scholarly journals Effect of TPA on ion fluxes and DNA synthesis in vascular smooth muscle cells.

1985 ◽  
Vol 101 (2) ◽  
pp. 454-459 ◽  
Author(s):  
N E Owen
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Vascular Smooth Muscle Cells ◽  
Egf Receptor ◽  
Phorbol Esters ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
A431 Cells

Previous reports have suggested that phorbol esters can decrease the affinity of epidermal growth factor (EGF) for its cellular receptors. Investigations of the consequences of the interaction between phorbol esters and EGF, however, have been limited to EGF-stimulated Na/H exchange in A431 cells (Whitely, B., D. Cassel, Y.-X. Zuang, and L. Glaser, 1984, J. Cell Biol., 99:1162-1166). In the present study, the effect of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) on EGF-stimulated ion transport and DNA synthesis was determined in cultured vascular smooth muscle cells (A7r5). It was found that TPA stimulated Na/H exchange when added alone (half-maximal stimulatory concentration, 25 nM). However, when cells were pretreated with TPA and then challenged with EGF, TPA significantly inhibited EGF-stimulated Na/H exchange (78%; half-maximal inhibition [Ki] at 2.5 nM). Subsequently the effects of TPA on Na/K/Cl co-transport were measured. TPA was observed to inhibit Na/K/Cl co-transport (half-maximal inhibitory concentration, 50 nM) and also to inhibit EGF-stimulated Na/K/Cl co-transport (100%; Ki at 5 nM). Finally, the effects of TPA on DNA synthesis were assessed. TPA had a modest stimulatory effect on DNA synthesis (half-maximal stimulatory concentration, 6 nM), but had a significant inhibitory effect on EGF-stimulated DNA synthesis (56%; Ki at 5 nM). These findings suggest that the inhibitory effect of TPA on EGF-receptor functions goes beyond previously reported effects on Na/H exchange in A431 cells and extends to EGF-stimulation of Na/K/Cl co-transport and DNA synthesis in vascular smooth muscle cells.

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.

Altered L-type Ca2+ channel currents in vascular smooth muscle cells from experimental diabetic rats

2000 ◽  
Vol 278 (3) ◽  
pp. H714-H722 ◽  
Author(s):  
Rui Wang ◽  
Yuejin Wu ◽  
Guanghua Tang ◽  
Lingyun Wu ◽  
Salma Toma Hanna
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Vascular Complications ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
Diabetic Rats ◽  
High Concentration ◽  
Voltage Dependent

Vascular complications of diabetes are associated with abnormal Ca2+ handling by vascular smooth muscle cells (SMCs) in which the alteration in L-type voltage-dependent Ca2+ channel (VDCC) currents may play an important role. In the present study, the characteristics of L-type VDCC currents in tail artery SMCs from streptozotocin-induced diabetic rats were examined. The densities, but not the voltage dependence, of L-type VDCC currents were reduced as diabetes progressed from 1 wk to 3 mo. The inhibitory effect of dibutyryl-cAMP on L-type VDCC currents was greater in diabetic SMCs than in age-matched control cells ( P < 0.01). Both the stimulatory effect of BAY K 8644 and the inhibitory effect of nifedipine on L-type VDCC currents were significantly enhanced in diabetic cells. The diabetes-related abnormalities in L-type VDCC currents were mimicked by culturing SMCs with a high concentration of glucose. Our results suggest that the properties of L-type VDCC in diabetic vascular SMCs were significantly altered, partially related to the increased L-type VDCC sensitivity to cAMP and hyperglycemia.

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