ABSTRACT
Minimally modified low density lipoprotein (mmLDL) is a well-known risk factor for coronary artery disease. Upregulation of vascular endothelin type B (ET(B)) receptors on the vascular smooth muscle cells is predicted to be the molecular mechanism that leads to cardiovascular pathogenesis. The objective of the present study was to examine the hypothesis that mmLDL upregulates ET(B) receptors in rat coronary artery. The contractile responses to sarafotoxin 6c (ET(B) receptor agonist) were studied using a sensitive myograph. ET(B) receptor mRNA and protein expression was determined using real-time PCR and Western blot analysis. The results showed that organ culture increased the contractile responses induced by sarafotoxin 6c and the levels of ET(B) receptor mRNA and protein. This increase was further enhanced by the addition of mmLDL (10µg/mL). Specific ERK1/2 inhibitors (SB386023 and U0126) and an NF-κB inhibitor (wedelolactone) attenuated the mmLDL-increased ET(B) receptor-mediated contraction and ET(B) receptor mRNA and protein levels. Wedelolactone significantly attenuated the mmLDL-decreased IκB(α) protein expression. Consistent with this result, IκB(α) protein expression was significantly decreased by culture with mmLDL compared to the level of expression in the organ culture group. However, the JNK inhibitor, SP600125 or p38 pathway inhibitor, SB203580 did not inhibit mmLDL-enhanced effects. The PKC inhibitor, staurosporine attenuated only culture-alone-increased effects. In conclusion, mmLDL upregulates the ET(B) receptors in rat coronary arterial smooth muscle cells, mainly via activation of the ERK1/2 MAPK and the downstream transcriptional factor NF-κB.
Subject(s)
Coronary Vessels/drug effects , Lipoproteins, LDL/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NF-kappa B/metabolism , Receptor, Endothelin B/metabolism , Animals , Blotting, Western , Butadienes/pharmacology , Coronary Vessels/metabolism , Coronary Vessels/physiology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Myography , NF-kappa B/antagonists & inhibitors , Nitriles/pharmacology , Organ Culture Techniques , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Endothelin B/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Staurosporine/pharmacology , Up-Regulation/drug effects , Vasoconstriction/drug effects , Vasoconstrictor Agents/pharmacology , Viper Venoms/pharmacologyABSTRACT
The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1/2 expression level in cultured rat TG, with its actions similar to the effective concentration of sumatriptan succinate, flunarizine hydrochloride and specific ERK1/2 pathway blocker. The intracellular signaling transduction ERK1/2 pathway may be involved in the gastrodin reducing CGRP up-regulation in rat TG after organ culture.
Subject(s)
Animals , Male , Rats , Benzyl Alcohols , Pharmacology , Butadienes , Pharmacology , Calcitonin Gene-Related Peptide , Genetics , Metabolism , Dose-Response Relationship, Drug , Flavonoids , Pharmacology , Flunarizine , Pharmacology , Gastrodia , Chemistry , Glucosides , Pharmacology , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Nitriles , Pharmacology , Organ Culture Techniques , Plants, Medicinal , Chemistry , RNA, Messenger , Rats, Sprague-Dawley , Sumatriptan , Pharmacology , Trigeminal Ganglion , Metabolism , Vasoconstrictor Agents , Pharmacology , Vasodilator Agents , PharmacologyABSTRACT
<p><b>AIM</b>To determine the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in the expression of endothelin receptor type B (ETB) during culture.</p><p><b>METHODS</b>SB386023, a specific inhibitor for ERK1/2 pathway, was used to define the intracellular signaling pathway for the upregulation of ETB receptors and sarafotoxin 6c (S6c), a selective agonist for ETB receptors, induced contraction in isolated rat superior mesenteric arteries. The contraction was recorded by a sensitive in vitro myograph and the receptor mRNA was quantified by a real-time PCR. The phosphorylated ERK1/2 proteins were analyzed by phosphoELISA assay.</p><p><b>RESULTS</b>S6c induced strong contractile responses of the artery after culture for 24 h, while there was no response to S6c in fresh vessel segments. The enhanced contractile response to S6c paralleled with an increase of mRNA for ETB receptors. The phosphorylated ERK1/2 proteins significantly increased after culture for 3 h. After co-culture with SB386023 for 24 h, S6c-induced contractions significantly decreased with reduction of Emax from (217 +/- 14) % to (127 +/- 23) % (P <0.01). This response paralleled with a decreased level of ETB receptor mRNA.</p><p><b>CONCLUSION</b>ERK1/2 pathway was involved in the up-regulation of ETB receptors on smooth muscle cells isolated from rat mesenteric arteries during culture.</p>
Subject(s)
Animals , Male , Rats , Cells, Cultured , Mesenteric Arteries , Cell Biology , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Muscle Contraction , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Organ Culture Techniques , Phosphorylation , RNA, Messenger , Genetics , Rats, Sprague-Dawley , Receptor, Endothelin B , Genetics , Signal Transduction , Up-Regulation , Vasoconstrictor Agents , Pharmacology , Viper Venoms , PharmacologyABSTRACT
<p><b>AIM</b>To study the vasodilation effect of atropine and its mechanism.</p><p><b>METHODS</b>Isometric tension was recorded in isolated rat super mesenteric arteries precontracted by noradrenaline (NE) to study the vasodilation effect of atropine, and to investigate the role of endothelial cell and vascular smooth muscle cell on vasodilation.</p><p><b>RESULTS</b>Atropine was shown to significantly dilate the endothelium-intact and endothelium-denuded arteries precontracted by NE. Nomega-Nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhabitor), indomethacin (cyclooxygenase inhibitor), propranolol (general beta adrenoceptor antagonist) and glibenclamide (ATP sensitive potassium channel inhibitor) showed no effect on vasodilation of atropine. Atropine did not affect the concentration-contraction curve of K+. However, atropine suppressed the contraction induced by NE and CaCl2, but not that by caffeine in the Ca+ -free Krebs solution.</p><p><b>CONCLUSION</b>Atropine showed significant vasodilation effect which may derive, in part, from endothelium. Besides, atropine could inhibit the receptor-mediated Ca2+ -influx and Ca2+ -release, which was inferred to the mechanism of atropine on vasodilation.</p>