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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.

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Related in: MedlinePlus

Top: Blot of 5-HT-induced stimulation of the p38 pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific p38 antibody. Blot is representative of four experiments. Total anti-p38 antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Bottom: Densitometry measurements for p38 kDa. Bar graphs represent means and vertical bars the SEM for N = 4. * = statistical difference for response in vehicle-incubated cell cultures.
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Figure 2: Top: Blot of 5-HT-induced stimulation of the p38 pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific p38 antibody. Blot is representative of four experiments. Total anti-p38 antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Bottom: Densitometry measurements for p38 kDa. Bar graphs represent means and vertical bars the SEM for N = 4. * = statistical difference for response in vehicle-incubated cell cultures.

Mentions: The p38 pathway has demonstrated a different time course of activation via G-protein coupled receptors than the ERK pathway [24]. Using the time point (30 min) at which angiotensin II demonstrates maximal stimulation of the JNK pathway we performed a concentration response curve (10-9 – 10-5 M) to 5-HT in vascular smooth muscle cells. Western analysis using phosphospecific antibodies was used to measure activation the p38 pathway. A total or non-phosphospecific antibody was used to ensure equal loading of p38 protein. After thirty-minutes of incubation with 5-HT (10-9 – 10-5 M), there was no enhanced phosphorylation of p38 (figure 2, bottom). In contrast, incubation with anisomycin (500 ng/ml) induced significant phosphorylation of p38 (2-fold above basal). These data suggest that 5-HT does not activate the p38 pathway in rat vascular smooth muscle cells despite the ability of other agonists to activate the pathway in these cells.


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 p38 pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific p38 antibody. Blot is representative of four experiments. Total anti-p38 antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Bottom: Densitometry measurements for p38 kDa. Bar graphs represent means and vertical bars the SEM for N = 4. * = statistical difference for response in vehicle-incubated cell cultures.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Top: Blot of 5-HT-induced stimulation of the p38 pathway in rat aortic vascular smooth muscle cells as measured by use of a phosphospecific p38 antibody. Blot is representative of four experiments. Total anti-p38 antibody recognizes nonphosphorylated forms as well as phosphorylated protein. Bottom: Densitometry measurements for p38 kDa. Bar graphs represent means and vertical bars the SEM for N = 4. * = statistical difference for response in vehicle-incubated cell cultures.
Mentions: The p38 pathway has demonstrated a different time course of activation via G-protein coupled receptors than the ERK pathway [24]. Using the time point (30 min) at which angiotensin II demonstrates maximal stimulation of the JNK pathway we performed a concentration response curve (10-9 – 10-5 M) to 5-HT in vascular smooth muscle cells. Western analysis using phosphospecific antibodies was used to measure activation the p38 pathway. A total or non-phosphospecific antibody was used to ensure equal loading of p38 protein. After thirty-minutes of incubation with 5-HT (10-9 – 10-5 M), there was no enhanced phosphorylation of p38 (figure 2, bottom). In contrast, incubation with anisomycin (500 ng/ml) induced significant phosphorylation of p38 (2-fold above basal). These data suggest that 5-HT does not activate the p38 pathway in rat vascular smooth muscle cells despite the ability of other agonists to activate the pathway in these cells.

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
Related in: MedlinePlus