Abstract 507: G Proteins are Required for Lipoxygenase EDHF Activity of Rabbit Arterial Smooth Muscle Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Kathryn M Gauthier ◽  
J. R Falck ◽  
William B Campbell
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
G Proteins ◽  
Muscle Cells ◽  
Specific Structure ◽  
Inhibitory Peptide ◽  
Receptor Binding Site ◽  
Channel Activation ◽  
Sk Channel ◽  
Vascular Activity

Arachidonic acid 15-lipoxygenase (15-LO) metabolites function as endothelium-derived hyperpolarizing factors in rabbit and human arteries. In rabbit arteries, LO metabolites mediate nitric-oxide and prostaglandin-independent relaxations to acetylcholine and AA. Previously, we characterized 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) as a major vasoactive 15-LO metabolite in rabbit arteries. 11,12,15-THETA requires a specific structure for vascular activity. 11(R),12(S),15(S)-THETA causes concentration-related relaxation whereas 11(R),12(R),15(S)-THETA is without activity. The specific structure requirement suggests a role for a receptor. Therefore, we examined the role of G proteins in 11(R),12(S),15(S)-THETA vascular activity. Western immunoblot verified protein expression of Gαs, Gαi and a Gαo in rabbit endothelial and smooth muscle cells. 11(R),12(S),15(S)-THETA increased GTPγ35S binding to rabbit arterial membranes 280±25% while 11(R),12(S),15(S)-THETA was without effect. In cell-attached patches of rabbit smooth muscle, 11(R),12(S),15(S)-THETA (100 nM) increased mean open time of apamin-sensitive, calcium-activated, small conductance potassium (SK) channels from 0.0001±0.0001 to 0.0015±0.0006. In inside-out patches, 11(R),12(S),15(S)-THETA did not increase channel opening (0.0001±0.0001) unless GTP was present (0.0051±0.0023). In the presence of GTP, an antibody against Gαs and a Gαs inhibitory peptide inhibited 11(R),12(S),15(S)-THETA SK channel activation (0.0007±0.0005, 0.0013±0.0012, respectively) whereas an antibody against Gαi was without effect (0.0042±0.0018). A cell-permeant, penetratin-linked Gαs inhibitory peptide also inhibited 11(R),12(S),15(S)-THETA SK channel activation in cell-attached patches (0.0005±0.0002) and blocked 11(R),12(S),15(S)-THETA relaxations in rabbit aorta (max relaxations = 74±6%, 23±7% for control and permeant peptide, respectively). These studies indicate that 11,12,15-THETA-induced SK channel activation and vascular relaxation are mediated by a Gs-coupled mechanism and that 11,12,15-THETA acts via a stereo-specific G protein coupled receptor/binding site.

scholarly journals Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells

2007 ◽  
Vol 582 (1) ◽  
pp. 41-61 ◽  
Author(s):  
Takashi Sakamoto ◽  
Toshihiro Unno ◽  
Takio Kitazawa ◽  
Tetsuro Taneike ◽  
Masahisa Yamada ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Signalling Pathways ◽  
Channel Activation ◽  
Cationic Channel

scholarly journals Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries

2007 ◽  
Vol 293 (1) ◽  
pp. H204-H214 ◽  
Author(s):  
T. Kamishima ◽  
T. Burdyga ◽  
J. A. Gallagher ◽  
J. M. Quayle
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Removal Rate ◽  
Muscle Cells ◽  
Amino Acid Residues ◽  
Resistance Arteries ◽  
Inhibitory Peptide ◽  
Caveolin 1 ◽  
Domain Peptide ◽  
Cerebral Resistance

The role of caveolins, signature proteins of caveolae, in arterial Ca2+ regulation is unknown. We investigated modulation of Ca2+ homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca2+ transients were simultaneously measured with voltage-clamped single cells. Membrane depolarization triggered Ca2+ current and increased intracellular Ca2+ concentration ([Ca2+]i). After repolarization, elevated [Ca2+]i returned to the resting level. Ca2+ removal rate was determined from the declining phase of the Ca2+ transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to amino acid residues 82–101 of caveolin-1, significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55–74 of caveolin-3, also significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, and cyclosporine A, inhibitors for calmodulin, Ca2+/calmodulin-dependent protein kinase II, and calcineurin, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca2+ ATPase inhibitor, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca2+ removal of resistance artery smooth muscle cells.

Src-Dependence and Pertussis-Toxin Sensitivity of Urokinase Receptor-Dependent Chemotaxis and Cytoskeleton Reorganization in Rat Smooth Muscle Cells

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 649-662 ◽  
Author(s):  
Bernard Degryse ◽  
Massimo Resnati ◽  
Shafaat A. Rabbani ◽  
Antonello Villa ◽  
Francesca Fazioli ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Cell Shape ◽  
Muscle Cells ◽  
Soluble Form ◽  
Integrin Subunit ◽  
Cytoskeleton Reorganization

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) vβ3, β1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.

scholarly journals Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

2010 ◽  
Vol 136 (3) ◽  
pp. 283-291 ◽  
Author(s):  
Guiling Zhao ◽  
Zachary P. Neeb ◽  
M. Dennis Leo ◽  
Judith Pachuau ◽  
Adebowale Adebiyi ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Bkca Channel ◽  
Ip3 Receptors ◽  
Arterial Smooth Muscle ◽  
Bkca Channels ◽  
Channel Activation ◽  
Arterial Smooth Muscle Cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.

Sign in / Sign up

Export Citation Format

Share Document