β-Adrenoceptor activation and PKA regulate delayed rectifier K+ channels of vascular smooth muscle cells

1998 ◽  
Vol 275 (2) ◽  
pp. H448-H459 ◽  
Author(s):  
E. Alejandro Aiello ◽  
A. Todd Malcolm ◽  
Michael P. Walsh ◽  
William C. Cole
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Portal Vein ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Catalytic Subunit ◽  
Delayed Rectifier ◽  
Rabbit Portal Vein

Macroscopic 4-aminopyridine (4-AP)-sensitive, delayed rectifier K+ current of vascular smooth muscle cells is increased during β-adrenoceptor activation with isoproterenol via a signal transduction pathway involving adenylyl cyclase and cAMP-dependent protein kinase (PKA) (Aiello, E. A., M. P. Walsh, and W. C. Cole. Am. J. Physiol. 268 ( Heart Circ. Physiol. 37): H926–H934, 1995.). In this study, we identified the single delayed rectifier K+(KDR) channel(s) of rabbit portal vein myocytes affected by treatment with isoproterenol or the catalytic subunit of PKA. 4-AP-sensitive KDR channels of 15.3 ± 0.6 pS ( n = 5) and 14.8 ± 0.6 pS ( n = 5) conductance, respectively, were observed in inside-out (I-O) and cell-attached (C-A) membrane patches in symmetrical KCl recording conditions. The kinetics of activation (time constant of 10.7 ± 3.02 ms) and inactivation (fast and slow time constants of 0.3 and 2.5 s, respectively) of ensemble currents produced by these channels mimicked those reported for inactivating, 4-AP-sensitive whole cell KDR current of vascular myocytes. Under control conditions, the open probability ( NP o) of KDR channels of C-A membrane patches at −40 mV was 0.014 ± 0.005 ( n = 8). Treatment with 1 μM isoproterenol caused a significant, approximately threefold increase in NP o to 0.041 ± 0.02 ( P < 0.05). KDR channels of I-O patches exhibited rundown after ∼5 min, which was not affected by ATP (5 mM) in the bath solution. Treatment with the purified catalytic subunit of PKA (50 nM; 5 mM ATP) restored KDRchannel activity and caused NP o to increase from 0.011 ± 0.003 to 0.138 ± 0.03 ( P < 0.05; n = 11). These data indicate that small-conductance, 15-pS KDRchannels are responsible for inactivating the macroscopic delayed rectifier K+ current of rabbit portal vein myocytes and that the activity of these channels is enhanced by a signal transduction mechanism involving β-adrenoceptors and phosphorylation by PKA at a membrane potential consistent with that observed in the myocytes in situ.

Abstract 486: Artificial miRNA Based Adenoviral Adam17 Silencing Reveals Essential Role of This Metalloprotease For EGF Receptor Transactivation In Primary Cultured Vascular Smooth Muscle Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Katherine Elliott ◽  
Allison Bourne ◽  
Takehiko Takayanagi ◽  
Akira Takaguri ◽  
Kunie Eguchi ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Gene Silencing ◽  
Vascular Smooth Muscle Cells ◽  
Egf Receptor ◽  
Muscle Cells ◽  
Vascular Cells ◽  
Receptor Transactivation

siRNA mediated gene silencing has been recently utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to the transient nature of silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture. To overcome such weakness of the siRNA based silencing and in order to establish reliable gene silencing in vascular cells, we devised an adenoviral-encoded miRNA based gene silencing system. Here we report the results of silencing ADAM17 in cultured rat vascular smooth muscle cells (VSMCs) and its functional consequences in angiotensin II (AngII) signal transduction. Four distinct miRNA sequences targeting rat ADAM17 were chosen based on recommendations from Invitrogen’s Block-iT RNAi Designer algorithm. The miRNA sequences were inserted into a mammalian expression vector, pcDNA 6.2-GW/EmGFP-miR, and the effective silencing by these vectors was confirmed in HEK cells expressing HA-tagged rat ADAM17. The 4 cassettes carrying the miRNAs were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Greater than 95% silencing of ADAM17 was achieved when VSMC were infected with 100-200 moi of the ADAM17 miRNA encoding adeonvirus for 72 h with enhancement of infection by fugene6. Relatively linear time and concentration dependencies were observed between 1 to 3 days and 10 to 100 moi of the infection. A miR-ADAM17 (100 moi) but not miR-control (100 moi) completely inhibited 100 nM AngII-induced HB-EGF shedding in VSMCs as assessed by a reporter assay. A miR-ADAM17 but not miR-control also inhibited AngII-induced EGF receptor transactivation and subsequent ERK1/2 activation in VSMCs as assessed by immunoblotting with phospho-selective antibodies. In conclusion, ADAM17 was found to be a major sheddase for HB-EGF contributing to the growth promoting signals induced by AngII in VSMCs. An artificial miRNA-base adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

2004 ◽  
Vol 287 (3) ◽  
pp. C807-C813 ◽  
Author(s):  
Mizuo Mifune ◽  
Haruhiko Ohtsu ◽  
Hiroyuki Suzuki ◽  
Gerald D. Frank ◽  
Tadashi Inagami ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Vascular Remodeling ◽  
Muscle Cells ◽  
Egf Family ◽  
And Migration

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling.

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