Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1

2006 ◽  
Vol 291 (6) ◽  
pp. C1326-C1335 ◽  
Author(s):  
Yulia Shakirova ◽  
Johan Bonnevier ◽  
Sebastian Albinsson ◽  
Mikael Adner ◽  
Bengt Rippe ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Rho Kinase ◽  
Signaling Proteins ◽  
Muscarinic Agonist ◽  
Smooth Muscle Contractility ◽  
Caveolin 1 ◽  
Femoral Arteries ◽  
Kinase C ◽  
Arterial Contraction ◽  
Relative Mrna Expression

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca2+ sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were ∼15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPγS was increased in KO compared with WT as shown using a pull-down assay. Following α-toxin permeabilization, no difference in Ca2+ sensitivity or in Ca2+ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased α1-adrenergic contraction. Following inhibition of PKC, α1-adrenergic contraction was normalized. PDBu-induced Ca2+ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca2+ sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca2+ sensitivity.

scholarly journals Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

2012 ◽  
Vol 303 (6) ◽  
pp. G713-G722 ◽  
Author(s):  
Jagmohan Singh ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Rho Kinase ◽  
Muscle Layer ◽  
Relative Distribution ◽  
Muscarinic Agonist ◽  
Circular Smooth Muscle ◽  
Kinase C ◽  
Basal Tone

Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca2+, KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K+depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10−5M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs′ SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS.

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle

2007 ◽  
Vol 293 (6) ◽  
pp. L1406-L1418 ◽  
Author(s):  
Reinoud Gosens ◽  
Gerald L. Stelmack ◽  
Gordon Dueck ◽  
Mark M. Mutawe ◽  
Martha Hinton ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscarinic Receptor ◽  
Airway Smooth Muscle ◽  
Isometric Force ◽  
Signaling Proteins ◽  
Muscarinic Agonist ◽  
Caveolin 1 ◽  
Intracellular Ca ◽  
Domain Peptide

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca2+ homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca2+ ([Ca2+]i) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M3 receptors (M3R) and Gαq/11 cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with β-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-β-cyclodextrin (mβCD) reduced sensitivity but not maximum [Ca2+]i induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mβCD disrupted the colocalization of caveolae-1 and M3R, but [ N-methyl-3H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca2+]i flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mβCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca2+]i mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca2+]i mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action

2002 ◽  
Vol 282 (3) ◽  
pp. G461-G469 ◽  
Author(s):  
Ya-Ping Fan ◽  
Rajinder N. Puri ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Ang Ii ◽  
Kinase C ◽  
Rho Kinase Inhibitor ◽  
Isolated Smooth Muscle Cells

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT1antagonist losartan but not AT2 antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca2+ channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p44/42mitogen-activating protein kinase (MAPK44/42) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT1 and AT2receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT1 receptors at the SMC and involves multiple intracellular pathways, influx of Ca2+, and activation of PKC, Rho kinase, and MAPK44/42.

scholarly journals Protein Kinase C-ε Induces Caveolin-Dependent Internalization of Vascular Adenosine 5′-Triphosphate–Sensitive K + Channels

Hypertension ◽  
2008 ◽  
Vol 52 (3) ◽  
pp. 499-506 ◽  
Author(s):  
Jundong Jiao ◽  
Vivek Garg ◽  
Baofeng Yang ◽  
Terry S. Elton ◽  
Keli Hu
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Cell Surface ◽  
Small Interfering Rna ◽  
Caveolin 1 ◽  
Kinase C ◽  
Interfering Rna ◽  
Pkc Activation

Vascular ATP-sensitive K + (K ATP ) channels are critical regulators of arterial tone and, thus, blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is known that protein kinase C (PKC) activation inhibits K ATP channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Here we report that caveolin-dependent internalization is involved in PKC-ε–mediated inhibition of vascular K ATP channels (Kir6.1 and SUR2B) by phorbol 12-myristate 13-acetate or angiotensin II in human embryonic kidney 293 cells and immortalized human saphenous vein vascular smooth muscle cells. We showed that Kir6.1 substantially overlapped with caveolin-1 at the cell surface. Cholesterol depletion with methyl-β-cyclodextrin significantly reduced, whereas overexpression of caveolin-1 largely enhanced, PKC-induced inhibition of Kir6.1/SUR2B currents. Importantly, we demonstrated that activation of PKC-ε caused internalization of K ATP channels, the effect that was blocked by depletion of cholesterol with methyl-β-cyclodextrin, expression of dominant-negative dynamin mutant K44E, or knockdown of caveolin-1 with small interfering RNA. Moreover, patch-clamp studies revealed that PKC-ε–mediated inhibition of the K ATP current induced by PMA or angiotensin II was reduced by a dynamin mutant, as well as small interfering RNA targeting caveolin-1. The reduction in the number of plasma membrane K ATP channels by PKC activation was further confirmed by cell surface biotinylation. These studies identify a novel mechanism by which the levels of vascular K ATP channels could be rapidly downregulated by internalization. This finding provides a novel mechanistic insight into how K ATP channels are regulated in vascular smooth muscle cells.

scholarly journals Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

2007 ◽  
Vol 204 (10) ◽  
pp. 2373-2382 ◽  
Author(s):  
Takahisa Murata ◽  
Michelle I. Lin ◽  
Yan Huang ◽  
Jun Yu ◽  
Phillip Michael Bauer ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Epithelial Cells ◽  
Pulmonary Arteries ◽  
Mitogen Activated Protein Kinase ◽  
No Production ◽  
Smooth Muscle Contractility ◽  
Caveolin 1 ◽  
Multiple Phenotypes ◽  
Mitogen Activated Protein

Caveolin-1 (Cav-1) is the principal structural component of caveolae organelles in smooth muscle cells, adipocytes, fibroblasts, epithelial cells, and endothelial cells (ECs). Cav-1–deficient (Cav-1 knockout [KO]) mice are viable and show increases of nitric oxide (NO) production in vasculature, cardiomyopathy, and pulmonary dysfunction. In this study, we generated EC-specific Cav-1–reconstituted (Cav-1 RC) mice and reexamined vascular, cardiac, and pulmonary phenotypes. Cav-1 KO pulmonary arteries had decreased smooth muscle contractility and increased endothelial NO synthase activation and hypotension; the latter two effects were rescued completely in Cav-1 RC mice. Cav-1 KO mice exhibited myocardial hypertrophy, pulmonary hypertension, and alveolar cell hyperproliferation caused by constitutive activation of p42/44 mitogen-activated protein kinase and Akt. Interestingly, in Cav-1 RC mice, cardiac hypertrophy and pulmonary hypertension were completely rescued, whereas alveolar hyperplasia was partially recovered because of the lack of rescue of Cav-1 in bronchiolar epithelial cells. These results provide clear physiological evidence supporting the important role of cell type–specific Cav-1 expression governing multiple phenotypes in the vasculature, heart, and lung.

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