- Top of page
- Statement of Authorship
Female sexual arousal is defined as the ability to reach and maintain a lubrication–swelling response over the sexual intercourse. In this context, increased genital blood flow during arousal promotes clitoral tumescence and congestion of the lower third of the vagina while producing fluids transudation through the vaginal wall [1, 2].
As demonstrated in a number of previous in vivo animal models, the arousal-related vasodilation of the vaginal arteries and the tumescence of the clitoral erectile tissue is regulated by the central and peripheral nervous systems [3-8]. Indeed, female genital tract is innervated by adrenergic, cholinergic, peptidergic, and nitric oxide (NO) synthase-containing nerves; the great heterogeneity of these multiple transmitter systems has been scantily studied in detail, especially in relation to the pathway of relaxation of vascular tissue as well as the responses of local vasocongestion [1, 2].
Numerous preclinical studies support a role for NO and its intracellular second messenger cyclic guanosine monophosphate (cGMP) in the female genital vascular function. Moreover, inhibition of the cGMP-degrading phosphodiesterase type 5 inhibitors (PDE5) either in isolated tissues or in animals revealed to enhance the relaxant responses of both the clitoris and the vagina [5-18]. Cyclic adenosine monophosphate (cAMP) is a further intracellular signaling molecule that is associated with regulation of vascular and nonvascular smooth muscle tonus of the urogenital tract . The cellular cAMP concentration increases upon production by adenylate cyclase (AC) that, in turn, is regulated by G-protein-coupled receptors (GPCRs). GPCRs are activated by extracellular stimuli, including, for example, catecholamines, neuropeptides, and prostaglandins . Among others, the receptors for the vasoactive intestinal polypeptide (VIP) and prostaglandin E1 (PGE1) are GPCRs. Activation of GPRCs eventually increases the intracellular levels of cAMP, thus leading to smooth muscle relaxation [16, 21]. Both in animals and in humans studies suggest that VIP may act as a transmitter that is able to regulate the female genital vascular response, whereas PGE1 is a vasodilator widely used to treat male erectile dysfunction and potentially useful in women with genital arousal disorder [2, 22-25]. VIP has been shown to relax female genital tissues with a concomitant increase in the tissue levels of cAMP. Inhibition of the degradation of VIP was recently demonstrated to enhance nerve-induced vaginal and clitoral blood flows in rabbits [1, 2, 20, 22, 26].
Phosphodiesterases carry out an important function in lowering cyclic nucleotide tissue levels. Among them, phosphodiesterase 4 (PDE4) is the cAMP-specific degrading enzyme that provides a main part of the cAMP hydrolyzing activity . Rolipram, a PDE4 selective inhibitor, was shown to relax both male erectile tissue [28-30] and female vaginal tissue . To the best of our knowledge, no data are available regarding the effects of rolipram on female genital blood flow in vivo. The aim of this preclinical study was to investigate the effect of the rolipram-dependent inhibition of the cAMP-degrading PDE4 over vaginal and clitoral blood flow responses to the activation of the dorsal clitoral nerve (DCN) in rats. Likewise, topical administration of PGE1 was evaluated in order to (i) obtain an internal validation of the model; (ii) to explore the role of cAMP on genital blood flow without any DCN stimulation; and (iii) to study whether rolipram may eventually improve the effect of PGE1.
- Top of page
- Statement of Authorship
The current study shows that the PDE4 inhibitor rolipram enhances clitoral and vaginal blood flow responses during activation of the DCN or to topical administration of PGE1. In addition, similar to the human, female rat expresses PDE4 in the vasculature of the vagina and clitoris that are supplied by VIP-containing nerves.
Previously, larger mammals such as dogs or rabbits have been used to simultaneously record clitoral and vaginal blood flows, whereas in rats, the vaginal and clitoral blood flow have been recorded separately [3-5, 18, 24, 26]. In this study, simultaneous changes in the blood flow of the clitoris and the vagina of rats were recorded (figure 1) and these responses were similar to what has previously been reported for either the clitoris or the vagina of dogs, rabbits, and rats [3-5, 18, 24, 33]. In most investigations, the pelvic plexus has been stimulated to induce female genital blood flow responses via efferent parasympathetic pathways [3, 5, 18, 33]. Similar to the current investigation, activation of the DCN was used by Vachon et al. for clitoral blood flow responses in the rat . The DCN not only conveys afferent information from the clitoris to the spinal cord but also receives efferent parasympathetic nerve branches from the vaginal nervous plexus and the pelvic nerve [34, 35]. Hence, stimulation of the DCN is likely to support blood flow responses of the female genitalia by direct action of efferent neurons supplying the clitoris and also via a spinal reflex by activation of preganglionic pelvic neurons that supply the clitoris and vagina [34-38]. It cannot be excluded that efferent branches that supply the vagina are also activated via the connections between the DCN and the vaginal plexus.
The activity of PDE4 is endogenously coordinated with AC and the cAMP-dependent protein kinase A via effector and feedback signals to govern the cAMP levels . Rolipram is a selective inhibitor of PDE4 that has been demonstrated to relax isolated nonvascular female genital smooth muscle tissue with concomitant increases in cAMP . The present findings show that rolipram effectively increased DCN-induced blood flow responses of the rat vagina and clitoris and that similar to the human female genitalia , vascular structures of the rat vagina and clitoris express immunoreactivity for PDE4. Rolipram not only increased the peak genital blood flows but also the rate of blood flows (figures 2-5), suggesting that the drug reduced resistance in the genital vasculature. These findings suggest a role for PDE4 in regulating blood flow to the vagina and clitoris and support that cAMP-related signals are of importance for the female genital vascular response.
Sildenafil caused similar effects on blood flow responses to DCN activation as rolipram, and the current results with sildenafil corresponds to previous findings that this drug enhances female genital blood flow during pelvic nerve stimulation in rats . Inhibition of the NO synthesis with L-NNA counteracted effects by sildenafil on genital blood flow. This was expected as both compounds act directly in the NO/cGMP pathway. It was not expected that L-NNA substantially counteracted the increase of genital blood flow in rats during selective inhibition of the cAMP-degrading PDE4 with rolipram (figures 2-5). These findings may suggest convergent signals between the cAMP/PDE4 pathway and the cGMP/PDE5 system. Cross talk between cAMP and cGMP was previously hypothesized to occur in the human vagina. Functional convergence between cyclic nucleotides and protein kinases was reported for some tissues [10, 37]. In vascular functions, relevant cross-activation of the cGMP-dependent protein kinase G by cAMP has been demonstrated . In the current study, L-NNA reduced rolipram-induced increases in genital blood flow to a lesser extent than in rats treated with sildenafil (figures 2-4). The inhibitory activity of sildenafil on PDE5 corresponds to a large effect by upstream inhibition of the NO synthesis by L-NNA that subsequently reduces the production of cGMP.
Neuronal signals initiate vasorelaxation with increased blood flow and physical expansion of the penile vasculature and erectile tissues . The subsequent stress on the endothelium activates endothelium-dependent signals that prolong vascular responses even after nerve stimulation is ended . In our study, we found that nerve-induced vascular genital responses persisted some time after nerve stimulation was stopped (figure 1). Taken together with reports of endothelium-dependent and NO- or cGMP-sensitive relaxant responses of the clitoral erectile tissue or subepithelial vaginal arteries [14, 39, 40], a similar cooperative function of nerves and endothelium may occur also for the female genital vascular response. This may explain the observed inhibitory effects by L-NNA on rolipram-induced increases in rat clitoral and vaginal blood flows. Hence, activation of the DCN induces release not only of NO but presumably also of other effectors, e.g., VIP, that via cAMP and PDE4-sensitive mechanisms also induce vasorelaxation. The subsequent increase in blood flow may in turn activate endothelial NO/cGMP signals that are sensitive to L-NNA.
Inhibition of the synthesis of NO (systemic or local) reduces intrapenile pressure increases to activation of the cavernous nerve by up to 80–90% and commonly abolishes erectile responses [41-43]. This does not seem to be the case for neurovascular responses of the female genitalia. Previous investigations in rats, dogs, and rabbits have found that systemic NOS inhibition, at similar or higher doses than those used in male models, reduces female genital blood flow responses to activation of nerves by only 25–65% [5, 17, 18]. Similarly, we demonstrated that ip L-NNA, after treatment with sildenafil or rolipram, did not reduce blood flow responses of the vagina or clitoris to DCN activation below control responses before addition of drugs. These findings suggest that the female genital neurovascular response is less dependent on NO and cGMP-mediated signals as compared with male penile erection.
PGE1 is an activator of the cAMP pathway widely available to treat erectile dysfunction. Positive effects by PGE1 on subjective arousal were reported also in pre- or postmenopausal women with sexual arousal disorder . We used PGE1 as a tool to assess cAMP-induced vascular genital responses of the female rat after inhibition of the NO synthesis. In rats treated with sildenafil and L-NNA, clitoral and vaginal blood flow responses to topical PGE1 administration reached similar peak values as obtained during DCN activation in the presence of either rolipram or sildenafil (figures 2, 4, and 6). The PGE1-induced responses of these rats were twofold larger than control blood flow responses during activation of the DCN at baseline (no drugs). In rats treated with rolipram and L-NNA, the PGE1-induced responses were larger (four- or fivefold the baseline control clitoral or vaginal peak blood flows to DCN activation). Hence, after blockade of the NO synthesis, PGE1 effectively increases blood flow of the female rat genitalia. This activity can be further increased by rolipram, presumably by inhibiting PDE4-mediated degradation of cAMP. In summary, our findings suggest that the cAMP and PDE4 system may be an important pathway for neurovascular genital responses of the female rat.
Some limitations of our study should be acknowledged. Blood flow records with Laser Doppler may exhibit variability, and small movements of the animal or the probes may disturb recordings with various artifacts . This may affect end points unless the technique is appropriately handled and data are properly interpreted. In the current study, stage of estrous cycle of rats were not confirmed which may have increased the interanimal variability.