Characterization and confocal imaging of calponin in gastrointestinal smooth muscle

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.

Download Full-text

Agonist-induced association of tropomyosin with protein kinase Cα in colonic smooth muscle

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00330.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. G268-G276 ◽

Cited By ~ 19

Author(s):

Sita Somara ◽

Haiyan Pang ◽

Khalil N. Bitar

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Thin Filament ◽

Regulatory Protein ◽

Direct Interaction ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Particulate Fraction ◽

Calcium Dependent

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCα in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCα in the particulate fraction may be due to acetylcholine-induced translocation of PKCα to the particulate fraction. To investigate whether the association of PKCα with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCα indicated direct interaction of tropomyosin with PKCα. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCα with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

Download Full-text

Estrogenic Action on Arterial Smooth Muscle: Permissive for Maintenance of CRHR2 Expression

Endocrinology ◽

10.1210/en.2011-1939 ◽

2012 ◽

Vol 153 (4) ◽

pp. 1915-1924 ◽

Cited By ~ 11

Author(s):

Shan Wang ◽

Xiaoyan Zhu ◽

Binhai Cong ◽

Xingji You ◽

Yangkai Wang ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Smooth Muscle Cells ◽

Protein Kinase A ◽

Muscle Cells ◽

Sham Operation ◽

Sprague Dawley ◽

Ovx Rats ◽

Kinase A

Urocortin (Ucn), a member of CRH family, has been implicated to be one of the endogenous regulators in the cardiovascular system and exerts its effects locally via an autocrine/paracrine fashion. Previous studies have shown the gender difference in CRH-induced vasodilation in human skin, which is related to the concentration of estrogens during the menstrual cycle. The aim of this study was to investigate whether estrogens modulate Ucn/CRH receptor type 2 (CRHR2) expression in vascular smooth muscle, thereby leading to vasodilation. We performed sham operation or bilateral ovariectomy (OVX) on female Sprague Dawley rats. OVX rats were sc administered 17β-estradiol (E2) at a dose of 30 μg/kg·d or with placebo for 12 wk. Primary smooth muscle cells of aorta were used for the in vitro study. It was found that the Ucn-induced vasodilation and CRHR2 expression were decreased in OVX rats and restored by E2 replacement treatment for 12 wk. E2 increased the expression of CRHR2 in cultured smooth muscle cells, which was blocked by estrogen receptor-β antagonist. Ucn significantly suppressed the phenylephrine-induced phospholipase Cβ3 activation, inositol 1,4,5-trisphosphate (IP3) production, and intracellular Ca2+ elevation. Ucn stimulated the expression of active GTP-bound Gαs protein and cAMP production. The suppressive effects of Ucn on phenylephrine-induced IP3 production and intracellular Ca2+ elevation were blocked by the inhibitors of adenylate cyclase and protein kinase A. Our results demonstrate that estrogen maintains the expression of CRHR2 in aorta smooth muscle, thereby enhancing vasodilator actions of Ucn. Ucn exerts its vasorelaxant effects via Gαs-cAMP-protein kinase A signaling, leading to down-regulation of the phospholipase Cβ-IP3-Ca2+ signaling pathway.

Download Full-text

Cannabidiol Attenuates Pulmonary Arterial Hypertension by Normalizing the Mitochondrial Function in Vascular Smooth Muscle Cells

10.1101/2020.11.04.364547 ◽

2020 ◽

Author(s):

Xiaohui Lu ◽

Jingyuan Zhang ◽

Huijiao Liu ◽

Wenqiang Ma ◽

Leo Yu ◽

...

Keyword(s):

Smooth Muscle ◽

Pulmonary Artery ◽

Smooth Muscle Cells ◽

Oxidant Stress ◽

Muscle Cells ◽

Pulmonary Artery Hypertension ◽

Independent Manner ◽

Mitochondrial Energy Metabolism

AbstractPulmonary artery hypertension (PAH) is a chronic disease associated with enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and dysfunctional mitochondria, which was with limited therapeutic options. It has been proved that cannabidiol (CBD) had antioxidant effects in many cardiovascular diseases, whereas the efficacy of CBD in PAH is unknown. To defined the effect of CBD in PAH, we explored the functions of CBD in both PASMCs proliferation test in vitro, and preventive and therapeutic PAH rodent models in vivo. The roles of CBD in mitochondria function and the oxidant stress were assessed in human PASMCs and PAH mice. We found that CBD significantly inhibited hyperproliferation of hypoxia-induced PASMCs, and intragastrically administered CBD could reverse the pathological changes in both Sugen-hypoxia and MCT-induced PAH mice models. Mechanical analysis demonstrated that CBD alleviated PAH by recovering mitochondrial energy metabolism, normalizing the hypoxia-induced oxidant stress, inhibiting abnormal glycolysis and lactate accumulation in cannabinoids receptors-independent manner. Thus, CBD could be a potential drug for PAH.Graphical Abstracts

Download Full-text

Ductus arteriosus smooth muscle cell migration on collagen: dependence on laminin and its receptors

Journal of Cell Science ◽

10.1242/jcs.107.4.1007 ◽

1994 ◽

Vol 107 (4) ◽

pp. 1007-1018

Author(s):

R.I. Clyman ◽

J. Tannenbaum ◽

Y.Q. Chen ◽

D. Cooper ◽

P.D. Yurchenco ◽

...

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Ductus Arteriosus ◽

Muscle Cells ◽

Collagen I ◽

Dependent Manner ◽

Independent Manner ◽

Alpha V Beta 3

During permanent closure of the ductus arteriosus, smooth muscle cells migrate through the extracellular matrix (ECM) to form intimal mounds that occlude the vessel's lumen. Smooth muscle cells (SMC) migrate over surfaces coated with collagen in vitro. During the migration SMC also synthesize fibronectin (FN) and laminin (LN). Antibodies against FN and LN inhibit migration on collagen by 30% and 67%, respectively. Because of the apparent importance of LN in migration, we examined how SMC interact with LN and LN fragments (P1, E8, P1′, E1′, E3, E4, and G). Ductus SMC adhere to high concentrations of LN and two fragments of the molecule: P1 and E8. They use a unique set of integrin receptors to bind to LN (alpha 1 beta 1, alpha 6 beta 1 and alpha v beta 3), to P1 (alpha 1 beta 1, alpha v beta 3), and to E8 (alpha 6 beta 1, alpha v beta 3). The alpha v beta 3 integrin binds to the P1 fragment of LN in an RGD peptide-dependent manner, and to the E8 fragment in an RGD-independent manner; the RGD site on the P1 fragment probably is not available to the cell in intact LN. Antibodies against beta 1 integrins completely inhibit SMC adhesion to LN; antibodies against the alpha v beta 3 integrin do not block SMC adhesion to LN, but do prevent cell spreading. LN is also capable of interfering with SMC adhesion to other ECM components. The antiadhesive effect of LN is located in the E1′ domain. Both exogenous and endogenous LN increase SMC motility on collagen I. The locomotion-promoting activity of LN resides in the E1′ antiadhesive domain, and not in its adhesive (P1, E8) domains. LN causes a decrease in the number of focal contacts on collagen I. This might enable SMC to alter their mobility as they move through the extracellular matrix to occlude the ductus arteriosus lumen.

Download Full-text

Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells

Biochemical Journal ◽

10.1042/bj2770445 ◽

1991 ◽

Vol 277 (2) ◽

pp. 445-450 ◽

Cited By ~ 14

Author(s):

D Y Zhao ◽

M D Hollenberg ◽

D L Severson

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Inhibitory Effect ◽

Kinase C ◽

Direct Inhibitory Effect ◽

Ic50 Values

The protein kinase C (PKC) activator phorbol 12,13-dibutyrate stimulated the phosphorylation of a 75 kDa protein (p75) in intact cultured A10 smooth-muscle cells and sonicated cell preparations; p75 was the only major substrate for endogenous PKC in sonicated A10 cells. The Ca(2+)-dependent phosphorylation of p75 in vitro was dramatically decreased in PKC-down-regulated A10 cells; however, p75 from identical sonicated cell preparations was still phosphorylated by an exogenous aortic PKC preparation. Calmodulin inhibited the phosphorylation of p75 by PKC, but not the phosphorylation of other PKC substrates (platelet P47 protein and histone). The addition of calmodulin after the phosphorylation reaction was started prevented further phosphorylation, but did not decrease the extent of phosphorylation of p75 that was reached before the addition of calmodulin. The inhibition of p75 phosphorylation was concentration-dependent, with IC50 values (concn. giving 50% inhibition) ranging from less than 0.5 to 10 micrograms of calmodulin/ml, and was Ca(2+)-dependent, requiring a free Ca2+ concentration of 10 microM or greater. These results suggest that the inhibition of the PKC-catalysed phosphorylation of p75 by calmodulin may be due to its interaction with the substrate, rather than a direct inhibitory effect on the enzyme, and that this inhibition could be regulated by intracellular Ca2+ concentration. Therefore, p75 may be a physiological link between the PKC and Ca2+/calmodulin pathways.

Download Full-text

α-Tocopherol specifically inactivates cellular protein kinase C α by changing its phosphorylation state

Biochemical Journal ◽

10.1042/bj3340243 ◽

1998 ◽

Vol 334 (1) ◽

pp. 243-249 ◽

Cited By ~ 191

Author(s):

Roberta RICCIARELLI ◽

Andrea TASINATO ◽

Sophie CLÉMENT ◽

Nesrin K. ÖZER ◽

Daniel BOSCOBOINIK ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Smooth Muscle Cells ◽

Protein Phosphatase ◽

Antioxidant Properties ◽

Muscle Cells ◽

Cellular Protein ◽

Kinase C

The mechanism of protein kinase C (PKC) regulation by α-tocopherol has been investigated in smooth-muscle cells. Treatment of rat aortic A7r5 smooth-muscle cells with α-tocopherol resulted in a time- and dose-dependent inhibition of PKC. The inhibition was not related to a direct interaction of α-tocopherol with the enzyme nor with a diminution of its expression. Western analysis demonstrated the presence of PKCα, β, δ, ε, ζ and µ isoforms in these cells. Autophosphorylation and kinase activities of the different isoforms have shown that only PKCα was inhibited by α-tocopherol. The inhibitory effects were not mimicked by β-tocopherol, an analogue of α-tocopherol with similar antioxidant properties. The inhibition of PKCα by α-tocopherol has been found to be associated with its dephosphorylation. Moreover the finding of an activation of protein phosphatase type 2A in vitro by α-tocopherol suggests that this enzyme might be responsible for the observed dephosphorylation and subsequent deactivation of PKCα. It is therefore proposed that PKCα inhibition by α-tocopherol is linked to the activation of a protein phosphatase, which in turn dephosphorylates PKCα and inhibits its activity.

Download Full-text

269.Estrogen and progesterone receptor expression is significantly reduced in cultured myometrial and fibroid smooth muscle cells

Reproduction Fertility and Development ◽

10.1071/srb04abs269 ◽

2004 ◽

Vol 16 (9) ◽

pp. 269

Author(s):

M. Zaitseva ◽

P. A. W. Rogers

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Internal Control ◽

Cultured Cells ◽

Progesterone Receptors ◽

Muscle Cells ◽

Receptor Expression ◽

Isolated Cells ◽

Large Variability

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial and fibroid smooth muscle cells (MSMC & FSMC) have been widely used as a model for the study of fibroid growth. However, there is ongoing controversy regarding expression levels of estrogen and progesterone receptors (ER and PR) in cultured cells vs tissue. The aim of the present study was to measure levels of mRNA for ER� and PR in myometrium and fibroid, and in cultured MSMC & FSMC. Myometrium and fibroids were collected from hysterectomy specimens (n = 8). Part of the tissue was snap frozen and the rest was used to isolate SMC, which were cultured for 3 passages and collected for RNA at P0 (2 weeks in culture) and P3 (5 weeks in culture). ERα and PR levels were quantified using real-time PCR and normalized using 18S rRNA as an internal control. Both ERα and PR were detected in all samples. Large variability in receptor levels between different isolates was detected. Surprisingly, despite large differences between the means, none of comparisons of tissue v. P0 cells were significant by non-parametric tests. However, there was a statistically significant reduction in both ERα and PR expression between whole tissue and isolated cells at P3 (Table 1, see PDF file). This is the first study to provide objective data to support a significant decline in ERα and PR expression in cultured MSMC and FSMC. Despite this decline, detectable levels of ERα and PR mRNA were present at both P0 and P3, potentially explaining why some published studies have been able to demonstrate in vitro response to steroids in these cells.

Download Full-text