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

Edvinsson L, Povlsen GK, Ahnstedt H, Waldsee R - J Neuroinflammation (2014)

Bottom Line: Application of KN93 at 1 h and every 12 h post-SAH significantly reduced vascular CaMKII immunoreactivity at 72 h.KN93 treatment significantly attenuated the contraction induced by ET-1 and 5-CT.The present study has shown that SAH is associated with increased contractile responses to ET-1 and 5-CT in cerebral arteries and enhanced early activation of CaMKII.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Vascular Research, Department of Clinical Sciences, Lund University and Lund University Hospital, Lund, SE-221 84, Sweden. lars.edvinsson@med.lu.se.

ABSTRACT

Background: 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.

Methods: SAH was induced in adult rats by injection of 250 μL autologous blood into the basal cisterns. The CaMKII activity in cerebral vessels was studied by Western blot and immunohistochemistry. The vasomotor responses of middle cerebral and basilar arteries were measured in a sensitive myograph system. The functional outcome was examined by the rotating pole test 2 and 3 days after SAH.

Results: SAH induced a rapid early increase in phosphorylated CaMKII protein at 1 h that was attenuated by cisternal administration of the CaMKII inhibitor KN93 (0.501 μg/kg) 45 min prior and immediately after SAH as evaluated by Western blot. Application of KN93 at 1 h and every 12 h post-SAH significantly reduced vascular CaMKII immunoreactivity at 72 h. In addition, contractile responses of cerebral arteries to endothelin-1 (ET-1) and 5-hydroxycarboxamide (5-CT) were increased at this time-point. KN93 treatment significantly attenuated the contraction induced by ET-1 and 5-CT. Importantly, treatment with the CaMKII inhibitor prevented SAH-induced deficits in neurological function, as evaluated by the rotating pole test, and similar sensorimotor scores were seen in sham-operated animals.

Conclusions: The present study has shown that SAH is associated with increased contractile responses to ET-1 and 5-CT in cerebral arteries and enhanced early activation of CaMKII. Treatment with the CaMKII inhibitor KN93 attenuated the contractile responses and prevented impaired sensorimotor function after SAH.

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Cerebrovascular contractile responses to ET-1 at 72 h post-SAH. Concentration-response curves were elicited by the cumulative application of ET-1 to (A) MCA and (B) basilar arteries. In both arteries, the first phase of the concentration-response curve represents ET-1-induced contraction via ETB receptors and the second phase contraction mediated by ETA receptors. Previous studies have characterized these responses in detail [4,9,28]. Values are expressed as percentage of the K+-induced contraction and are presented as mean ± SEM. ***P <0.001. Number of animals in each group, n = 6–8.
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Fig4: Cerebrovascular contractile responses to ET-1 at 72 h post-SAH. Concentration-response curves were elicited by the cumulative application of ET-1 to (A) MCA and (B) basilar arteries. In both arteries, the first phase of the concentration-response curve represents ET-1-induced contraction via ETB receptors and the second phase contraction mediated by ETA receptors. Previous studies have characterized these responses in detail [4,9,28]. Values are expressed as percentage of the K+-induced contraction and are presented as mean ± SEM. ***P <0.001. Number of animals in each group, n = 6–8.

Mentions: Potassium (63.5 mM) induced a potent vasoconstriction in all vessels and was used as an internal control set to 100%. There was no difference in the potassium-induced responses between the groups studied. Contractile responses to cumulative administration of ET-1 (10−14 to 10−7 M) are shown in Figure 4. In cerebral arteries from sham-operated rats, ET-1 induced sigmoidal concentration-response curves in both MCA and BA. SAH (untreated or placebo) resulted in leftward shifts, and a change from sigmoidal to biphasic concentration-response curves. This is in accordance with our earlier studies, which demonstrated that the first phase of the ET-1 concentration-response curves represents contraction mediated by contractile ETB receptors upregulated in the smooth muscle cells after SAH, whereas the second phase of the curve represents vasoconstriction mediated by ETA receptors [9]. The change into a biphasic curve together with the increased Emax values after SAH suggests upregulation of both ETA and ETB receptors after SAH. The detailed pharmacological characterizations have been published before [4,9,28]. Treatment with KN93 totally abolished the elevation of the first phase but not the second phase, thus yielding ET-1 concentration-response curves similar to the vascular contractile response seen in arteries from sham operated animals. This indicates that KN93 prevents the expression of ETB receptors after SAH.Figure 4


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

Edvinsson L, Povlsen GK, Ahnstedt H, Waldsee R - J Neuroinflammation (2014)

Cerebrovascular contractile responses to ET-1 at 72 h post-SAH. Concentration-response curves were elicited by the cumulative application of ET-1 to (A) MCA and (B) basilar arteries. In both arteries, the first phase of the concentration-response curve represents ET-1-induced contraction via ETB receptors and the second phase contraction mediated by ETA receptors. Previous studies have characterized these responses in detail [4,9,28]. Values are expressed as percentage of the K+-induced contraction and are presented as mean ± SEM. ***P <0.001. Number of animals in each group, n = 6–8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269841&req=5

Fig4: Cerebrovascular contractile responses to ET-1 at 72 h post-SAH. Concentration-response curves were elicited by the cumulative application of ET-1 to (A) MCA and (B) basilar arteries. In both arteries, the first phase of the concentration-response curve represents ET-1-induced contraction via ETB receptors and the second phase contraction mediated by ETA receptors. Previous studies have characterized these responses in detail [4,9,28]. Values are expressed as percentage of the K+-induced contraction and are presented as mean ± SEM. ***P <0.001. Number of animals in each group, n = 6–8.
Mentions: Potassium (63.5 mM) induced a potent vasoconstriction in all vessels and was used as an internal control set to 100%. There was no difference in the potassium-induced responses between the groups studied. Contractile responses to cumulative administration of ET-1 (10−14 to 10−7 M) are shown in Figure 4. In cerebral arteries from sham-operated rats, ET-1 induced sigmoidal concentration-response curves in both MCA and BA. SAH (untreated or placebo) resulted in leftward shifts, and a change from sigmoidal to biphasic concentration-response curves. This is in accordance with our earlier studies, which demonstrated that the first phase of the ET-1 concentration-response curves represents contraction mediated by contractile ETB receptors upregulated in the smooth muscle cells after SAH, whereas the second phase of the curve represents vasoconstriction mediated by ETA receptors [9]. The change into a biphasic curve together with the increased Emax values after SAH suggests upregulation of both ETA and ETB receptors after SAH. The detailed pharmacological characterizations have been published before [4,9,28]. Treatment with KN93 totally abolished the elevation of the first phase but not the second phase, thus yielding ET-1 concentration-response curves similar to the vascular contractile response seen in arteries from sham operated animals. This indicates that KN93 prevents the expression of ETB receptors after SAH.Figure 4

Bottom Line: Application of KN93 at 1 h and every 12 h post-SAH significantly reduced vascular CaMKII immunoreactivity at 72 h.KN93 treatment significantly attenuated the contraction induced by ET-1 and 5-CT.The present study has shown that SAH is associated with increased contractile responses to ET-1 and 5-CT in cerebral arteries and enhanced early activation of CaMKII.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Vascular Research, Department of Clinical Sciences, Lund University and Lund University Hospital, Lund, SE-221 84, Sweden. lars.edvinsson@med.lu.se.

ABSTRACT

Background: 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.

Methods: SAH was induced in adult rats by injection of 250 μL autologous blood into the basal cisterns. The CaMKII activity in cerebral vessels was studied by Western blot and immunohistochemistry. The vasomotor responses of middle cerebral and basilar arteries were measured in a sensitive myograph system. The functional outcome was examined by the rotating pole test 2 and 3 days after SAH.

Results: SAH induced a rapid early increase in phosphorylated CaMKII protein at 1 h that was attenuated by cisternal administration of the CaMKII inhibitor KN93 (0.501 μg/kg) 45 min prior and immediately after SAH as evaluated by Western blot. Application of KN93 at 1 h and every 12 h post-SAH significantly reduced vascular CaMKII immunoreactivity at 72 h. In addition, contractile responses of cerebral arteries to endothelin-1 (ET-1) and 5-hydroxycarboxamide (5-CT) were increased at this time-point. KN93 treatment significantly attenuated the contraction induced by ET-1 and 5-CT. Importantly, treatment with the CaMKII inhibitor prevented SAH-induced deficits in neurological function, as evaluated by the rotating pole test, and similar sensorimotor scores were seen in sham-operated animals.

Conclusions: The present study has shown that SAH is associated with increased contractile responses to ET-1 and 5-CT in cerebral arteries and enhanced early activation of CaMKII. Treatment with the CaMKII inhibitor KN93 attenuated the contractile responses and prevented impaired sensorimotor function after SAH.

Show MeSH
Related in: MedlinePlus