Adenosine A₁receptor signaling inhibits BK channels through a PKCα-dependent mechanism in mouse aortic smooth muscle

Abstract

Adenosine receptors (AR; A1, A2A, A2B, and A3) contract and relax smooth muscle through different signaling mechanisms. Deciphering these complex responses remains difficult because relationships between AR subtypes and various end-effectors (e.g., enzymes and ion channels) remain to be identified. A1AR stimulation is associated with the production of 20–hydroxyeicosatetraenoic acid (20–HETE) and activation of protein kinase C (PKC). 20–HETE and PKC can inhibit large conductance Ca2+/voltage-sensitive K+ (BK) channels that regulate smooth muscle contraction. We tested the hypothesis that activation of A1AR inhibits BK channels via a PKC-dependent mechanism. Patch clamp recordings and Western blots were performed using aortae of wild type (WT) and A1AR knockout (A1KO) mice. There were no differences in whole-cell K+ current or α and β1 subunits expression between WT and A1KO. 20–HETE (100 nmol/L) inhibited BK current similarly in WT and A1KO mice. NECA (5′–N–ethylcarboxamidoadenosine; 10 μmol/L), a nonselective AR agonist, increased BK current in myocytes from both WT and A1KO mice, but the increase was greater in A1KO (52 ± 15 vs. 17 ± 3%; P < 0.05). This suggests that A1AR signaling negatively regulates BK channel activity. Accordingly, CCPA (2–chloro–N(6)-cyclopentyladenosine; 100 nmol/L), an A1AR-selective agonist, inhibited BK current in myocytes from WT but not A1KO mice (81 ± 4 vs. 100 ± 7% of control; P < 0.05). Gö6976 (100 nmol/L), a PKCα inhibitor, abolished the effect of CCPA to inhibit BK current (99 ± 3% of control). These data lead us to conclude that, in aortic smooth muscle, A1AR inhibits BK channel activity and that this occurs via a mechanism involving PKCα.

Angiotensin II stimulation alters vasomotor response to adenosine in mouse mesenteric artery: role for A1 and A2B adenosine receptors

“…In addition, CYP4A contributes to the development of angiotensin II‐dependent hypertension (McGiff and Laniado‐Schwartzman, ; Harder et al, ; Haller et al, ; McGiff et al, ; Muthalif et al, ; ; Zaman et al, ). PLC, 20‐HETE and ERK1/2 pathways are involved in the regulation of vascular tone by adenosine through the activation of A 1 receptors (Rogel et al, ; Jacobson and Gao, ; Ponnoth et al, ; Kunduri et al, ; Kunduri et al, ). Thus, we believe that acute angiotensin II stimulation may alter adenosine receptor‐dependent signalling, establishing an important role for adenosine in the regulation of vascular tone in MAs.…”

“…Moreover, in chronic angiotensin II and L-NAME hypertension mouse models, genetic deletion of A 1 receptors has been shown to attenuate arteriolar and BP responses (Gao et al, 2011;Lee et al, 2012). Previously, we demonstrated that CYP4A and ERK1/2 are involved in the A 1 receptordependent contractile response in mouse aorta (Ponnoth et al, 2012a;Kunduri et al, 2013b;Kunduri et al, 2013a). In agreement, our data revealed that enhanced vasoconstriction to NECA in MAs following angiotensin II stimulation was completely dependent on the A 1 receptor and mediated through CYP4A and ERK1/2 (Figures 4 and 5).…”

“…Furthermore, A 1 receptor KO mice were used to determine the role of the A 1 receptor. To study the effect of CYP4A, MAs were pre‐incubated with HET0016 (10 μM) (Ponnoth et al, ; Kunduri et al, ), an inhibitor of CYP4A and 20‐HETE production. In addition, the role of ERK1/2 was determined by using the inhibitor PD98059 (1 μM) (Ponnoth et al, ; El‐Awady et al, ; Kunduri et al, ).…”

“…A 1 and A 3 receptor activation causes vasoconstriction, while stimulation of A 2A and A 2B receptors produces vasodilatation (Ohisalo, ; Mubagwa et al, ; Olanrewaju and Mustafa, ; Olanrewaju et al, ; Mubagwa and Flameng, ; Jacobson and Gao, ; Hein et al, ). In mouse aorta, A 1 receptor‐dependent smooth muscle contraction involves cytochrome P450 epoxygenase type 4A (CYP4A), PKCα and MAPKs, mainly p44/42 MAPK (ERK1/2) (Ansari et al, ; Sanjani et al, ; Kunduri et al, ; Kunduri et al, ). Moreover, A 1 receptor‐mediated vasoconstriction occurs through the activation of PKCα, leading to ERK1/2 phosphorylation in coronary artery smooth muscle cells (Tawfik et al, ; Ansari et al, ).…”

Western blot analysis of BK channelβ1-subunit expression should be interpreted cautiously when using commercially available antibodies

“…We tested the specificity and sensitivity of commercially available BK b1-subunit antibodies using tissues from WT and BK b1-KO mice, and found that under denaturing conditions these antibodies lacked either specificity or sensitivity for BK b1-subunit in BK b1-subunit enriched tissues from C57BL/6 mice. The antibodies evaluated in this study have been used previously in different tissues including blood vessels, tracheal smooth muscle cells, and kidney from rats, mice, and humans (Matharoo-Ball et al 2003;Chang et al 2006;Grimm et al 2007Grimm et al , 2009Yang et al 2009Yang et al , 2013Albarwani et al 2010;Xie et al 2010;Zhang et al 2010;Howitt et al 2011;Ahn et al 2012;Loot et al 2012;Lu et al 2012;Evseev et al 2013;Kunduri et al 2013;Shi et al 2013a,b;Zheng et al 2013;Evanson et al 2014;Leo et al 2014;Nystoriak et al 2014;Pabbidi et al 2014;Yi et al 2014). However, our study is the first to test the specificity and sensitivity of all commercially available antibodies for detection of the BK b1subunit using BK b1-subunit KO mice test.…”