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

Abstract

Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ET(B) 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; ET(A) and ET(B) receptor agonist) and sarafotoxin 6c (S6c; ET(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ET(A) and ET(B) 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 ET(B) receptor increased during organ culture, but there was no change in the expression of the ET(A) 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 ET(B) 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 and MEK1/2 inhibition time-dependently modify inflammatory signaling in rat cerebral arteries during organ culture

“…High levels of activated CaMKII were found in freshly isolated and non-incubated arteries (Figure 2A) in accordance with an earlier study [22]. The p-CaMKII expression decreased with incubation time to a significant difference at 6 and 24 hours ( P <0.05).…”

“…CaMKII and MEK1/2 have been shown to be involved in cerebrovascular receptor upregulation after cerebral ischemia [8,22]. The exact mechanisms involved in vascular inflammation are, however, poorly understood [1].…”

“…To evaluate the role of CaMKII or MEK/ERK1/2 in caspase-3 activation, basilar artery segments were cultured for 24 hours in the presence or absence of KN93 (10 −5  M) or U0126 (10 −5  M) added at 0 or 6 hours after initiating incubation (Figure 1B). The concentrations have been evaluated previously [21,22]. The vessel segments were immediately snap-frozen and stored at -80°C until use.…”

“…In addition, CaMKII involvement has been shown in neuronal apoptosis [19]. Previous organ culture studies have demonstrated activation of MAPK kinases (MEK/ERK1/2) in middle cerebral arteries (MCAs) [20,21] and CaMKII in basilar arteries [22] in parallel to endothelin receptor B (ET B ) receptor upregulation. These studies revealed a time-dependent effect of MEK/ERK1/2 and CaMKII inhibitors on ET B receptor upregulation at protein and functional levels.…”

Permanent Distal Occlusion of Middle Cerebral Artery in Rat Causes Local Increased ET_B, 5-HT_1B and AT₁ Receptor-Mediated Contractility Downstream of Occlusion

“…Concentration-response curves were obtained by cumulative application of the agonists endothelin-1 (ET-1; an endogen ET A and ET B receptor-specific agonist) and sarafotoxin 6c (S6c; a selective ET B receptor agonist; NeoMPS, Strasbourg, France) in the concentration range 10 -14 to 10 -7 M , and angiotensin II (Ang II; an endogen AT receptor-specific agonist; NEoMPS) and 5-carboxamidotryptamine (5-CT; a 5-HT analogue selective for 5-HT 1B/1D receptors; Sigma) in the concentration range of 10 -12 to 10 -6.5 M . A specific ET A receptor-mediated response from ET-1 was ensured by the pre-run with S6c [24], and a specific AT 1 receptor-mediated response was insured by application of the AT 2 receptor antagonist PD123319 (10 -5.5 M , Sigma) to the myograph bath 30 min prior to concentration response curves for Ang II. To confirm specific ET B receptor-mediated contraction, the specific competitive ET B receptor antagonist BQ788 (Sigma) was added in control experiments at a concentration of 0.9 µ M 30 min prior to cumulative application of S6c in 24-hour downstream segment samples (n = 3).…”