Inhibition of PKC activity blocks the increase of ET(B )receptor expression in cerebral arteries

BACKGROUND: Previous studies have shown that there is a time-dependent upregulation of contractile endothelin B (ET(B)) receptors in middle cerebral arteries (MCA) after organ culture. This upregulation is dependent on mitogen-activated protein kinases and possibly protein kinase C (PKC). The aim of this study was to examine the effect of PKC inhibitors with different profiles on the upregulation of contractile ET(B )receptors in rat MCA. Artery segments were incubated for 24 hours at 37°C. To investigate involvement of PKC, inhibitors were added to the medium before incubation. The contractile endothelin-mediated responses were measured and real-time PCR was used to detect endothelin receptor mRNA levels. Furthermore, immunohistochemistry was used to demonstrate the ET(B )receptor protein distribution in the MCA and Western blot to measure which of the PKC subtypes that were affected by the inhibitors. RESULTS: The PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and PKC inhibitor 20–28 attenuated the ET(B )receptor mediated contractions. Furthermore, Ro-32-0432 and bisindolylmaleimide I decreased ET(B )receptor mRNA levels while PKC inhibitor 20–28 reduced the amount of receptor protein on smooth muscle cells. PKC inhibitor 20–28 also decreased the protein levels of the five PKC subtypes studied (α, βI, γ, δ and ε). CONCLUSION: The results show that PKC inhibitors are able to decrease the ET(B )receptor contraction and expression in MCA smooth muscle cells following organ culture. The PKC inhibitor 20–28 affects the protein levels, while Ro-32-0432 and bisindolylmaleimide I affect the mRNA levels, suggesting differences in activity profile. Since ET(B )receptor upregulation is seen in cerebral ischemia, the results of the present study provide a way to interfere with the vascular involvement in cerebral ischemia

Smoking particles enhance endothelin A and endothelin B receptor-mediated contractions by enhancing translation in rat bronchi

BACKGROUND: Smoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP) from cigarette smoke, to nicotine and to DMSO, respectively. METHODS: Isolated rat bronchial segments were cultured for 24 hours in the presence or absence of DSP, nicotine or DMSO alone. Contractile responses to sarafotoxin 6c (a selective agonist for ET(B )receptors) and endothelin-1 (an ET(A )and ET(B )receptor agonist) were studied by use of a sensitive myograph. Before ET-1 was introduced, the ET(B )receptors were desensitized by use of S6c. The remaining contractility observed was considered to be the result of selective activation of the ET(A )receptors. ET(A )and ET(B )receptor mRNA expression was analyzed using real-time quantitative PCR. The location and concentration of ET(A )and ET(B )receptors were studied by means of immunohistochemistry together with confocal microscopy after overnight incubation with selective antibodies. RESULTS: After being cultured together with DSP for 24 hours the bronchial segments showed an increased contractility mediated by ET(A )and ET(B )receptors, whereas culturing them together with nicotine did not affect their contractility. The up-regulation of their contractility was blunted by cycloheximide treatment, a translational inhibitor. No significant change in the expression of ET(A )and ET(B )receptor mRNA through exposure to DMSO or to nicotine exposure alone occurred, although immunohistochemistry revealed a clear increase in ET(A )and ET(B )receptors in the smooth muscle after incubation in the presence of DSP. Taken as a whole, this is seen as the presence of a translation mechanism. CONCLUSION: The increased contractility of rat bronchi when exposed to DSP appears to be due to a translation mechanism