In vivo experimental stroke and in vitro organ culture induce similar changes in vasoconstrictor receptors and intracellular calcium handling in rat cerebral arteries

Abstract

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries after stroke. Here, we evaluate changes of ET(B) and 5-HT(1B) receptors, intracellular calcium levels, and calcium channel expression in rat middle cerebral artery (MCA) after focal cerebral ischemia and in vitro organ culture, a proposed model of vasoconstrictor receptor changes after stroke. Rats were subjected to 2 h MCA occlusion followed by reperfusion for 1 or 24 h. Alternatively, MCAs from naïve rats were cultured for 1 or 24 h. ET(B) and 5-HT(1B) receptor-mediated contractions were evaluated by wire myography. Receptor and channel expressions were measured by real-time PCR and immunohistochemistry. Intracellular calcium was measured by FURA-2. Expression and contractile functions of ET(B) and 5-HT(1B) receptors were strongly upregulated and slightly downregulated, respectively, 24 h after experimental stroke or organ culture. ET(B) receptor-mediated contraction was mediated by calcium from intracellular and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential canonical calcium channels, but not voltage-operated calcium channels.

Upregulation of 5-Hydroxytryptamine Receptor Signaling in Coronary Arteries after Organ Culture

“…Increased protein levels may account for the increased functional contractile responses observed after depletion of intracellular Ca 2+ . Hoel et al reported that expression of 5-HT 1B/1D receptors is upregulated after organ culture in rat cerebral arteries [15], which resembles the alterations in SMC function after subarachnoidal hemorrhage [16], [17]. There is an increased level of 5-HT 2A receptor mRNA and of contractile 5-HT 2A receptors after organ culture of rat mesenteric artery ring segments in which the endothelium has been removed [21].…”

“…Organ culture also induces downregulation of the contractility of angiotensin-II receptors, thereby reflecting the phenotypic changes in human coronary arteries from patients with ischemic heart disease [13], [14]. Expression of 5-HT 1B/1D receptors is also upregulated after organ culture of rat cerebral arteries, which resembles the alterations in SMC function after subarachnoidal hemorrhage [15], [16], [17]. So far, this model of vascular disease has been applied to study the pharmacological characteristics and underlying molecular and cellular mechanism of vascular receptor alterations.…”

Low density lipoprotein induces upregulation of vasoconstrictive endothelin type B receptor expression

“…More interestingly, there is induced upregulation of vasoconstrictive ET B receptor expression on porcine coronary artery smooth muscle cells in diet-induced hypercholesterolemia (Hasdai et al, 1997), in smoke exposed rats and in hypertension (Li et al, 2007), ischemic cardiovascular disease (Wackenfors et al, 2004), ischemic stroke (Povlsen et al, 2012) as well as in suspected acute coronary syndrome in patients (Dimitrijevic et al, 2009). Both ET-1 overexpression and vasoconstrictive ET B receptor upregulation contribute to vasospasms and decrease in local blood flow (Beg et al, 2006).…”

CaMKII and MEK1/2 inhibition time-dependently modify inflammatory signaling in rat cerebral arteries during organ culture

“…The inhibition of CaMKII and MEK1/2 results in attenuated ET B receptor upregulation and improved neurological outcome after SAH [29]. The present study was designed to evaluate the time-dependent effects of CaMKII and MEK1/2 inhibitors on signaling kinases and proteins involved in cerebrovascular inflammatory responses using an in vitro approach that mimics several of the ischemic-like vascular wall changes [5,9]. …”

“…Experimental middle cerebral artery occlusion (MCAO) and subarachnoid hemorrhage (SAH) have been shown to be associated with inflammation in the walls of cerebral arteries and in the microvasculature [4-7]. We have developed a method of isolated cerebral artery culture which simulates some aspects of cerebrovascular inflammation [8,9]. A multitude of stimuli have been identified that induce inflammation and apoptosis in vascular smooth muscle cells (VSMCs) including nitric oxide donors [10], serum starvation [11], tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1) [12].…”