Erectile dysfunction (ED), the inability to achieve an erection firm enough for satisfactory sexual intercourse, is a common comorbidity in males with diabetes mellitus (DM). Approximately 50% of diabetic men develop ED.¹ Treatments such as phosphodiesterase type 5 (PDE5) inhibitors, intracavernous injection therapy, and vacuum constriction devices have varying success. Between 60% and 70% of ED patients cease PDE5 treatment due to adverse effects.² Around 50% of DM patients with ED do not respond to PDE5 inhibitors.³ As the prevalence of DM in younger males is increasing, more men risk developing ED earlier in life.⁴ Therefore, there is a need to investigate novel targets for the treatment of ED.

The interconnected vascular sinuses of the corpus cavernosum (CC), fed by the helicine arteries, are separated by trabeculae consisting of connective tissue and smooth muscle cells (SMCs). Detumescence is maintained by tonic contraction of terminal arterioles and trabecular SMCs, restricting blood flow through the penis. Conversely, erection is achieved by vasodilation of penile arteries, increasing blood flow through the penis. This increase in blood volume is accommodated by the relaxation of trabecular SMCs.⁵ Therefore, the coordinated contraction and relaxation of CC SMCs is critical to erectile function. Previously, the Ca²⁺-activated Cl^- channel anoctamin-1 (Ano1) has been shown to play a role in penile SMC function. Antagonists of Ano1 inhibited spontaneous CC contractions and inhibited Cl^- currents and Ca²⁺ activity in freshly isolated cells.⁶ Neurotransmitters that modulate CC activity are thought to target SMCs directly as SMCs express soluble guanylate cyclase, the receptor for nitric oxide (NO).⁷ However, as the CC is sparsely innervated, a cellular mediator could be present between nerves and SMCs. Furthermore, the specific cell type that expresses Ano1 has not been identified as previous studies have relied on morphological identification of SMCs alone. Cells expressing platelet derived growth factor receptor-α (PDGFRα) have been identified in visceral smooth muscles and have been shown to mediate inhibitory neurotransmission.⁸ In the renal pelvis, atypical SMCs that express PDGFRα and myosin heavy chain (SmMHC) also express Ano1. These cells are thought to generate the rhythmic contractions of the renal pelvis to transport urine away from the kidney.⁹ The aim of this study was to identify and characterize the cell type that expresses Ano1 in the CC and to determine if these cells could also mediate neurotransmission.

To investigate whether SMCs in the CC express Ano1, CC cells were isolated from a mouse that expresses the fluorescent reporter eGFP in SMCs (SMC-eGFP mouse) and sorted using fluorescence activated cell sorting (FACS). FACS purification of SMC-eGFP⁺ cells yielded a single population of cells (Figure 1, Aa). Preliminary gene expression studies revealed that these cells expressed Myh11, a specific marker of SMCs, as well as high expression of Pdgfra and low expression of Ano1 (Figures 1, Ab and 2, A). As SMCs are considered the direct targets of neurotransmitters, the expression of soluble guanylate cyclase (Gucy1a1) and PKG (Prkg1), critical NO pathway proteins, was examined. SMC-eGFP⁺ cells expressed high levels of Gucy1a1 and low levels of Prkg1 (Figure 2, A). As SMC-eGFP⁺ cells also expressed high levels of Pdgfra (Figure 1, A), CC cells were isolated from a mouse expressing eGFP in PDGFRα⁺ cells (PDGFRα-eGFP mouse). FACS of PDGFRα⁺ cells revealed 2 populations of eGFP⁺ cells, a bright and dim population (Figure 1, Ba). qPCR of cell-specific markers showed that while the bright population expressed Pdgfra, the dim population expressed both Pdgfra and Myh11 (Figure1, Bb) and also more highly expressed Ano1 (Figure 2, B). This population also expressed high levels of Gucy1a1 and Prkg1. To confirm the above findings, immunofluorescent labelling of PDGFRα-eGFP CC tissues with antibodies targeting SmMHC was carried out. This revealed eGFP⁺ cells that also labeled positively for SmMHC, representing the dim population of PDGFRα-eGFP⁺ and SmMHC-eGFP⁺ cells (Figure 3).

These preliminary data suggest that caution should be exercised when using traditional molecular markers for SMCs ie Myh11 as a novel population of atypical SMCs expressing Myh11, Pdgfra, and Ano1 is present in the mouse CC. These cells also express NO pathway signaling genes and therefore could be crucial targets for inhibitory neurotransmitters. Increased intracellular Ca²⁺ signaling would lead to continuous activation of Ano1, maintaining a depolarized membrane potential and contraction of the CC. NO stimulation would ultimately relax the CC by lowering intracellular Ca²⁺ concentrations, preventing the continuous activation of Ano1 and membrane hyperpolarization. Therefore, these novel atypical SMCs represent a potential future therapeutic target for the treatment of ED.