Protein kinase mediated upregulation of endothelin A, endothelin B and 5-hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery

1 Organ culture has been shown to upregulate both endothelin (ET) and 5-hydroxytryptamine 1B/1D (5-HT1B/1D) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process. 2 The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhitor), RO 31-7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET-1 (unspecific ETA and ETB agonist), S6c (specific ETB agonist) and 5-CT (5-HT1 agonist). Levels of mRNA coding for the ETA, ETB, 5-HT1B and 5-HT1D receptors were analysed using real-time RT-PCR. 3 Classical PKC's are critically involved in the appearance of the ETB receptor; co-culture with RO 31-7549 abolished the contractile response (6.9+/-1.8%) and reduced the ETB receptor mRNA by 44+/-4% as compared to the cultured control. Correlation between decreased ETB receptor mRNA and abolished contractile function indicates upstream involvement of PKC. 4 Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7-25% increase in E-max in response to ET-1, S6c and 5-CT as compared to the cultured control. 5 Staurosporine inhibited the culture induced upregulation of the response of both the ETA and the 5-HT1B/1D receptors, but had no significant effect on the mRNA levels of these receptors. This lack of correlation indicates an additional downstream involvement of protein kinases.