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Protein kinase C inhibitors decrease endothelin ET(B) receptor mRNA expression and contraction during organ culture
Erik Uddman1, Mikael Adner, Lars Edvinsson
1Department of Internal Medicine, Lund University Hospital, Lund, Sweden. Erik.Uddman@med.lu.se
Abstract:
The effect of protein kinase C (PKC) inhibitors on the induction of endothelin ET(B) receptors during organ culture was examined in isolated segments of the rat mesenteric artery. After 24 h of organ culture, the endothelin ET(B) receptor agonist sarafotoxin 6c (S6c) induced a strong contraction compared to fresh segments. The contractile response after 24-h organ culture to S6c was studied in presence (30-min preincubation) or absence, after 24-h treatment, of the PKC inhibitors staurosporine, K252a and Ro31-7549. Exposure to staurosporine or K252a in presence and after 24-h treatment reduced the S6c contraction. In contrast, presence of 2-1[1-3(aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (Ro31-7549), did not affect the S6c-induced contraction, whereas 24-h treatment abolished the increase of contraction. The PKA inhibitor N-(2-[bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H89) did not affect the S6c responses. The mRNA expressions of endothelin ET(B) receptors (analysed with real-time PCR) were abolished after 24-h treatment with the PKC inhibitors. These results suggest that PKC is involved in the endothelin ET(B) receptor upregulation following organ culture.
Insights
Protein kinase C (PKC) inhibitors reduce endothelin ET(B) receptor upregulation in rat arteries cultured ex vivo. This suggests PKC plays a role in increasing these receptors during organ culture.
Area of Science:
- Vascular Biology
- Pharmacology
- Molecular Biology
Background:
- Endothelin ET(B) receptors mediate vasoconstriction.
- Organ culture can alter receptor expression in arterial segments.
- Protein kinase C (PKC) is a key signaling pathway in cells.
Purpose of the Study:
- To investigate the role of PKC in endothelin ET(B) receptor upregulation during organ culture of rat mesenteric arteries.
- To determine the effect of specific PKC inhibitors on receptor-mediated contraction and mRNA expression.
Main Methods:
- Isolated rat mesenteric artery segments were maintained in organ culture for 24 hours.
- Contractile responses to endothelin ET(B) receptor agonist sarafotoxin 6c (S6c) were measured.
- PKC inhibitors (staurosporine, K252a, Ro31-7549) were applied during preincubation or 24-hour treatment.
- Endothelin ET(B) receptor mRNA expression was analyzed using real-time PCR.
Main Results:
- Organ culture significantly increased S6c-induced contraction, indicating ET(B) receptor upregulation.
- PKC inhibitors staurosporine and K252a reduced this enhanced contraction.
- The PKC inhibitor Ro31-7549 abolished the upregulation after 24-hour treatment.
- PKC inhibition also abolished the increased mRNA expression of endothelin ET(B) receptors.
Conclusions:
- PKC signaling is involved in the upregulation of endothelin ET(B) receptors in rat mesenteric arteries during organ culture.
- PKC inhibitors can prevent the increase in receptor expression and function under these conditions.