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

Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

<p>Abstract</p> <p>Background</p> <p>Late cerebral ischemia carries high morbidity and mortality after subarachnoid hemorrhage (SAH) due to reduced cerebral blood flow (CBF) and the subsequent cerebral ischemia which is associated with upregulation of contractile receptors in the vascular smooth muscle cells (SMC) via activation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide a therapeutic window for treatment of cerebral ischemia after SAH.</p> <p>Results</p> <p>Here we demonstrate that SAH increases the phosphorylation level of ERK1/2 in cerebral vessels and reduces the neurology score in rats in additional with the CBF measured by an autoradiographic method. The intracisternal administration of SB-386023-b, a specific inhibitor of raf, given 6 h after SAH, aborts the receptor changes and protects the brain from the development of late cerebral ischemia at 48 h. This is accompanied by reduced phosphorylation of ERK1/2 in cerebrovascular SMC. SAH per se enhances contractile responses to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and angiotensin II (Ang II), upregulates ET_B, 5-HT_1Band AT₁receptor mRNA and protein levels. Treatment with SB-386023-b given as late as at 6 h but not at 12 h after the SAH significantly decreased the receptor upregulation, the reduction in CBF and the neurology score.</p> <p>Conclusion</p> <p>These results provide evidence for a role of the ERK1/2 pathway in regulation of expression of cerebrovascular SMC receptors. It is suggested that raf inhibition may reduce late cerebral ischemia after SAH and provides a realistic time window for therapy.</p