P2Y₂ receptor activation decreases blood pressure via intermediate conductance potassium channels and connexin 37

Abstract

Aims: Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y₂ receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y₂ receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response.

Methods: We compared the effects of a P2Y₂/₄ receptor agonist in wild-type (WT) mice and in mice lacking KCa3.1, KCa1.1, Cx37 or Cx40 under anaesthesia, while monitoring intra-arterial blood pressure and heart rate.

Results: Acute activation of P2Y₂/₄ receptors (0.01-3 mg kg(-1) body weight i.v.) caused a biphasic blood pressure response characterized by a dose-dependent and rapid decrease in blood pressure in WT (maximal response % of baseline at 3 mg kg(-1) : -38 ± 1%) followed by a consecutive increase in blood pressure (+44 ± 11%). The maximal responses in KCa3.1(-/-) and Cx37(-/-) were impaired (-13 ± 5, +17 ± 7 and -27 ± 1, +13 ± 3% respectively), whereas the maximal blood pressure decrease in response to acetylcholine at 3 μg kg(-1) was not significantly different (WT: -53 ± 3%; KCa3.1(-/-) : -52 ± 3; Cx37(-/-) : -53 ± 3%). KCa1.1(-/-) and Cx40(-/-) showed an identical biphasic response to P2Y2/4 receptor activation compared to WT.

Conclusions: The data suggest that the P2Y2/4 receptor activation elicits blood pressure responses via distinct mechanisms involving KCa3.1 and Cx37.

Keywords: K+ channels; P2 receptors; gap junction; hyperpolarization; myogenic tone; signalling.

Figure 1

Maximal responses in mean arterial blood pressure (MAP) to acute bolus application of INS45973 (P2Y_2/4 agonist) in wild-type (WT, n = 8) and P2Y₂ receptor knockout mice (P2Y₂^−/−, n = 8). Original recording of INS45973-induced blood pressure effects at 3 mg kg⁻¹ body weight (bar = 25 s) in a WT mouse (left). In WT, application of INS45973 dose-dependently and rapidly decreased blood pressure (middle, early phase), which started to partially recover during drug application and then continued into a dose-dependent increase in blood pressure (right side, late phase). In contrast, INS45973 in P2Y₂^−/− mice dose-dependently and rapidly increased blood pressure, which was sustained during drug application and thereafter recovered to baseline within 1–2 min, consistent with the short half-life of INS45973 [for an original blood pressure trace see Rieg et al. (2011)]. Some error bars are covered. *P < 0.05 vs. WT (two-way anova with repeated measures followed by Dunnett’s test).

Figure 2

Maximal responses in mean arterial blood pressure (MAP) to acute bolus application of INS45973 (P2Y_2/4 agonist) in endothelial NO synthase knockout mice (eNOS^−/−, n = 5). In eNOS^−/− mice, application of INS45973 induced a dose-dependent, rapid decrease in blood pressure (left side, early phase). The dose-dependent rise in blood pressure above basal values following the initial decrease in eNOS^−/− mice was significantly impaired compared to WT mice (right side, late phase). Some error bars are covered. *P < 0.05 vs. WT (two-way anova with repeated measures followed by Dunnett’s test).

Figure 3

Maximal responses in mean arterial blood pressure (MAP) to acute bolus application of INS45973 (P2Y_2/4 agonist) in intermediate-conductance potassium channel knockout mice (KCa3.1^−/−, n = 5). In KCa3.1^−/− mice, application of INS45973 induced a dose-dependent, rapid decrease in blood pressure (left side, early phase), which was significantly impaired compared to wild-type (WT) mice. The blood pressure decrease in response to acetylcholine showed a right shift of the dose–response curve in KCa3.1^−/− compared to WT mice; however, the maximum response was unaffected. The blood pressure decrease caused by acetylcholine is not followed by an acute blood pressure increase above basal values. Inset: original recording of acetylcholine-induced blood pressure effects at 3 µg kg⁻¹ body weight in a WT mouse (bar = 25 s). The dose-dependent rise in blood pressure in response to INS45973 above basal values following the initial decrease in KCa3.1^−/− mice was also significantly impaired compared to WT mice (right side, late phase). Some error bars are covered. *P < 0.05 vs. WT (two-way anova with repeated measures followed by Dunnett’s test).

Figure 4

Maximal responses in mean arterial blood pressure (MAP) to acute bolus application of INS45973 (P2Y_2/4 agonist) in big-conductance potassium channel knockout mice (KCa1.1^−/−, n = 4). In KCa1.1^−/− mice, application of INS45973 induced a dose-dependent, rapid decrease in blood pressure (left side, early phase). The dose-dependent rise in blood pressure above basal values following the initial decrease in KCa1.1^−/− mice was comparable to WT mice (right side, late phase). Some error bars are covered. *P < 0.05 vs. WT (two-way anova with repeated measures followed by Dunnett’s test).

Figure 5

Maximal responses in mean arterial blood pressure (MAP) to acute bolus application of INS45973 (P2Y_2/4 agonist) in connexin 37 knockout mice (Cx37^−/−, n = 5). In Cx37^−/− mice, application of INS45973 induced a dose-dependent, rapid decrease in blood pressure (left side, early phase) which was significantly impaired compared to wild-type (WT) mice. The dose-dependent rise in blood pressure above basal values following the initial decrease in Cx37^−/− mice was also significantly impaired compared to WT mice (right side, late phase). The dose-dependent blood pressure decrease in response to acetylcholine was comparable between Cx37^−/− and WT mice and not followed by an acute blood pressure increase above basal values. Some error bars are covered. *P < 0.05 vs. WT (two-way anova with repeated measures followed by Dunnett’s test).

Figure 6

Maximal responses in mean arterial blood pressure (MAP) to acute bolus application of INS45973 (P2Y_2/4 agonist) in connexin 40 knockout mice (Cx40^−/−, n = 4). In Cx40^−/− mice, application of INS45973 induced a dose-dependent, rapid decrease in blood pressure (left side, early phase). The dose-dependent rise in blood pressure above basal values following the initial decrease in Cx40^−/− mice was comparable to wild-type (WT) mice (right side, late phase). Some error bars are covered. *P < 0.05 vs. WT (two-way anova with repeated measures followed by Dunnett’s test).

Figure 7

A proposed model of murine blood pressure responses to P2Y_2/4 receptor activation. Our previous data implicated that the acute vasodilatory response to INS45973, a P2Y_2/4 agonist, is mediated by P2Y₂ receptor activation on endothelial cells. This hypothesis is supported by experiments in P2Y₂ receptor knockout mice which lack the observed initial blood pressure decrease and show instead an immediate increase in blood pressure (Rieg et al. 2011). The blood pressure increase possibly results as a consequence of P2Y₄ receptor activation directly on vascular smooth muscle cells resulting in vasoconstriction (Bar et al. 2008). The fact that endothelial NO synthase knockout mice responded with a comparable vasodilation implicated a role for endothelial derived hyperpolarization (EDH) in the acute blood pressure decrease. We speculate that P2Y₂ receptor activation, possibly via an increase in intracellular calcium, activates calcium-dependent intermediate-conductance potassium channels, KCa3.1, which induces EDH-type vasodilations. Lack of KCa3.1 impairs this response (red circle). The role of small-conductance potassium channels (KCa2.3) in response to P2Y_2/4 receptor activation needs to be determined. Calcium-dependent big-conductance potassium channels (KCa1.1) on vascular smooth muscle cells are not part of P2Y_2/4 receptor-initiated blood pressure responses. Gap junction proteins including connexin 37 (Cx37) and connexin 40 (Cx40) contribute to myoendothelial gap junction (MEGJ) communication and electrically conduct EDH activity. Our data suggest that Cx37 is part of such a communication because Cx37 knockout mice show an impaired blood pressure response to P2Y_2/4 receptor activation (red circle). In contrast to the distinct response observed in Cx37^−/− mice, Cx40 is not part of the P2Y_2/4 receptor-initiated blood pressure responses. Endothelial cells and vascular smooth muscle cells are speculated to endogenously release ATP and/or UTP via pannexin 1 (Panx1) channels (Lohman & Isakson 2014) which could consecutively activate P2Y_2/4 receptors and contribute to regulation of vascular tone.