Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle

2002 ◽  
Vol 282 (4) ◽  
pp. H1304-H1310 ◽  
Author(s):  
Wang-Xian Tang ◽  
Ya-Fei Chen ◽  
Ai-Ping Zou ◽  
William B. Campbell ◽  
Pin-Lan Li
Keyword(s):  
Smooth Muscle ◽  
Gradient Centrifugation ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
Accessory Protein ◽  
Open Probability ◽  
Arterial Smooth Muscle ◽  
Fk 506 ◽  
Intracellular Ca

cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca2+in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01–10 μM) significantly increased the open probability ( NP O) of reconstituted RyR/Ca2+release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca2+ release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1–10 μM) markedly increased the activity of RyR/Ca2+ release channels. In the presence of FK-506, cADPR did not further increase the NP O of RyR/Ca2+ release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP O of RyR/Ca2+ release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca2+ release channels from the SR of BCASM.

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

2011 ◽  
Vol 300 (6) ◽  
pp. H2016-H2026 ◽  
Author(s):  
Neerupma Silswal ◽  
Nikhil K. Parelkar ◽  
Michael J. Wacker ◽  
Marco Brotto ◽  
Jon Andresen
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Arterial Smooth Muscle Cells

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P2] is a newly identified phosphoinositide that modulates intracellular Ca2+ by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca2+, we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca2+ stores PI(3,5)P2 would increase intracellular Ca2+ in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P2 was present in the mouse aorta and that exogenously applied PI(3,5)P2 readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P2 elevated intracellular Ca2+, and it also contracted aortic rings. Both the rise in intracellular Ca2+ and the contraction caused by PI(3,5)P2 were prevented by antagonizing RyRs, while the majority of the PI(3,5)P2 response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca2+ stores with thapsigargin or caffeine and/or ryanodine blunted the Ca2+ response and greatly attenuated the contraction elicited by PI(3,5)P2. The removal of extracellular Ca2+ or addition of verapamil to inhibit voltage-dependent Ca2+ channels reduced but did not eliminate the Ca2+ or contractile responses to PI(3,5)P2. We also found that PI(3,5)P2 depolarized aortic smooth muscle cells and that LaCl3 inhibited those aspects of the PI(3,5)P2 response attributable to extracellular Ca2+. Thus, full and sustained aortic contractions to PI(3,5)P2 required the release of SR Ca2+, probably via the activation of RyR, and also extracellular Ca2+ entry via voltage-dependent Ca2+ channels.

cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle

2001 ◽  
Vol 280 (1) ◽  
pp. H208-H215 ◽  
Author(s):  
Pin-Lan Li ◽  
Wang-Xian Tang ◽  
Hector H. Valdivia ◽  
Ai-Ping Zou ◽  
William B. Campbell
Keyword(s):  
Smooth Muscle ◽  
Lipid Bilayers ◽  
Ryanodine Receptors ◽  
Fold Increase ◽  
Ruthenium Red ◽  
Dependent Manner ◽  
High Concentration ◽  
Open Probability ◽  
Arterial Smooth Muscle ◽  
Concentration Dependent Manner

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca2+release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca2+ release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs+ current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NPo) of the channels in the presence of ryanodine (0.01–1 μM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 μM) and ruthenium red (40–80 μM) substantially inhibited the activity of RYR/Ca2+ release channels. Caffeine (0.5–5 mM) markedly increased the NPo of these Ca2+release channels of the SR, but d- myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NPo of these Ca2+release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 μM) into the cis bath solution produced a 2.9-fold increase in the NPo of these RYR/Ca2+release channels. An eightfold increase in the NPo of the RYR/Ca2+ release channels (0.0056 ± 0.001 vs. 0.048 ± 0.017) was observed at a concentration of cADPR of 1 μM. The effect of cADPR was completely abolished by ryanodine (50 μM). In the presence of cADPR, Ca2+-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca2+ release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca2+ release through the activation of RYRs on the SR of these smooth mucle cells.

scholarly journals Kv2.1 channels play opposing roles in regulating membrane potential, Ca2+ channel function, and myogenic tone in arterial smooth muscle

2020 ◽  
Vol 117 (7) ◽  
pp. 3858-3866 ◽  
Author(s):  
Samantha C. O’Dwyer ◽  
Stephanie Palacio ◽  
Collin Matsumoto ◽  
Laura Guarina ◽  
Nicholas R. Klug ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Myogenic Tone ◽  
Arterial Smooth Muscle ◽  
Membrane Hyperpolarization ◽  
Intracellular Ca ◽  
Specific Regulation

The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2–CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.

scholarly journals Role of Ryanodine Receptor Subtypes in Initiation and Formation of Calcium Sparks in Arterial Smooth Muscle: Comparison with Striated Muscle

2009 ◽  
Vol 2009 ◽  
pp. 1-15 ◽  
Author(s):  
Kirill Essin ◽  
Maik Gollasch
Keyword(s):  
Smooth Muscle ◽  
Calcium Release ◽  
Striated Muscle ◽  
Ryanodine Receptors ◽  
Calcium Content ◽  
Receptor Subtypes ◽  
Calcium Stores ◽  
Calcium Sparks ◽  
Arterial Smooth Muscle

Calcium sparks represent local, rapid, and transient calcium release events from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In arterial smooth muscle cells (SMCs), calcium sparks activate calcium-dependent potassium channels causing decrease in the global intracellular[Ca2+]and oppose vasoconstriction. This is in contrast to cardiac and skeletal muscle, where spatial and temporal summation of calcium sparks leads to global increases in intracellular[Ca2+]and myocyte contraction. We summarize the present data on local RyR calcium signaling in arterial SMCs in comparison to striated muscle and muscle-specific differences in coupling between L-type calcium channels and RyRs. Accordingly, arterial SMCCav1.2L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux though RyRs. Downregulation of RyR2 up to a certain degree is compensated by increased SR calcium content to normalize calcium sparks. This indirect coupling betweenCav1.2and RyR in arterial SMCs is opposite to striated muscle, where triggering of calcium sparks is controlled by rapid and direct cross-talk betweenCav1.1/Cav1.2L-type channels and RyRs. We discuss the role of RyR isoforms in initiation and formation of calcium sparks in SMCs and their possible molecular binding partners and regulators, which differ compared to striated muscle.

Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca2+ signaling in single resistance arterial smooth muscle cells

2008 ◽  
Vol 295 (6) ◽  
pp. C1590-C1601 ◽  
Author(s):  
Carl P. Nelson ◽  
Jonathon M. Willets ◽  
Noel W. Davies ◽  
R. A. John Challiss ◽  
Nicholas B. Standen
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Critical Role ◽  
Green Fluorescence Protein ◽  
Acetoxymethyl Ester ◽  
Arterial Smooth Muscle ◽  
Surface Receptors ◽  
Simultaneous Imaging ◽  
Intracellular Ca ◽  
Activate Cell

Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca2+ concentration ([Ca2+]i) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca2+ signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes α, δ, and ε; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCα mirrored the rise in [Ca2+]i, while that of eGFP-PKCδ or -ε occurred more slowly. Endothelin-induced translocation of eGFP-PKCε was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca2+]i increases using 1,2-bis-( o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCα translocation, while eGFP-PKCδ translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca2+ and PKC responses to different vasoconstrictors in ASM.

scholarly journals Effect of lysophosphatidylglycerol on intracellular free Ca2+ concentration in A10 vascular smooth muscle cells

2017 ◽  
Vol 95 (10) ◽  
pp. 1283-1288 ◽  
Author(s):  
Ying Zhang ◽  
Jing-Dian Zhang ◽  
Ming-Qin Zhu ◽  
Ming Zhang ◽  
Yan-Jun Xu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Elevated Level ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
The Other ◽  
Ip3 Receptor ◽  
Acetoxymethyl Ester ◽  
Vascular Abnormalities ◽  
Intracellular Ca

Although plasma levels of lysophosphatidylglycerol (LPG) are increased in hypertension, its role in the pathogenesis of vascular defects is not clear. In view of the importance of Ca2+ overload in causing vascular smooth muscle (VSM) dysfunction, the action of LPG on [Ca2+]i in cultured A10 VSM cell line was examined by using Fura 2-AM acetoxymethyl ester technique. LPG was found to induce a concentration-dependent increase in [Ca2+]i in VSM cells. This change was dependent both on the extracellular and intracellular Ca2+ sources, as it was reduced by 30% by EGTA, an extracellular Ca2+ chelator, and 70% by thapsigargin, a sarcoplasmic reticulum (SR) Ca2+-pump inhibitor. However the increase in [Ca2+]i due to LPG was not altered by caffeine or ryanodine, which affect Ca2+-release through the ryanodine receptors in the SR. On the other hand, LPG-induced change in [Ca2+]i was suppressed by 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, a phospholipase C (PLC) inhibitor, as well as by xestospongin and 2-aminoethoxydiphenyl borate, two inositol trisphosphate (IP3) receptor inhibitors in the SR. These observations support the view that LPG-induced increase in [Ca2+]i in VSM cells is mainly a result of Ca2+ release from Ca2+ pool in the SR through PLC/IP3-sensitive signal transduction mechanism. Furthermore, it is suggested that the elevated level of LPG may induce intracellular Ca2+ overload and thus play a critical role in the development of vascular abnormalities.

FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells

2004 ◽  
Vol 286 (3) ◽  
pp. C538-C546 ◽  
Author(s):  
Yong-Xiao Wang ◽  
Yun-Min Zheng ◽  
Qi-Bing Mei ◽  
Qinq-Song Wang ◽  
Mei Lin Collier ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Ryanodine Receptors ◽  
Regulatory Mechanisms ◽  
Contraction Coupling ◽  
Fk506 Binding Protein ◽  
Cyclic Adp Ribose ◽  
Intracellular Ca ◽  
Relationship Of

Intracellular Ca2+ release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous Ca2+ release at lower concentrations and produces macroscopic Ca2+ release at higher concentrations; neurotransmitter-evoked Ca2+ release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated Ca2+ release in smooth muscle.

Role of capacitative Ca2+ entry in bronchial contraction and remodeling

2002 ◽  
Vol 92 (4) ◽  
pp. 1594-1602 ◽  
Author(s):  
Michele Sweeney ◽  
Sharon S. McDaniel ◽  
Oleksandr Platoshyn ◽  
Shen Zhang ◽  
Ying Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transient Receptor Potential ◽  
Bronchial Hyperresponsiveness ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Wall Thickening ◽  
Bronchial Wall ◽  
Intracellular Ca ◽  
Transient Receptor

Asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and airway obstruction by bronchospasm and bronchial wall thickening due to smooth muscle hypertrophy. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) may serve as a shared signal transduction element that causes bronchial constriction and bronchial wall thickening in asthma. In this study, we examined whether capacitative Ca2+ entry (CCE) induced by depletion of intracellular Ca2+ stores was involved in agonist-mediated bronchial constriction and bronchial smooth muscle cell (BSMC) proliferation. In isolated bronchial rings, acetylcholine (ACh) induced a transient contraction in the absence of extracellular Ca2+ because of Ca2+ release from intracellular Ca2+ stores. Restoration of extracellular Ca2+in the presence of atropine, an M-receptor blocker, induced a further contraction that was apparently caused by a rise in [Ca2+]cyt due to CCE. In single BSMC, amplitudes of the store depletion-activated currents ( I SOC) and CCE were both enhanced when the cells proliferate, whereas chelation of extracellular Ca2+ with EGTA significantly inhibited the cell growth in the presence of serum. Furthermore, the mRNA expression of TRPC1, a transient receptor potential channel gene, was much greater in proliferating BSMC than in growth-arrested cells. Blockade of the store-operated Ca2+channels by Ni2+ decreased I SOC and CCE and markedly attenuated BSMC proliferation. These results suggest that upregulated TRPC1 expression, increased I SOC, enhanced CCE, and elevated [Ca2+]cyt may play important roles in mediating bronchial constriction and BSMC proliferation.

Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury

2014 ◽  
Vol 307 (3) ◽  
pp. H337-H345 ◽  
Author(s):  
Lara Gotha ◽  
Sang Yup Lim ◽  
Azriel B. Osherov ◽  
Rafael Wolff ◽  
Beiping Qiang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Critical Role ◽  
Arterial Injury ◽  
Side Chains ◽  
Wild Type ◽  
Injury Response ◽  
Migratory Response

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2Δ3/Δ3 (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type ( P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB ( P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

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