Activation of Erk mitogen-activated protein kinase proteins by vascular serotonin receptors.

We previously demonstrated that 5-hydroxytryptamine 2A (5-HT 2A ) receptor-mediated rat arterial contraction was dependent on activation of tyrosine kinases, including mitogen-activated protein kinase (MAPK) kinase. In the current study, we examined arterial smooth muscle for the presence of serotonin (5-hydroxytryptamine, 5-HT) 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, and 5-HT 7 receptor mRNA and hypothesized that, if present, activation of these receptors would stimulate the extracellular signal-regulated kinase (Erk) MAPK pathway and an Erk MAPK-dependent contraction. RT-PCR analyses of rat aortic smooth muscle cells, cultured and fresh, indicated the presence of 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, and 5-HT 7 receptor mRNA. The 5-HT 1B agonists RU24969 and CGS12066B, 5-HT 1B/1D/1F receptor agonist sumatriptan, and 5-HT 2B receptor agonist BW723C86 (10(-9) - 10(-4) M ) did not contract the aorta, nor did the 5-HT 7 receptor antagonist LY215840 leftward shift 5-HT-induced contraction. The 5-HT 1E/1F receptor agonist BRL54443 induced contraction, but this was abolished by the 5-HT 2A/2C receptor antagonist ketanserin (10 nM ); contraction was not observed with a different 5-HT 1F receptor agonist, LY344864. 5-HT and alpha-methyl-5-HT produced a concentration-dependent increase in Erk MAPK activity in cultured aortic smooth muscle cells and in aorta contracted with these agonists. All other agonists were inactive; a high concentration of BRL54443 (10 microM ) stimulated Erk MAPK activation (150% basal). Thus, while mRNA and possibly protein for multiple 5-HT receptors are present in aortic smooth muscle, only the 5-HT 2A receptor plays a significant role in directly modulating contractility and activating the Erk MAPK pathway.

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

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.

Enhanced contraction to 5-hydroxytryptamine is not due to "unmasking" of 5-hydroxytryptamine(1b) receptors in the mesenteric artery of the deoxycorticosterone acetate-salt rat.

5-Hydroxytryptamine(1B) (5-HT(1B)) receptors have been implicated in mediating arterial contraction to 5-HT. Additionally, the 5-HT(1B) receptor has been reported to be "unmasked" by depolarizing stimuli. We hypothesized that 5-HT(1B) receptors in arteries from hypertensive animals, arteries reported to have a depolarized resting membrane potential in smooth muscle cells, are unmasked and participate in the supersensitivity observed to 5-HT in hypertension. We used the isolated tissue bath apparatus and examined the response of superior mesenteric arteries from normotensive sham and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. The 5-HT(1B) agonists CP93129 and sumatriptan (10(-9) to 10(-5) mol/L) caused a maximal contraction (50+/-12% of phenylephrine [10(-5) mol/L] contraction) in arteries from DOCA-salt rats; no contraction was observed in arteries from normotensive rats. The 5-HT(1B) receptor antagonist GR55562 (100 nmol/L) inhibited both the 5-HT- (4-fold rightward shift) and CP93129-induced (11-fold rightward shift) contractions in mesenteric arteries from hypertensive DOCA-salt rats. In other experiments, arteries from normotensive rats were incubated with 15 mmol/L KCl, as a depolarizing stimulus, and then exposed to 5-HT and CP93129. In the presence of KCl, a small leftward shift to 5-HT was observed. However, the presence of a depolarizing stimulus was unable to produce changes in the 5-HT maximal response to resemble that of arteries from DOCA-salt rats, nor was contraction to CP93129 observed. These data support the conclusions that 5-HT(1B) receptors mediate contraction in mesenteric arteries from hypertensive rats and that this enhanced response to 5-HT is not due to membrane depolarization alone.