Human cerebrovascular contractile receptors are upregulated via a B-Raf/MEK/ERK-sensitive signaling pathway

<p>Abstract</p> <p>Background</p> <p>Cerebral ischemia results in a rapid increase in contractile cerebrovascular receptors, such as the 5-hydroxytryptamine type 1B (5-HT_1B), angiotensin II type 1 (AT₁), and endothelin type B (ET_B) receptors, in the vessel walls within the ischemic region, which further impairs local blood flow and aggravates tissue damage. This receptor upregulation occurs via activation of the mitogen-activated protein kinase pathway. We therefore hypothesized an important role for B-Raf, the first signaling molecule in the pathway. To test our hypothesis, human cerebral arteries were incubated at 37°C for 48 h in the absence or presence of a B-Raf inhibitor: SB-386023 or SB-590885. Contractile properties were evaluated in a myograph and protein expression of the individual receptors and activated phosphorylated B-Raf (p-B-Raf) was evaluated immunohistochemically.</p> <p>Results</p> <p>5-HT_1B, AT₁, and ET_Breceptor-mediated contractions were significantly reduced by application of SB-590885, and to a smaller extent by SB-386023. A marked reduction in AT₁receptor immunoreactivity was observed after treatment with SB-590885. Treatment with SB-590885 and SB-386023 diminished the culture-induced increase of p-B-Raf immunoreactivity.</p> <p>Conclusions</p> <p>B-Raf signaling has a key function in the altered expression of vascular contractile receptors observed after organ culture. Therefore, specific targeting of B-Raf might be a novel approach to reduce tissue damage after cerebral ischemia by preventing the previously observed upregulation of contractile receptors in smooth muscle cells.</p

Stroke, Sex and Vascular Receptor Changes in the Brain

Stroke is a severe cerebrovascular disease in which the neuronal tissue and vasculature of the brain undergo ischemia-evoked alterations. We have demonstrated an increased expression of cerebrovascular contractile receptors in the brain. This is hypothesized to mitigate cerebral blood flow and exacerbate tissue damage after stroke. An increased contractile property of these receptors has been demonstrated to occur by MEK/ERK1/2 signaling. The majority of pre-clinical studies on stroke are performed in young healthy male animals, despite the fact there is a difference in male and female stroke incidence. The present thesis therefore aimed to investigate the status of vascular receptor changes after experimental stroke and organ culture, with two focus areas that previously have been missing – studies on human material and female subjects. In human cerebral arteries, we found that increased vasoconstriction of 5-HT1B, AT1 and ETB receptors during organ culture, a model of ischemic-like receptor changes, is mediated by B-Raf/MEK/ERK1/2 signaling. Although, increased mRNA and protein of these receptors were found in arteries from both sexes, the contraction to Ang II and ET-1 was markedly lower in female arteries. Focal cerebral ischemia in female rats induced an enhanced contractile property of cerebrovascular ETB receptors, similar to previous observations in males. Ovariectomy, and thereby loss of progesterone and estrogen, resulted in less ischemia-induced ETB receptor upregulation. Hormone therapy with progesterone, but not estrogen, reversed these changes. The increased ETB receptor expression and vasoconstriction after cerebral ischemia in female rat was demonstrated to be mediated by MEK/ERK1/2 signaling. MEK1/2 inhibition attenuated the ETB receptor upregulation and improved the neurological outcome. The present thesis demonstrates for the first time sex differences in vascular function of human cerebral arteries. The underlying mechanism in decreased responsiveness of female arteries remains to be elucidated. This may involve differences in receptor coupling or signal transduction influenced by female sex steroid hormones, or biological sex. Our experimental studies on stroke suggest the loss of progesterone after ovariectomy suppresses ischemia-induced ETB receptor upregulation. Further, the signal transduction pathway involved in vascular receptor changes after cerebral ischemia is suggested to be similar in both sexes. MEK1/2 inhibition is therefore a promising therapeutic target for stroke therapy in both males and females

Differential localization and characterization of functional calcitonin gene-related peptide receptors in human subcutaneous arteries.

Calcitonin gene-related peptide (CGRP) and its receptor are widely distributed within the circulation and the mechanism behind its vasodilation not only differs from one animal species to another but is also dependent on the type and size of vessel. The present study examines the nature of CGRP-induced vasodilation, characteristics of the CGRP receptor antagonist telcagepant and localization of the key components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) of the CGRP receptor in human subcutaneous arteries

Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries

Waldsee R, Ahnstedt H, Eftekhari S, Edvinsson L. Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries. Am J Physiol Heart Circ Physiol 298: H823-H832, 2010. First published December 11, 2009; doi:10.1152/ajpheart.00759.2009.-Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ETB receptors in smooth muscle cells via increased transcription. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CAMK) in the transcriptional expression of endothelin receptors after organ culture. Rat basilar arteries were incubated for 24 h with or without the CAMK inhibitor KN93 or ERK1/2 inhibitor U0126. The contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ETA and ETB receptors and CAMKII were determined by real-time PCR, and their protein levels were evaluated by immunohistochemistry and Western blot. The mRNA levels of CAMKII and the ETB receptor increased during organ culture, but there was no change in the expression of the ETA receptor. This effect was abolished by coincubation with KN93 or U0126. In functional studies, both inhibitors attenuated the S6c-induced contraction. Incubating the arteries with KN93, but not U0126, decreased the amount of phosphorylated CAMKII. The inhibitors had no effect on the levels of myosin light chain during organ culture, as measured by Western blot. CAMKII is involved in the upregulation of the endothelin ETB receptor and interacts with the ERK1/2 pathway to enhance receptor expression. CAMKII has no effect on the contractile apparatus in rat cerebral arteries

CaMKII inhibition with KN93 attenuates endothelin and serotonin receptor-mediated vasoconstriction and prevents subarachnoid hemorrhage-induced deficits in sensorimotor function

It has been suggested that transcriptional upregulation of cerebral artery contractile endothelin (ETB) and 5-hydroxytryptamine (5-HT1B) receptors play an important role in the development of late cerebral ischemia and increased vasoconstriction after subarachnoid hemorrhage (SAH). We tested the hypothesis that inhibition of calcium calmodulin-dependent protein kinase II (CaMKII) may reduce cerebral vasoconstriction mediated by endothelin and serotonin receptors and improve neurological outcome after experimental SAH