Pannexin channel inhibition— An evolving target to lower blood pressure?

Abstract

More than 50 years after spironolactone has come on the market its mechanism of action continues to expand. In this issue of Circulation Research, Good et al. document the discovery that spironolactone is not only an inhibitor of the mineralocorticoid receptor, but also inhibits pannexin 1 channels.

Resistant hypertension (RH), defined as blood pressure >140/90 mm Hg despite the use of three antihypertensive agents, is well recognized and not rare¹. For drug treatment of RH, first-line drug choices for blood pressure control include (1) a diuretic (should be included unless not tolerated), (2) either an angiotensin-converting enzyme inhibitor or angiotensin-receptor blocker and (3) a calcium channel blocker and in certain patients a beta blocker². Based on several smaller clinical trials, guidelines recommend to add the mineralocorticoid receptor (MR) antagonists spironolactone or eplerenone when blood pressure remains uncontrolled². Recently the randomized controlled PATHWAY-2 trial showed a superior beneficial effect of spironolactone in patients on optimal three drug regimen in a double-blind crossover setting over the alternative drugs bisoprolol (beta receptor blocker) and doxazosin (alpha receptor blocker)³. Results showed that spironolactone lowered systolic blood pressure by −8.7 mmHg, while bisoprolol and doxazosin were also effective, but inferior with −4.5 mmHg and −4.0 mmHg reduction respectively.

Spironolactone’s well recognized mode of action is the antagonism of the MR in the distal tubule of the kidney. Activation of the MR e.g. by aldosterone leads to its translocation into the nucleus and subsequent changes in gene expression. The net effect on ion and water homeostasis is sodium retention, volume increase and potassium excretion. Antagonizing the MR has therefore the opposite effect, making spironolactone a potassium sparing diuretic.

While there seems to be a clear benefit of spironolactone in RH, the mechanistic basis for this has not been established. One common hypothesis is that by adding another diuretic, more effective diuresis/natriuresis is achieved than with one diuretic alone (Fig. 1). The MR is also expressed in the vessel wall in both endothelial cells and SMCs and its actions has been associated with effects on blood pressure^{4, 5}. Other “off target” effects of spironolactone may play a role as well, but clear demonstration that such effects contribute significantly to lower blood pressure has not been presented.

Fig. 1.

Multiple proposed actions of spironolactone (spiro) to lower blood pressure. The primary target is the mineralocorticoid receptor (MR) in the kidney which reduces sodium re-uptake from the distal tubule lumen causing increased sodium and water excretion (left). More recently MR inhibition in the vasculature is increasingly recognized to reduce vasoconstriction (middle), but has also been associated with additional benefits including e.g. anti-inflammatory and anti-fibrotic effects not depicted in the scheme. The new pathway proposed by Good et al. is marked with red arrows (right). Spironolactone blocks pannexin channels (Panx1) in SMCs which reduces ATP release during alpha adrenergic stimulation (α1). This subsequently diminishes alpha adrenergic vasoconstriction resulting in lower vascular tone.

In this issue of Circulation Research, Isakson, Ravichandran and coworkers report a novel target of spironolactone⁶. The investigators used a cellular assay to identify small molecule inhibitors of pannexin 1 channels and discovered that spironolactone and several of its metabolites block pannexin channels. Could this be one of the suspected “off target” effects of spironolactone?

Pannexin 1 is ubiquitously expressed in multiple tissue and cell-types including endothelial and smooth muscle cells (SMCs) of the vasculature. Prior work from the Isakson group established that pannexin channels amplify vasoconstriction in response to alpha-adrenergic stimulation via purinergic signaling⁷. Consistent with the earlier study, here the authors demonstrate that spironolactone blunted the response to the alpha1 agonist phenylephrine in isolated human and mouse arteries and thus acts as an apparent alpha adrenoceptor antagonist. They elegantly utilize genetic strategies to show that this inhibition is independent of MR inhibition, but dependent on pannexin channels in SMCs. The data are intriguing, did pannexin inhibition contribute all along unrecognized to the blood pressure lowering effects of spironolactone?

Then again, Good et al. do not provide direct evidence that pannexin channels are inhibited by spironolactone in RH patients and it seems premature to assume that this is the case. Foremost it is uncertain if pannexin inhibition is broadly achieved with routinely administered doses of spironolactone, e.g. in PATHWAY-2 patients received typical doses of 25–50 mg p.o. per day³. Directly corresponding plasma and tissue levels of spironolactone are not available, but plasma levels of total spironolactone metabolites after eight days of 100 mg spironolactone p.o. daily reach peak values that are just about approaching the IC₅₀ of 18.9 μmole/L for pannexin inhibition in isolated arteries⁸. In plasma spironolactone is almost entirely bound to plasma protein, which limits diffusion. Spironolactone concentrations in the arterial vessel wall have apparently not been investigated and might rely on surrounding tissue. Spironolactone and its metabolites enrich most notable in the intestine, the liver and the kidney, but e.g. in the brain and skeletal muscle tissue concentrations remain below plasma concentrations⁹. If we take the primary target of spironolactone, the MR, the IC₅₀ is around 24 nmole/L, i.e. spironolactone is close to 1000 fold more potent inhibiting the MR than pannexin channels¹⁰. Altogether, it requires additional evidence that pannexin channels in resistance arteries are inhibited by typical doses of spironolactone.

It should also be highlighted that Good et al. analyzed acute blood pressure changes within 45 min after i.p. injection of spironolactone. While that further emphasizes that the observed effects are independent of MR inhibition, it does not allow for conclusions about long-term blood pressure regulation. If pannexins in SMCs are inhibited, then pannexins in other cell types e.g. in endothelial cells and erythrocytes would be inhibited as well, which might reduce vasodilatory ATP release into the vessel lumen¹¹. It is also noteworthy that the role of pannexin channels in alpha adrenergic stimulation has been challenged¹². Last but not least, the study by Good et al. utilizes only male mice and recruited only male donors for human arteries. Therefore, strictly, results may not directly apply to the female sex.

At this point there are also ongoing controversies about some of the properties of the pannexin 1 channel that require clarification in the future^{13, 14}. Briefly, for pannexin 1 two open channel conformations have been proposed, one low and one large conductance state that is dependent on the mode of activation¹³. Voltage stimulation in the absence of other stimuli induces the chloride selective, low conductance conformation, while other stimuli e.g. high K+ induce the large pore that is permeable to ATP. In Good et al. the large conductance channel is never observed, consistent with previous reports from this group, while ATP release is reported separately in apoptotic Jurkat cells. While not of major concern for the conclusion of the study, independent confirmation of the inhibitory effect of spironolactone on the large conductance state of pannexin 1 and the corresponding IC₅₀ might be useful.

Despite these open questions, the discovery that spironolactone inhibits pannexin channels brings the pannexin field one step closer to an inhibitor that is in widespread clinical use. Spironolactone might be a new useful tool to probe the divers and exciting emerging roles of pannexin 1 channels. The results presented in Good et al. are compelling to suggest that pannexin channel inhibition is a potential new treatment modality for RH. Whether or not pannexin inhibition already contributes to long-term blood pressure lowering in hypertensive patients receiving spironolactone remains to be elucidated.

ABBREVIATIONS

ATP

adenosinetriphosphate

i.p

intraperitoneal

IC₅₀

half-maximal inhibitory concentration

mmHg

millimeter of mercury

MR

mineralcorticoid receptor

p.o

by mouth, taken orally

SMC

smooth muscle cell