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Inability of serotonin to activate the c-Jun N-terminal kinase and p38 kinase pathways in rat aortic vascular smooth muscle cells.

Banes AK, Loberg RD, Brosius FC, Watts SW - BMC Pharmacol. (2001)

Bottom Line: Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point.Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Ml 48824, USA. banesamy@msu.edu

ABSTRACT

Background: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells.

Results: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10(-9) - 10(-5) M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.

Conclusion: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.

Show MeSH
Top: Blot of 5-HT-induced stimulation of the ERK/MAPK pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific ERK/MAPK antibody. Densitometry measurements are for the 42 kDa band. Blot is representative of four experiments. Bottom: Total ERK/MAPK antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Densitometry measurements are for the 42 kDa band.
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Figure 1: Top: Blot of 5-HT-induced stimulation of the ERK/MAPK pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific ERK/MAPK antibody. Densitometry measurements are for the 42 kDa band. Blot is representative of four experiments. Bottom: Total ERK/MAPK antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Densitometry measurements are for the 42 kDa band.

Mentions: In agreement with previously published studies from our lab, 5-HT caused a concentration-dependent activation of the ERK pathway (figure 1). This activation occurs maximally at five minutes of stimulation and returns to basal levels by thirty minutes [23]. This time course of activation is consistent with that of other G-protein coupled receptors.


Inability of serotonin to activate the c-Jun N-terminal kinase and p38 kinase pathways in rat aortic vascular smooth muscle cells.

Banes AK, Loberg RD, Brosius FC, Watts SW - BMC Pharmacol. (2001)

Top: Blot of 5-HT-induced stimulation of the ERK/MAPK pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific ERK/MAPK antibody. Densitometry measurements are for the 42 kDa band. Blot is representative of four experiments. Bottom: Total ERK/MAPK antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Densitometry measurements are for the 42 kDa band.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC58586&req=5

Figure 1: Top: Blot of 5-HT-induced stimulation of the ERK/MAPK pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific ERK/MAPK antibody. Densitometry measurements are for the 42 kDa band. Blot is representative of four experiments. Bottom: Total ERK/MAPK antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Densitometry measurements are for the 42 kDa band.
Mentions: In agreement with previously published studies from our lab, 5-HT caused a concentration-dependent activation of the ERK pathway (figure 1). This activation occurs maximally at five minutes of stimulation and returns to basal levels by thirty minutes [23]. This time course of activation is consistent with that of other G-protein coupled receptors.

Bottom Line: Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point.Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Ml 48824, USA. banesamy@msu.edu

ABSTRACT

Background: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells.

Results: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10(-9) - 10(-5) M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.

Conclusion: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.

Show MeSH