Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone

Abstract

P anx1 (pannexin 1) channels have emerged as a major means for release of the nucleotides that mediate purinergic signaling, 1 which is involved in multiple physiological and pathophysiological processes such as apoptosis, 2 pyroptosis, 3 tumor cell metastasis, 4 vasoconstriction, 5 leukocyte migration, 6 insulin release from adipocytes, 7 N-methyl-D-aspartate receptor activation, 8 and neuronal survival. 9 Of particular relevance to this current work, Panx1 channels are activated by both caspase-dependent cleavage of the carboxyl terminus and cleavage-independent, receptor-mediated activation mechanisms. During apoptosis, caspase 3/7 cleaves the carboxyl terminus of Panx1, resulting in an irreversibly activated channel that can release ATP as a find-me signal to surrounding phagocytes.2 Alternatively, Panx1 channels, which are also expressed in smooth muscle cells (SMCs), are reversibly activated in a receptor-dependent mechanism. In vascular SMCs, we and others have demonstrated that sympathetic-mediated vasoconstriction and blood pressure are Panx1 dependent based on genetic and pharmacological perturbation.5,10-13 Specifically, activation of α1-adrenergic receptors results in opening of Panx1 channels that activate downstream purinergic receptors through release of ATP. Small ions and large molecules can permeate through open Panx1 channels, which allows for Panx1 channel activities to be assayed in several ways: by entry into cells of dyes, such as TO-PRO-3 or ethidium bromide; by release of nucleotides, such as ATP and UTP (uridine triphosphate); and by plasma membrane Panx1 channel currents (reviewed in [14][15][16] ). Given the paucity of selective compounds capable of targeting Panx1, we used different assays to identify and validate new Panx1 blockers that would be useful in these various pathophysiological contexts, such as treatmentresistant hypertension. Editorial, see p 543 In This Issue, see p 533 Meet the First Author, see p 534Original received November 12, 2017; revision received December 6, 2017; accepted December 12, 2017. In November 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.99 days. Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone.Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. Methods and Results:First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hyperte...

Pannexin 1 activation and inhibition is permeant‐selective

“…cells. This permeant-selective CBX inhibition profile aligns with earlier studies (Bruzzone et al 2005;Ma et al 2009;Bhaskaracharya et al 2014;Hansen et al 2014b), but conflicts with CBX-mediated inhibition of dye uptake in Panx1-expressing cell systems or ischemic neurons (Thompson et al 2006(Thompson et al , 2008Huang et al 2007;Good et al 2018). These reports suggest differential modes of Panx1 activation and/or that some Panx1-assigned effects may arise from CBX inhibition of other large-pore channels, such as the volume-regulated anion channel LRRC8, in which both ion conductance and osmolyte transport is blocked by CBX (Voss et al 2014;Planells-Cases et al 2015).…”

“…The novel Panx1 inhibitor spironolactone, likewise, showed no effect on the ethidium uptake, while efficiently inhibiting the membrane currents in Panx1-expressing oocytes. In contrast, spironolactone blocks membrane current, ATP release and TO-PRO-3 uptake in apoptotic Jurkat T cells (Good et al 2018). The well-established connexin hemichannel inhibitors lanthanum and flufenamic acid (Nielsen et al 2017) had little inhibitory effect on ethidium uptake and membrane currents in Panx1-expressing oocytes (Bruzzone et al 2005;Pelegrin & Surprenant, 2006;Ma et al 2009;Nielsen et al 2017).…”

“…; Good et al . ). These reports suggest differential modes of Panx1 activation and/or that some Panx1‐assigned effects may arise from CBX inhibition of other large‐pore channels, such as the volume‐regulated anion channel LRRC8, in which both ion conductance and osmolyte transport is blocked by CBX (Voss et al .…”

“…In contrast, spironolactone blocks membrane current, ATP release and TO‐PRO‐3 uptake in apoptotic Jurkat T cells (Good et al . ). The well‐established connexin hemichannel inhibitors lanthanum and flufenamic acid (Nielsen et al .…”

“…2D). The novel Panx1 inhibitor spironolactone, which has been demonstrated to inhibit current, ATP release and TO-PRO uptake (Good et al 2018), caused a reduction of Panx1-mediated membrane current (n = 9) with no effect (or even a slight enhancement) on ethidium permeation (n = 3, Fig. 2E).…”