Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways

Abstract

Background: Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.

Methods: Human internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists.

Results: The endothelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 microM bisindolylmaleimide I and 10 microM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 microM SB203580, 10 microM PD98059 and 10 microM SP600125, respectively).

Conclusion: In conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.

Figure 1

(A) Endothelin-1 contractions (after ET_B receptor desensitisation) and (B) ET_A receptor mRNA levels in cultured and non-cultured human internal mammary arteries, examined using in vitro pharmacology (n = 27) and real time PCR (n = 8) experiments. The results are shown as mean values ± S.E.M. Statistical analyses, comparing cultured with non-cultured, were performed using Student's t-test. There were no significant differences.

Figure 2

Contractile responses elicited by cumulative application of sarafotoxin 6c in non-cultured segments of human internal mammary arteries in the absence (control) and presence of 0.1 μmol/l BQ788. The results are shown as mean values ± S.E.M of six experiments. Statistical analyses of the maximum contraction, comparing control with BQ 788, was performed using Student's t-test, where P < 0.05 (*) was considered significant.

Figure 3

Contractile responses elicited by cumulative application of the endothelin ET_B receptor agonist sarafotoxin 6c in segments of human internal mammary arteries. The arterial segments were either not cultured or cultured in the absence or presence of the protein kinase C inhibitors (A) Ro-32-0432 or (B) bisindolylmaleimide I. The results are shown as mean values ± S.E.M of six experiments. Statistical analysis was performed using ANOVA with Dunnett's post-test for multiple comparisons. P < 0.05 (*) and P < 0.01 (**) was considered significant. Comparisons were made between the results from arteries exposed to culture with and without Ro-32-0432 or bisindolylmaleimide I.

Figure 4

(A) The levels of endothelin ET_B receptor mRNA expression in human internal mammary arteries, examined using real time PCR. (B) Endothelin ET_B receptor protein expression in human internal mammary arteries, examined using Western blot. The arteries were either not cultured or cultured in the absence or presence of the protein kinase C inhibitors Ro-32-0432 or bisindolylmaleimide I. The results are shown as mean values ± S.E.M of six experiments. Endothelin ET_B mRNA levels in cultured and non-cultured arteries were compared using Student's t-test, where P < 0.05 (*) was considered significant.

Figure 5

Contractile responses elicited by cumulative application of the endothelin ET_B receptor agonist sarafotoxin 6c in segments of human internal mammary arteries. The arterial segments were either not cultured or cultured in the absence or the presence of mitogen-activated kinase (MAPK) pathway inhibitors; (A) the P38 MAPK inhibitor SB203580, (B) the ERK1/2 inhibitor PD98059 or (C) the JNK inhibitor SP600125. The results are shown as mean values ± S.E.M of six experiments. Statistical analysis was performed using ANOVA with Dunnett's post-test for multiple comparisons. P < 0.05 (*) and P < 0.01 (**) was considered significant. Comparisons were made between the results from arteries exposed to culture with and without SB203580, PD98059 or SP600125.

Figure 6

(A) The levels of endothelin ET_B receptor mRNA expression in human internal mammary arteries, examined using real time PCR. (B) Endothelin ET_B receptor protein expression in human internal mammary arteries, examined using Western blot. The arteries were either not cultured or cultured in the absence or presence of mitogen-activated kinase (MAPK) pathway inhibitors; the P38 MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059 or the JNK inhibitor SP600125. The results are shown as mean values ± S.E.M of six experiments. Endothelin ET_B mRNA levels in cultured and non-cultured arteries were compared using Student's t-test, where P < 0.05 (*) and P < 0.01 (**) was considered significant.