Penile erection is mediated by relaxation of corpus cavernosum smooth muscle (CCSM), primarily driven by cyclic nucleotide signalling. Current therapies for erectile dysfunction (ED) target the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) axis, with phosphodiesterase-5 inhibitors (PDE5Is) enhancing cGMP signalling to promote relaxation. However, 30-40% of patients, particularly those with diabetes or vascular disease, respond poorly due to impaired NO bioavailability and reduced cGMP formation, limiting efficacy ^[1].

The parallel cyclic adenosine monophosphate (cAMP) pathway represents an alternative target, traditionally attributed to protein kinase A (PKA)-mediated reductions in intracellular Ca²⁺. Clinically, this is exploited via intracavernosal prostaglandin E1 (PGE1; alprostadil). PKA also modulates ion channels, including K_v7.5 channels, expressed in mouse CCSM cells ^[2]. More recently, exchange protein directly activated by cAMP (EPAC) has emerged as a distinct cAMP effector, inducing smooth muscle relaxation via Rap1/2 signalling, though its role in CCSM remains poorly defined.

Using selective activators of PKA (6-MB) and EPAC (007-AM), we investigated their contributions in mouse CCSM. EPAC-1 expression was confirmed at transcript (RT-PCR, qPCR) and protein levels (immunoreactivity in isolated CCSM cells), with antibody validation in mouse kidney cortex, which highly expresses EPAC-1 ^[3].

In isometric tension studies, CCSM pre-contracted with phenylephrine (PE; 3×10^-7M) exhibited sustained phasic activity. EPAC activation (007-AM, 1×10^-5M) produced modest but significant reductions in contraction frequency (6.87±1.47 min^-1 to 5.17±1.07 min^-1; P=0.0156) and amplitude (0.99±0.16 mN to 0.86±0.16 mN; P=0.0276). Similarly, PKA activation (6-MB, 1×10^-4M) reduced contraction frequency (6.70±0.59 min^-1 to 4.57±0.47 min^-1; P=0.0001). However, co-activation abolished phasic contraction frequency (5.83±0.94 min^-1 to 0.00±0.0 min^-1; P=0.0016) and amplitude (0.87±0.14 mN to 0.00±0.0 mN; P=0.0015).

At the cellular level, PE induced Ca²⁺ oscillations in isolated CCSM cells. 007-AM significantly reduced oscillation frequency (20.0±4.0 min^-1 to 0.5±0.5 min^-1; P=0.0087) at high PE (1×10^-5M), previously identified as membrane potential-independent ^[2]. In contrast, 6-MB had no effect under these conditions (25.7±4.2 min^-1 to 25.2±3.5 min^-1; P=0.8560) but abolished oscillations (9.5±0.9 min^-1 to 0.0±0.0 min^-1; P=0.0445) at low PE (1×10^-7M), which are membrane potential-sensitive. This effect was prevented (8.0±1.0 min^-1 to 15.5±2.1 min^-1; P=0.4896) by the K_v7 blocker XE-991, implicating K_v7 channels in PKA-mediated responses.

Collectively, these data show that EPAC-1 is expressed in CCSM and inhibits Ca²⁺ signalling, potentially via suppression of sarcoplasmic reticulum Ca²⁺ release, while PKA may promote relaxation through K_v7.5 channel activation and membrane hyperpolarisation. Although co-application of 007-AM and 6-MB abolished contractions, the ability of EPAC activation alone to suppress Ca²⁺ oscillations suggests a dominant role for EPAC rather than simple additive effects. Targeting cAMP effectors, particularly EPAC, alone or alongside PKA, may therefore offer a strategy to restore CCSM relaxation in patients unresponsive to PDE5Is.