Evidence of an excitatory purinergic innervation in mouse corpus cavernosum smooth muscle
Authors: Lim Xin Rui , Mercer Mitchell , Harraz Osama F , Hollywood Mark A , Sergeant Gerard P , Thornbury Keith D 
Published: September 2024

Abstract

Background
Evidence suggests that the corpus cavernosum smooth muscle (CCSM) cells of several species, including humans, express purinergic P2X receptors, but it is not known if the corpus cavernosum has an excitatory purinergic innervation.

Aim
In this study we aimed to determine if the mouse CCSM has a functional purinergic innervation.

Methods
Mouse CCSM myocytes were enzymatically isolated and studied using the perforated patch configuration of the patch clamp technique. Isometric tension was measured in whole cavernosum tissue subjected to electrical field stimulation (EFS) to evoke nerve-mediated responses.

Outcomes
The mouse CCSM myocytes expressed P2X1 receptors, and adenosine triphosphate (ATP) evoked inward currents in these cells. In addition, P2X1-mediated contractions were recorded in whole tissue in response to EFS.

Results
In cells held under a voltage clamp at −60 mV, ATP (1 μm) evoked large inward currents (mean approximately 900 pA). This current rapidly declined but was repeatable at 8-minute intervals. α,β-methylene ATP (10 μM), an agonist of P2X1 and P2X3 receptors, caused a similar current that also rapidly declined. Desensitization to α,β-methylene ATP negated the effect of ATP, but the ATP effect was restored 8 minutes after washout of α,β-methylene ATP. The effect of ATP was reversibly blocked by NF449 (1 μm), a selective antagonist of P2X1 receptors. In isometric tension experiments electrical field stimulation (EFS) at 0.5-8 Hz evoked frequency-dependent contractions in the presence of l-nitro arginine (l-NO-Arg) (100 μm). When phentolamine (3 μm) and atropine (1 μm) were applied, there remained a nonadrenergic, noncholinergic component of the response to EFS, consisting mainly of a transient contraction. This was significantly reduced by NF449 (1 μm). Finally, in immunocytochemistry experiments, isolated CCSM myocytes stained positively when exposed to an antibody raised against P2X1 receptors.

Clinical Implications
Previous studies have shown that P2X1 receptors in CCSM are upregulated in diabetes. These findings, taken together with the functional evidence presented here, indicate that P2X1 receptors may provide an alternative therapeutic target for treatment of erectile dysfunction in patients with diabetes, which is known to be relatively resistant to treatment with phosphodiesterase 5 inhibitors.

Strengths and Limitations
Strengths of this study are the use of a combination of functional experiments (patch clamp) and immunocytochemical analyses to show expression of P2X1 receptors on CCSM myocytes while also performing functional experiments to show that stimulation these receptors results in contraction of CCSM. A limitation of this study was the use of animal rather than human tissue.

Conclusion
This investigation provides evidence that mouse corpus cavernosum smooth muscle cells express P2X1 receptors and that these receptors are involved in mediating part of the contractile response to nerve stimulation evoked by EFS.