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Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric
Abstract:
1. We have studied the effect of endothelin-1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist. 2. Endothelin-1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration-dependent (1-100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and alpha-toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28-33, 56-61, 75-85 and 105-115 kDa. Endothelin-1-induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin A1. 3. In intact small arteries tyrphostin A23 inhibited the force developed to endothelin-1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin A1 inhibited the contractile response to endothelin-1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin-1 (100 nM) in the absence of extracellular calcium. 4. In rat alpha-toxin permeabilized mesenteric arteries endothelin-1 caused a concentration-dependent increase in force in the presence of 10 microM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin A1 did not affect endothelin-1-induced calcium sensitization of force. 5. We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin-1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.
Insights
Endothelin-1 rapidly increases protein tyrosine phosphorylation in rat mesenteric arteries, coinciding with contraction. Tyrosine kinase inhibition affects this response, suggesting its importance in vascular smooth muscle contraction.
Area of Science:
- Vascular Physiology
- Molecular Biology
- Biochemistry
Background:
- Endothelin-1 is a potent vasoconstrictor.
- Protein tyrosine phosphorylation plays a role in cellular signaling pathways.
- The precise mechanisms linking endothelin-1 to vascular contraction are not fully elucidated.
Purpose of the Study:
- To investigate the effect of endothelin-1 on protein tyrosine phosphorylation in rat small mesenteric arteries.
- To determine the role of tyrosine kinase inhibition in endothelin-1-induced vascular contraction.
Main Methods:
- Studied protein tyrosine phosphorylation levels in response to endothelin-1 in rat mesenteric arteries.
- Utilized tyrosine kinase inhibitors (tyrphostin A23 and A1) to assess their impact on contractile responses.
- Examined responses in both intact and alpha-toxin permeabilized arterial preparations.
Main Results:
- Endothelin-1 caused a rapid and sustained increase in protein tyrosine phosphorylation, temporally linked to contraction.
- Phosphorylation occurred in specific protein clusters (28-33, 56-61, 75-85, 105-115 kDa) and was calcium-independent.
- Tyrosine kinase inhibition (tyrphostin A23) attenuated endothelin-1-induced contraction and altered its profile, while also inhibiting calcium sensitization in permeabilized arteries.
Conclusions:
- Increased protein tyrosine phosphorylation is crucial for endothelin-1-mediated contraction in small mesenteric arteries.
- This signaling pathway activation is implicated in the tonic phase of contraction, potentially by modulating calcium sensitivity of the contractile apparatus.