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Keywords:

  • Blood Flow;
  • Clitoris;
  • Vagina;
  • Doppler;
  • PDE4;
  • cAMP;
  • VIP

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

Introduction.

Cyclic adenosine 3′5′ monophosphate (cAMP) is produced by adenylate cyclase after activation by, e.g., vasoactive intestinal polypeptide or prostaglandin E1 (PGE1). The cAMP-degrading phosphodiesterase 4 (PDE4) is expressed in the vagina and clitoris, but no information is available on the functional role for PDE4-related signals in the female neurovascular genital response.

Aim.

The aim of this study is to study the effect of inhibition of PDE4 with rolipram on nerve- and PGE1-induced vaginal and clitoral blood flow responses of rat.

Methods.

Measure of clitoral and vaginal blood flow and blood pressure in anesthetized rats during activation of the dorsal clitoral nerve (DCN) before and after intraperitoneal administration of rolipram or sildenafil (phosphodiesterase type 5 inhibitors [PDE5]) and nitro-L-arginine (L-NNA) (nitric oxide synthase inhibitor). Effect by topical administration of PGE1 on genital blood flow was also evaluated.

Main Outcome Measure.

Blood flow was recorded as tissue perfusion units (TPU) by a Laser Doppler Flowmeter. Mean arterial blood pressure (MAP) was recorded (cmH2O) in the carotid artery. Blood flow responses are expressed as TPU/MAP. Unpaired t-test and an analysis of variance were used.

Results.

Compared with control stimulations, rolipram (0.3 mg/kg) caused a twofold increase in peak blood flow (P < 0.05) and fourfold increase of the rate of clitoral blood flow during activation of the DCN (P < 0.05). Simultaneously, a twofold increase in peak blood flow and threefold increase in rate of blood flow were noted in the vagina (P < 0.05). Similar effects were noted for sildenafil (0.2 mg/kg) (P < 0.05). Inhibitory effects by L-NNA (60 mg/kg) on blood flow responses to DCN activation were significantly lower for rats treated with rolipram than with sildenafil (P < 0.05). PGE1-induced (10 μg) blood flow responses were significantly higher (P < 0.05) in rats treated with rolipram than with sildenafil.

Conclusions.

These findings suggest that the cAMP/PDE4 system may be of similar functional importance as the nitric oxide/cyclic guanosine monophosphate/PDE5 pathway for neurovascular genital responses of the female rat.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

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 [19]. 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 [20]. 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 [27]. Rolipram, a PDE4 selective inhibitor, was shown to relax both male erectile tissue [28-30] and female vaginal tissue [16]. 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.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

Ethical Approval

The Animal Ethics Committee of San Raffaele University, Milan, Italy approved the study protocol.

Animals

Female Sprague-Dawley rats (N = 12; 250–300 mg) were used and housed in a 12–12 hours dark–light cycle environment under standard laboratory conditions with free access to water and food. Intraperitoneal (ip) ketamine (75 mg/kg) and xylazine (50 mg/kg) were used for anesthesia. Throughout the experiments, animals breathed spontaneously. Rats were sacrificed by CO2 asphyxia. Clitoral and vaginal tissue were harvested for immunofluorescence.

Surgical Procedures and In Vivo Experimental Protocol

During general anesthesia, a midline incision was performed in the neck and a heparinized (100 IU/mL) polyethylene (PE 50) catheter (Clay-Adams Company Inc., New York, NY, USA) was inserted into the carotid artery and connected to a P23 DC pressure transducer (Statham Instruments Inc., Ventura, CA, USA) for recordings of mean arterial pressure (MAP; cmH2O).

The clitoral glans and the DCNs were visualized as described by Vachon et al. [4]. A laser Doppler probe (Transonic Systems, New York, NY, USA) was placed on the surface of the clitoral glans, whereas another probe was positioned inside the vagina. A drop of paraffin oil clothed the probes to facilitate blood flow registration. Simultaneous records of clitoral and vaginal blood flow were acquired via an MP100 unit (Biopac Systems Inc., Goleta, CA, USA) during activation of the DCN with a stainless-steel bipolar electrode connected to a S48 stimulator (Grass Instruments, West Warwick, RI, USA). The pulse width was 0.2 ms with a train duration of 5 ms at 10 Hz. Each stimulation lasted 30 seconds. Blood flow was recorded continuously every 0.1 second. Voltage-response curves (2.5 V; 5 V; 7.5 V) were determined in each animal, and a submaximal voltage (5 V) was chosen for further investigations with drugs. Ip rolipram (0.3 mg/kg, N = 6) or sildenafil citrate (0.2 mg/kg, N = 6) was given; the selected doses were based on previous uses in rats [18, 31]. Activation of the DCN was repeated 20 minutes after administration of each drug. After these records, all animals were subjected to ip injection of a competitive inhibitor of nitric oxide synthase (NOS) with selectivity for the neuronal and endothelial isoforms of the enzyme (L-NNA 60 mg/kg, Sigma-Aldrich, St. Louis, MO, USA), and after 30 minutes another electrical stimulation of the DCN was performed. In order to obtain accurate values, three stimulations were performed every 4 minutes in each condition, and the average of these values was calculated for each animal.

At the end of the experiments, the effects by topical PGE1 (10 μg) [32] on clitoral or vaginal blood flow were assessed. We also tested the vehicles (ip and topically) in six rats.

The following parameters were evaluated: baseline blood flow (before activation of the DCN) and peak blood flow (maximal blood flow values during activation of the DCN—baseline blood flow). Blood flow values were normalized to MAP. The rate of blood flow per second was calculated at 50% of peak blood flow and expressed as tissue perfusion units (TPU)/second.

Immunofluorescence

Genital tissues were immediately placed in an ice-cold solution of 4% formaldehyde in phosphate-buffered saline (pH 7.4) and further processed as described previously [10]. Tissue sections (10 μm) were incubated overnight with a rabbit antibody for PDE4 (1:250, FabGennix, Frisco, TX, USA) and a guinea pig antibody for VIP (1:1,000; Eurodiagnostica, Malmo, Sweden). After rinsing, secondary species-directed IgG Goat anti-Rat 488 Alexa Fluor (1:2,000) was applied to sections for 2 hours. Sections were analyzed in a Leica fluorescence microscope (Leica Microsystems, Milano, Italy), and images were captured and visualized digitally in a Leica Image station (Leica Microsystems).

Drugs

All drugs were purchased from Sigma-Aldrich. Working stock solutions (1 mg/mL) of L-NNA, PGE1, and sildenafil citrate were prepared freshly in saline on the day of the experiment. Rolipram was initially dissolved in ethanol at (10 mg/mL) and then further diluted in saline to 1 mg/mL.

Statistical Analyses

An analysis of variance (post hoc test Student-Newman–Keuls) was used for comparisons of repeated measures. For comparison of effects between two measures, an unpaired t-test was used. All calculations are based on the number of individual animals. Significance level was accepted for P < 0.05. Values are expressed as mean ±standard error of the mean.

Result

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

In Vivo Blood Flow Recordings

Electrical stimulation of the DCN did not affect MAP (P > 0.05). There were no differences in baseline blood flow values between the different groups of rat. The mean baseline vaginal and clitoral blood flows were 0.16 ± 0.009 TPU/MAP (N = 12) and 0.19 ± 0.01 TPU/MAP (N = 12), respectively. Mean baseline arterial pressure was 69 ± 2 cmH2O and 70 ± 1 cmH2O for rats treated with sildenafil (N = 6) or rolipram (N = 6), respectively.

In all rats, activation of the DCN induced similar voltage-dependent and reproducible increases in blood flow of the clitoris and the vagina. Control stimulations at 2.5 V, 5 V, and 7.5 V resulted in peak clitoral blood flows of 0.06 ± 0.006 TPU/MAP, 0.1 ± 0.009 TPU/MAP, and 0.17 ± 0.009 TPU/MAP (N = 12), respectively. Corresponding values for vaginal blood flow were 0.05 ± 0.003 TPU/MAP, 0.09 ± 0.008 TPU/MAP, and 0.12 ± 0.01 TPU/MAP (N = 12).

No changes in clitoral or vaginal blood flow were recorded during stimulation of the DCN after administration of vehicles (P > 0.05).

After ip. pretreatment with rolipram, peak blood flow responses of the clitoris were increased during activation of the DCN (5 V) to 0.21 ± 0.04 TPU/MAP (N = 6; P < 0.05, Figure 1A). Rolipram increased the rate of clitoral blood flow from 0.32 ± 0.05 TPU/second (baseline) to 1.27 ± 0.2 TPU/second (P < 0.05, Figure 2A). After addition of L-NNA following rolipram administration, the peak and rate of clitoral blood flow during DCN activation were reduced to 0.11 ± 0.04 TPU/MAP (P < 0.05, Figure 1A) and 0.53 ± 0.12 TPU/second (P < 0.05, Figure 2A). Mean clitoral peak blood flow response after sildenafil was 0.21 ± 0.03 TPU/MAP (N = 6; P < 0.05, Figure 1B), and the rate of blood flow was increased from 0.16 ± 0.03 TPU/second (baseline) to 1.32 ± 0.51 TPU/second (P < 0.05, Figure 2B). L-NNA reduced the sildenafil-induced increase of peak clitoral blood flow to 0.07 ± 0.009 TPU/MAP (P < 0.05, Figure 1B) and also the rate of blood flow to 0.18 ± 0.07 TPU/second (P < 0.05, Figure 2B). The effect by L-NNA on peak and rate of clitoral blood flow was lower (P < 0.05) in rats treated with rolipram than sildenafil (Figures 1 and 2C).

figure

Figure 1. Graphs depicting the clitoral peak blood flow during activation of the dorsal clitoral nerve (DCN). (A) At baseline, after intraperitoneal rolipram (0.3 mg/kg, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). (B) Clitoral peak blood flow at baseline, after sildenafil (0.2 mg/kg, ip, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). The effect by L-NNA (C) on peak clitoral blood flow was lower in rats treated with rolipram than rats treated with sildenafil. *P < 0.05; **P < 0.01 anova; §P < 0.05 t-test. L-NNA = nitro-L-arginine; TPU = tissue perfusion units; MAP = mean arterial pressure; anova = analysis of variance

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figure

Figure 2. Graphs depicting the clitoral rate of blood flow during activation of the dorsal clitoral nerve (DCN). (A) At baseline, after intraperitoneal rolipram (0.3 mg/kg, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). In (B), clitoral rate of blood flow at baseline, after sildenafil (0.2 mg/kg, ip, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). The effect by L-NNA (C) on the rate of clitoral blood flow was lower in rats treated with rolipram than rats treated with sildenafil. *P < 0.05, **P < 0.01 anova. §P < 0.05 t-test. L-NNA = nitro-L-arginine; TPU = tissue perfusion units; anova = analysis of variance

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In the vagina, rolipram increased peak blood flows to 0.18 ± 0.02 TPU/MAP (N = 6; P < 0.05, Figures 3 and 4A), and blood flow rates were increased from 0.56 ± 0.11 TPU/second (baseline) to 1.82 ± 0.26 TPU/second (P < 0.05, Figure 5A) during activation of the DCN. L-NNA reduced the peak and rate of the vaginal blood flow to 0.13 ± 0.008 TPU/MAP (P < 0.05, Figure 2A) and 0.81 ± 0.23 TPU/second (P < 0.05, Figure 5A).

figure

Figure 3. Original tracings of mean arterial blood pressure (MAP) and blood flow recordings from the clitoris and vagina during 30 seconds activation of the dorsal clitoral nerve (DCN) in two rats before and after intraperitoneal pretreatment (20 minutes) with (A) rolipram 0.3 mg/kg or (B) sildenafil 0.2 mg/kg. TPU = tissue perfusion units

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figure

Figure 4. Graphs depicting the vaginal peak blood flow during activation of the dorsal clitoral nerve (DCN). (A) At baseline, after intraperitoneal rolipram (0.3 mg/kg, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). In (B), vaginal peak blood flow at baseline, after sildenafil (0.2 mg/kg, ip, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). The effect by L-NNA (C) on peak vaginal blood flow was lower in rats treated with rolipram than rats treated with sildenafil. *P < 0.05, **P < 0.01 anova; §P < 0.05 t-test. L-NNA = nitro-L-arginine; TPU = tissue perfusion units; MAP = mean arterial pressure; anova = analysis of variance

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figure

Figure 5. Graphs depicting the vaginal rate of blood flow during activation of the dorsal clitoral nerve (DCN). (A) At baseline, after intraperitoneal rolipram (0.3 mg/kg, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). In (B), vaginal rate of blood flow at baseline, after sildenafil (0.2 mg/kg, ip, 20 minutes), and after addition of L-NNA (60 mg/kg, 20 minutes). The effect by L-NNA (C) on the rate of vaginal blood flow was not different in rats treated with rolipram or with sildenafil. *P < 0.05, **P < 0.01 anova; §P < 0.05 t-test. L-NNA = nitro-L-arginine; TPU = tissue perfusion units; anova = analysis of variance

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Vaginal peak blood flow was increased to 0.17 ± 0.01 TPU/MAP (P < 0.05) by sildenafil (Figures 3 and 4B). The rate of blood flow was increased from 0.43 ± 0.03 TPU/second (baseline) to 0.77 ± 0.04 TPU/second (P < 0.05, Figure 5B). The inhibitory effect by L-NNA on peak vaginal blood flow was larger (P < 0.05, Figure 4C) in rats treated with sildenafil than rolipram, but no differences were noted for the rate of blood flow (Figure 5C).

Without activation of the DCN, topical administration of PGE1 in rats treated with rolipram and L-NNA induced peak clitoral and vaginal blood flows that were 0.38 ± 0.05 TPU/MAP and 0.49 ± 0.06 TPU/MAP (Figures 6 and 7), respectively. In sildenafil- and L-NNA-treated rats, the PGE1-induced peak blood flows were lower than in rolipram rats, and these were 0.21 ± 0.04 TPU/MAP (P < 0.05) and 0.20 ± 0.06 TPU/MAP (P < 0.05, Figure 7), respectively.

figure

Figure 6. Original tracing of mean arterial blood pressure (MAP) and blood flow recordings from the clitoris and vagina during a PGE1-induced genital blood flow response in one rat. TPU = tissue perfusion units; PGE1 = prostaglandin E1

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figure

Figure 7. Graphs depicting the (A) clitoral or (B) vaginal blood flow response (without activation of the dorsal clitoral nerve) to topical prostaglandin E1 (PGE1; 10 μg) after intraperitoneal administration of rolipram and L-NNA, or sildenafil and L-NNA. *P < 0.05 t-test. L-NNA = nitro-L-arginine; TPU = tissue perfusion units; MAP = mean arterial pressure

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Ip. administration of sildenafil, rolipram, or L-NNA did not have any effect on the MAP or on the baseline flow of the clitoris or vagina. Topical addition of PGE1 did not have any effect on MAP.

Immunofluorescence

Vaginal arteries and arterioles that were supplied by VIP-immunoreactive (IR) nerve fibers also expressed PDE4-immunoreactivity in the vascular wall (Figure 8). The clitoral erectile tissue expressed PDE4-IR in cavernosal and arteriolar walls. Interspersed in the clitoral erectile tissue, rich amounts of VIP-IR nerves and terminals were visualized (Figure 8).

figure

Figure 8. Immunofluorescence. Vasoactive intestinal polypeptide (VIP) immunoreactive (IR) nerve fibers and varicose nerve terminals (Alexa Green) in clitoral (A) and vaginal (B) sections costained for phosphodiesterase 4 (PDE4, Alexa Red). (A) Clitoral erectile tissue with PDE4-IR sinusoidal smooth muscle surrounded by VIP-IR nerve fibers and terminal varicosities. Magnification 200×. (B) Subepithelial arterioles of the vaginal wall with PDE4-IR vascular smooth muscle that are supplied by VIP-IR nerve fibers and terminal varicosities, magnification 200×.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

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 [4]. 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 [27]. 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 [16]. 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 [16], 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 [18]. 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 [37]. 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 [38]. The subsequent stress on the endothelium activates endothelium-dependent signals that prolong vascular responses even after nerve stimulation is ended [38]. 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 [44]. 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 [45]. 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.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

Rolipram, a selective inhibitor of the cAMP-degrading PDE4, effectively increases rat clitoral and vaginal blood flow during activation of the DCN. Effects by rolipram were partly counteracted by L-NNA suggesting that cooperative functions between cAMP/PDE4- and NO/cGMP-signal occur in the female rat genital vascular tissue. After blockade of the NO-signal, rolipram effectively enhanced PGE1-induced blood flow responses of the clitoris and the vagina, further supporting an important function for the cAMP/PDE4 system in the female rat genital vascular function.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

The Swedish Medical Research Council, the Gester Foundation, and the Urological Research Institute supported the production of the manuscript.

Conflict of Interest: The authors have no conflicts of interest.

Statement of Authorship

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References

Category 1

  • (a)
    Conception and Design
    Fabio Castiglione; Petter Hedlund; Andrea Salonia; Patrizio Rigatti; Francesco Montorsi
  • (b)
    Acquisition of Data
    Fabio Castiglione; Petter Hedlund; Andrea Salonia; Alice Bergamini; Andrea Russo; Giovanni La Croce; Giulia Castagna; Giorgia Colciago
  • (c)
    Analysis and Interpretation of Data
    Fabio Castiglione; Petter Hedlund; Andrea Salonia; Alice Bergamini

Category 2

  • (a)
    Drafting the Article
    Fabio Castiglione; Petter Hedlund; Andrea Salonia; Alice Bergamini; Andrea Russo; Giovanni La Croce; Giulia Castagna; Giorgia Colciago
  • (b)
    Revising It for Intellectual Content
    Fabio Castiglione; Petter Hedlund; Andrea Salonia; Alice Bergamini; Patrizio Rigatti; Francesco Montorsi

Category 3

  • (a)
    Final Approval of the Completed Article
    Fabio Castiglione; Petter Hedlund; Andrea Salonia; Alice Bergamini; Andrea Russo; Giovanni La Croce; Giulia Castagna; Giorgia Colciago; Patrizio Rigatti; Francesco Montorsi

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Result
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Statement of Authorship
  10. References
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