ADP release, thromboxane A2 production and aggregation. GPIa/IIa plays a key role in p72 syk activation induced by collagen, but other collagen receptors may work in synergy to fully activate p72 syk . Actin polymerization is a prerequisite for both p72 syk activation and other intracellular signal transduction pathways.

scholarly journals Tyrosine Phosphorylation and p72syk Activation by an Anti-Glycoprotein Ib Monoclonal Antibody

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Mutsumasa Yanabu ◽  
Yukio Ozaki ◽  
Shosaku Nomura ◽  
Tetsuya Miyake ◽  
Yasuhiko Miyazaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Protein Tyrosine Phosphorylation ◽  
Fab Fragment ◽  
Glycoprotein Ib ◽  
Protein Tyrosine

Abstract NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ibα, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF ). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-FcγII receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab′ fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab′ fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the FcγII receptor. Cross-linking of the receptor-bound Fab′ fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5–induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle

1997 ◽  
Vol 273 (2) ◽  
pp. C509-C519 ◽  
Author(s):  
Y. X. Wang ◽  
M. I. Kotlikoff
Keyword(s):  
Signaling Pathway ◽  
Calcium Release ◽  
Kinase Inhibitor ◽  
Ruthenium Red ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Concentration Dependent Manner ◽  
Calmodulin Antagonist ◽  
Dependent Protein ◽  
Full Activation

We investigated the muscarinic activation of Ca(2+)-activated Cl- currents [ICl(Ca)] in voltage-clamped equine tracheal myocytes. The threshold of cytosolic free Ca2+ concentration ([Ca2+]i) required for activation of ICl(Ca) was 202 +/- 22 nM, and full activation of the current occurred at 771 +/- 31 nM. Hexahydro-sila-difenidol (M3 antagonist) inhibited the methacholine-induced phasic [Ca2+]i increase and ICl(Ca) in a concentration-dependent manner, whereas methoctramine (M2 antagonist) only slightly attenuated the [Ca2+]i increase and ICl(Ca) (14.8 and 21.4%, respectively), consistent with incomplete selectivity. Dialysis of heparin (10 mg/ml) blocked methacholine-induced [Ca2+]i and ICl(Ca) but had no effect on the caffeine-induced Ca2+ release or ICl(Ca); inositol 1,4,5-trisphosphate (100 microM) induced ICl(Ca) and blocked the methacholine current. Conversely, ruthenium red (50 microM) prevented the caffeine-induced [Ca2+]i release and ICl(Ca) but had no effect on methacholine-induced [Ca2+]i or current. Intracellular dialysis of the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7, 500 microM) or the Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (5 microM) had no effect on the [Ca2+]i increase or ICl(Ca). Pertussis toxin (0.5 mg/ml) did not affect the increase in [Ca2+]i or ICl(Ca). Dialysis with antibodies directed against the alpha-subunit of Gq/G11 (Gq alpha/ G alpha 11) blocked the methacholine-induced ICl(Ca) in a concentration-dependent manner, whereas anti-G alpha i-1/G alpha 1-2 antibodies (1:35) and anti-G alpha i-3/G(o) alpha antibodies (1:35) were without effect. The results indicate that stimulation of phospholipase C via M3/Gq proteins is the predominant signaling pathway for the activation of ICl(Ca); at high agonist concentrations, Ca(2+)-induced Ca2+ release does not appear to play a prominent role in muscarinic signaling.

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