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Minimally modified LDL upregulates endothelin type A receptors in rat coronary arterial smooth muscle cells.

ABSTRACT: Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. The present study investigated the effects of mmLDL on the expression of endothelin type A (ET(A)) receptors in coronary arteries. Rat coronary arteries were organ-cultured for 24 h. The contractile responses were recorded using a myographic system. ET(A) receptor mRNA and protein expressions were determined using real-time PCR and western blotting, respectively. The results showed that organ-culturing in the presence of mmLDL enhanced the arterial contractility mediated by the ET(A) receptor in a concentration-dependent and time-dependent manner. Culturing with mmLDL (10 μ g/mL) for 24 h shifted the concentration-contractile curves toward the left significantly with increased E(max) of 228% ± 20% from control of 100% ± 10% and significantly increased ET(A) receptor mRNA and protein levels. Inhibition of the protein kinase C, extracellular signal-related kinases 1 and 2 (ERK1/2), or NF- κ B activities significantly attenuated the effects of mmLDL. The c-Jun N-terminal kinase inhibitor or the p38 pathway inhibitor, however, had no such effects. The results indicate that mmLDL upregulates the ETA receptors in rat coronary arterial smooth muscle cells mainly via activating protein kinase C, ERK1/2, and the downstream transcriptional factor, NF- κ B.

SUBMITTER: Li J

PROVIDER: S-EPMC3703896 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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Minimally modified LDL upregulates endothelin type A receptors in rat coronary arterial smooth muscle cells.

Li Jie J Cao Lei L Xu Cang-Bao CB Wang Jun-Jie JJ Cao Yong-Xiao YX

Mediators of inflammation 20130619

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. The present study investigated the effects of mmLDL on the expression of endothelin type A (ET(A)) receptors in coronary arteries. Rat coronary arteries were organ-cultured for 24 h. The contractile responses were recorded using a myographic system. ET(A) receptor mRNA and protein expressions were determined using real-time PCR and western blotting, respectively. The results showed that organ-culturin ...[more]

PMID: 23861561

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Project description:Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca2+ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca2+ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH.

Project description:Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCdelta1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET(A)R) antagonist, BQ123. To characterize ET(A)R desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

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Minimally modified LDL upregulates endothelin type A receptors in rat coronary arterial smooth muscle cells.

Publications

Minimally modified LDL upregulates endothelin type A receptors in rat coronary arterial smooth muscle cells.

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