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Keywords:

  • Corpus Cavernosum;
  • ATP;
  • Cation Current;
  • Chloride Current;
  • Detumescence;
  • Intracellular Ca2+

Abstract

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

Introduction

Although adenosine triphosphate (ATP) has often been reported to relax the corpus cavernosum, this may be mediated by indirect effects, such as release of nitric oxide from the endothelium. Recent data suggest that P2X1 receptors may be up-regulated in diabetes, and these exert an anti-erectile effect by causing the corpus cavernosum smooth muscle cells (CCSMCs) to contract. However, to date, there is no functional evidence that ATP can directly stimulate CCSMC.

Aims

This study aims to (i) to directly examine the effect of ATP on membrane currents in freshly isolated CCSMC, where influences of endothelium and other cells are absent; and (ii) to determine the receptor subtypes, ionic currents, and Ca2+ signals stimulated by ATP.

Methods

CCSMCs were enzymatically dispersed from male New Zealand White rabbits for patch clamp recording and measurement of intracellular Ca2+ in fluo-4-loaded cells using spinning disk confocal microscopy.

Main Outcome Measures

Patch clamp recordings were made of ATP-evoked membrane currents and spontaneous membrane currents. Spinning disk confocal imaging of intracellular Ca2+ was performed, and the response to ATP was recorded.

Results

ATP evoked repeatable inward currents in CCSMC (1st application: −675 ± 101 pA; 2nd application: −694 ± 120 pA, N = 9, P = 0.77). ATP-induced currents were reduced by suramin from −380 ± 121 to −124 ± 37 pA (N = 8, P < 0.05), by α,β-methylene ATP from −755 ± 235 to 139 ± 49 pA (N = 5, P < 0.05), and by NF449 from −419 ± to −51 ± 13 pA (N = 6, P < 0.05). In contrast, MRS2500, a P2Y1 antagonist, had no effect on ATP responses (control: −838 ± 139 pA; in MRS2500: −822 ± 184 pA, N = 13, P = 0.84) but blocked inward currents evoked by 2-MeSATP, a P2Y1,12,13 agonist (control: −623 ± 166 pA; in MRS2500: −56 ± 25 pA, N = 6, P < 0.05). The ATP-evoked inward current was unaffected by changing the transmembrane Cl gradient but reversed in direction when extracellular Na+ was reduced, indicating that it was a cation current.

Conclusions

ATP directly stimulates CCSMC by evoking a P2X-mediated cation current. Doyle C, Sergeant GP, Hollywood MA, McHale NG, and Thornbury KD. ATP evokes inward currents in corpus cavernosum myocytes. J Sex Med 2014;11:64–74.


Introduction

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

The state of tumescence of the penis depends on a balance of the actions of inhibitory and excitatory agonists that modulate the degree of contraction of the smooth muscle cells of the corpus cavernosum and penile arteries. Inhibitory agonists, such as nitric oxide, prostaglandin E2, and vasoactive intestinal peptide are pro-erectile, relaxing the muscle so that the cavernous sinuses fill with blood. Conversely, excitatory agonists, such as noradrenaline, endothelins, and angiotensin II contract the muscle and are antierectile [1]. Adenosine triphosphate (ATP) is another agonist that can modulate the corpus cavernosum, though its role is controversial and its effects are complex. Within smooth muscle tissues, ATP may be released from several different sources including nerves, endothelium/epithelium, smooth muscle myocytes, platelets, red blood cells, and other cell types [2,3]. Depending on the situation and the tissue in question, it can either be excitatory or inhibitory. Thus, in the vas deferens, and some blood vessels, ATP behaves as an excitatory cotransmitter with noradrenaline, causing contraction by acting on P2X purinoceptors, which are a family of cation-permeable ligand-gated ion channels [4–6]. However, in arteries, ATP is also released from the endothelium as a result of shear stress, and this feeds back on the endothelial P2Y receptors, which are typically metabotropic receptors coupled to intracellular second messenger cascades, in this situation resulting in release of nitric oxide (NO) and relaxation of arterial smooth muscle [3,7]. Another factor is that ATP is rapidly broken down by ectonucleotidases to yield active metabolites, such as adenosine diphosphate (ADP) and adenosine, the latter stimulating P1 purinoceptors that may have different actions to those of either P2X or P2Y receptors.

Early reports suggested that in some species ATP caused contraction of the retractor penis, a muscle closely related to corpus cavernosum [8–10]. However, since then, numerous studies have found ATP and its metabolites to be pro-erectile, at least when applied exogenously, as they caused relaxation of the corpus cavernosum (reviewed by Phatarpekar et al. [11]). Although there is no overall consensus as to the receptor subtypes mediating this response, it is clear that it can partly be attributed to a combination of P2Y receptor-mediated release of NO from the endothelium and breakdown of ATP to adenosine, which causes relaxation by acting on P1 receptors. However, it is also possible that in some circumstances, ATP may be antierectile, as α,β-methylene ATP, a P2X receptor agonist, caused contraction of human corpus cavernosum in vitro [12] and ATP itself could cause contraction in the rabbit corpus cavernosum under conditions where resting tension was initially low [13]. The idea that ATP may have antierectile effects has been gradually gaining interest following the immunohistochemical identification of P2X1 receptors on the membranes of corpus cavernosum smooth muscle cells in rats [14], and the fact that several studies have suggested P2X receptors play a pathological role in the development of erectile dysfunction in diabetes [15,16]. Suadicani et al. [15] found that P2X1 receptor expression increased as erectile capacity decreased during development of diabetes induced by streptozotocin and also demonstrated a negative correlation between P2X1 expression and erectile capacity as normal healthy animals matured [15]. Also, PPADS, a P2X receptor antagonist, improved the otherwise compromised nerve-mediated relaxation responses in diabetic rats so that they resembled those of control rats [16].

Therefore, although the overall effect of exogenously applied ATP in corpus cavernosum is to cause smooth muscle relaxation, most likely by indirect actions, the potential for it to have direct contractile effects on the smooth muscle cells warrants further investigation. So far, there are no published studies demonstrating direct actions of ATP or other purinergic agonists on corpus cavernosum smooth muscle cells. These cells have previously been successfully isolated for electrophysiological studies, where they were found to generate spontaneous Ca2+-activated Cl currents [17,18]. The aim of the present study was to examine the action of ATP on isolated corpus cavernosum smooth muscle cells to determine if it could evoke direct excitatory effects.

Methods

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

Cell Isolation

All procedures were carried out in accordance with European Union legislation and ethical standards and approved by Dundalk Institute of Technology Animal Care and Use Committee. Male New Zealand White rabbits (16–20 weeks old) were euthanized with intravenous pentobarbitone (120 mg/kg), and the penises were removed. The tunica albuginea was opened bilaterally to expose both corpora cavernosa, and these were removed, placed in Ca2+ free Hanks solution and cut into 1 mm3 pieces with fine scissors. The pieces were then incubated in an enzyme medium containing (per 5 mL of Hanks Ca2+-free solution): collagenase 15 mg (type Ia), protease 1 mg (type XXIV), bovine serum albumin 10 mg, and trypsin inhibitor 10 mg for 5–10 minutes at 37°C. All of these chemicals were supplied by Sigma-Aldrich (Arklow, Ireland). The tissue pieces were then placed in Ca2+-free Hanks solution and stirred for a further 5–10 minutes to release viable smooth muscle cells as demonstrated previously [18]. The cell suspension was stored at 4°C for use within 8 hours.

Patch Clamp Recordings

Patch clamp recordings were made as described previously [18,19] using the amphotericin B perforated patch method, where electrical access between the pipette and cell interior is achieved by inclusion of the pore forming compound amphotericin B (600 μg/mL) in the pipette solution [20]. Voltage clamp commands were delivered via an Axopatch 1D patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) connected to a Digidata 1322A AD/DA converter (Molecular Devices) interfaced to a computer running pClamp software (Molecular Devices). Drugs were delivered via a pipette (tip diameter 200 μm) placed close to the cell. All experiments were carried out at 35–37°C.

For most experiments, the calculated equilibrium potential for Cl (ECl) was set to 0 mV (symmetrical Cl) by using solutions (2) & (3) below, but in some experiments, this was adjusted to −40 mV by using solutions (2) and (4).

Calcium Imaging

Intracellular Ca2+ was imaged as described previously [19]. Cells were plated and incubated in 0.4 μM fluo-4AM (Molecular Probes, Life Technologies, Grand Island, NY, USA) in “Hanks Ca2+-free solution” (see below) but with added 100 μM Ca2+ for 6–8 minutes at 20°C. They were then continuously perfused in physiological saline (solution 2, below) containing 1.8 mM Ca2+ and imaged using an iXon 887 EMCCD camera (Andor Technology, Belfast, UK) coupled to a CSU22 spinning disk confocal head (Yokogawa, Tokyo, Japan) and Nikon TE200-U inverted microscope with a ×60 oil immersion lens (Nikon Corporation, Tokyo, Japan). A krypton-argon laser (Melles Griot, Cambridge, UK) excited the fluo-4 at 488 nm, and emitted light was detected at wavelengths >510 nm. Images were acquired at 15 frames/s (fps) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Plots of the fluorescence (F) were normalized by dividing by F0, defined as the minimum fluorescence measured in the control period just before addition of ATP. Thus, the resting Ca2+, expressed as F/F0, had a value of unity. For display purposes in Figures 8, two-dimensional images of the movie were created by drawing a line down the long axis of the cell and invoking the “reslice” command in ImageJ. This command displays the pixel intensity along a series of lines, each of which corresponds to a single frame of the movie stack. These are then aligned vertically left to right so that the vertical direction (y) corresponds to distance along the cell, the horizontal direction corresponds to time (t), and the intensity (brightness) corresponds to the Ca2+ level. The intensity was pseudocoloured so that “hotter” colors correspond to increased Ca2+ levels.

Statistics and Analysis

Data were analyzed using Prism (GraphPad Software Inc., La Jolla, CA, USA) and are presented as the mean ± standard error of the mean. Statistical comparisons were made using Student's paired t-test (2-tailed) on paired data, or by repeated measures analysis of variance followed by the Bonferroni post hoc test for multiple comparisons, where three columns were compared (i.e., where wash out experiments were performed). In each case, P < 0.05 was regarded as significant. Changes in cytosolic Ca2+ were compared using the Wilcoxon signed-rank test, to test if F/F0 differed from the theoretical value of unity. Throughout, N refers to the number of cells in each experimental series. N was normally derived from ≥3 animals.

Solutions

The following solutions were made freshly each day from chemicals supplied by Fisher Scientific (Dublin, Ireland), unless stated otherwise. Concentrations are stated in mM: solution 1: Hanks Ca2+-free (cell dispersal): 141 Na+, 5.8 K+, 130 Cl, 15.5 HCO3, 0.34 HPO42−, 0.44 H2PO4, 10 dextrose, 2.9 sucrose, 10 HEPES (Melford) (pH adjusted to 7.4 with NaOH); physiological saline (in bath and drug delivery system); solution 2: 130 Na+, 5.8 K+, 135 Cl, 4.16 HCO3, 0.34 HPO32−, 0.44 H2PO4, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42−, 10 dextrose, 2.9 sucrose, 10 HEPES (pH adjusted to 7.4 with NaOH); solution 3: patch pipette solution (for ECl = 0 mV): 133 Cs+, 1 Mg2+, 135 Cl, 0.5 EGTA (Sigma), 10 HEPES (pH adjusted to 7.2 with CsOH); and solution 4: pipette solution in patch pipette (for ECl = −40 mV): Cs +133 mM, 1 Mg2+, 30 Cl, 105 aspartate, 0.5 EGTA, 10 HEPES (pH adjusted to 7.2 with CsOH).

In all of the electrophysiological experiments described in Figures 1-6, the bath solution was solution 2, and the pipette solution was solution 3. With this combination, ECl was 0 mV. In experiments presented in Figure 7B–D, solution 4 was used as the pipette solution so as to adjust ECl to −40 mV, when used with the bath solution (solution 2). In experiments described in Figure 7, where extracellular Na+ was reduced from 130 to 13 mM, this was achieved by equimolar substitution of NaCl with N-methyl-d-glucamine in solution 2 and pH adjusted with HCl. In experiments where Ca2+ was recorded, the bath solution was solution 2. In these experiments, the cells were not dialysed with pipette solution.

figure

Figure 1. Effect of ATP on activity of corpus cavernosum smooth muscle cells under voltage clamp. (A) A cell producing spontaneous inward Cl currents responded to a 20-second application of ATP by producing a large transient inward current lasting 4 seconds. (B) A minority of cells (16%) treated as in (A) produced multiple currents in response to ATP. (C) Time-dependent control showing that the effect of ATP (20 μM) was repeatable. (D) Summary of the effect of ATP (20 μM) applied on two occasions 3–5 minutes apart. The two responses were not significantly different (P = 0.77, N = 9).

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Figure 2. Effect of suramin on the response to ATP. (A) ATP (20 μM) evoked a large transient inward current. A second application of ATP in the presence of suramin (100 μM) failed to evoke any current distinguishable from the spontaneous Cl currents. The response to ATP was completely restored following wash out of suramin. (B) Summary of the effect of suramin on the response to ATP (N = 8, *P < 0.05).

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Figure 3. Effect of MRS2500 on the response to ATP. (A) ATP (20 μM) evoked a large transient inward current. A second application of ATP in the presence of MRS2500 (100 nM) evoked a similar response. (B) Summary of the effect of MRS2500 on the response to ATP. The two responses were not significantly different (N = 13, P = 0.84).

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Figure 4. Effect of MRS2500 on the response to 2-MeSADP. (A) 2-MeSADP (1 μM) evoked a large transient inward current similar to that evoked by ATP (see Figures 1-3). In contrast to the effect of ATP, the response to 2-MeSADP was greatly attenuated by MRS2500 (100 nM). (B) Summary of the effect of MRS2500 on the response to 2-MeSADP (N = 6, P < 0.05).

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Figure 5. Effect of α,β-methylene ATP on the response to ATP. (A) ATP (20 μM) evoked a large transient inward current. After several minutes, α,β-methylene ATP (1 μM) was applied. This also evoked a transient inward current greater in amplitude than the spontaneous Cl currents. In the continued presence of α,β-methylene ATP, a second application of ATP failed to evoke any current. On wash out of α,β-methylene ATP, the response to ATP was fully restored. (B) Summary of the effect of α,β-methylene ATP on the response to ATP. (N = 5, *P < 0.05, comparing ATP control with ATP in the presence of α,β-methylene ATP).

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Figure 6. Effect of NF449 on the response to ATP. (A) ATP (20 μM) evoked a large transient inward current. In the presence of NF449 (5 μM), a second application of ATP evoked little or no current, but the response returned fully on wash out of NF449. (B) Summary of the effect of NF449 on the response to ATP (N = 6, *P < 0.05).

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Figure 7. Effect of changing ionic gradients on the response to ATP. (A) The same conditions as in previous figures (holding potential = −60 mV, ECl = 0 mV, [Na]o = 130 mM), ATP (20 μM) evoked a large transient inward current. This was superficially similar to the current evoked by caffeine (10 mM). (B) When the transmembrane Cl gradient was changed so that ECl = −40 mV, and the cell held positive to this (−10 mV) the caffeine-evoked current became outward in direction, consistent with a Cl current, but the ATP-evoked current remained inward. (C) In the same cell shown in (B), the external Na+ concentration, [Na+], was reduced to 13 mM. This caused the ATP-evoked current to become outward. (D) Summary of the effect of changing [Na+] from 130 mM (left hand columns) to 13 mM (N = 4).

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Figure 8. The effect of ATP (20 μM) on intracellular Ca2+. (A) Upper panel shows Ca2+ events displayed as a distance (y) vs. time (t) plot derived from a movie (see Methods). Lower panel shows intensity vs. time plot of the changes in cytosolic [Ca2+] (averaged over the whole cell area) for the record in the upper panel. ATP was applied for 10 seconds as indicated by the blue bar. (B) Summary of the effect of ATP on cytosolic Ca2+. Basal Ca2+ was defined as the value during the quiescent period just before application of ATP (P < 0.05, N = 6). (C) Summary of the effect of ATP on cell length (*P < 0.05, N = 6).

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Drugs

Drugs were α,β-methylene ATP, ATP, suramin, (Sigma-Aldrich), MRS2500, 2-2-methylthio-ATP (MeSATP) and NF449 (Tocris Bioscience, Bristol, UK).

Results

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

When isolated corpus cavernosum smooth cells were held under voltage clamp at −60 mV, they often exhibited spontaneous Ca2+-activated Cl currents (IClCa, Figure 1A–C) as described previously [18,19]. In 84% of cells, a 20-second application of ATP (20 μM) evoked a single transient inward current that decayed despite the continued presence of ATP (Figure 1A). In 16% of cells, ATP evoked multiple currents (Figure 1B). The effect of ATP was also prone to desensitization; however, repeatable responses were obtained if >3 minutes was left between applications. As many of the following experiments involved giving repeated applications of ATP to compare the effects of ATP in the absence and presence of an antagonist, it was necessary to first establish the reproducibility of the response in a series of control experiments. An example of the effect of adding ATP on two occasions for 20 seconds, 4 minutes apart, is shown in Figure 1C, where it is clear that the effect was reproducible. This was confirmed in summary data from similar experiments where the first and second applications yielded mean contraction amplitudes of −675 ± 101 pA and −694 ± 120 pA, respectively (P = 0.77, N = 9, Figure 1D).

The effect of ATP was antagonized by suramin (100 μM), a P2 receptor blocker that does not select between P2X and P2Y receptor subtypes (Figure 2A). In contrast, suramin had no effect on spontaneous Cl currents. Figure 2A also shows that the antagonistic effect of suramin was completely reversible on wash out. Suramin reduced the ATP-induced inward current from −380 ± 121 pA to −124 ± 37 pA (P < 0.05, N = 8, Figure 2B) and the current returned to −393 ± 127 pA following wash out of suramin, which was not significantly different from control (P > 0.05).

The response to ATP was unaffected by MRS2500 (100 nM), a specific blocker of P2Y1 receptors (e.g., Figure 3A). This was confirmed in a total 13 paired experiments where no significant difference was found between applications of ATP alone, and in the presence of MRS2500 (mean response before MRS2500: −838 ± 139; mean response in presence of MRS2500: −822 ± 184, P = 0.84, N = 13, Figure 3B). However, MRS2500 effectively blocked responses to a P2Y agonist, 2-MeSADP (1 μM), although 2-MeSADP produced responses that were indistinguishable from ATP (Figure 4A, B). In Figure 4B, the mean response to 2-MeSADP was −623 ± 166 pA, whereas in the presence of MRS2500, this was reduced to −56 ± 25 pA (P < 0.05, N = 6). These experiments suggest that, although the corpus cavernosum myocytes possess P2Y1 receptors, the responses to ATP appear to be mediated mainly by other receptor subtypes.

To further explore the receptor subtype involved, the effect of the P2X receptor agonist, α,β-methylene ATP (1 μM) was examined. This compound characteristically stimulates P2X1 and P2X3 receptors before causing them to desensitize, rendering them temporarily unresponsive to other P2X agonists [6]. Figure 5A shows a record from a cell that was first exposed to ATP for 20 seconds, which evoked the usual large inward current. After a few minutes, α,β-methylene ATP was applied for several minutes, resulting in a transient inward current at the beginning of the application. ATP was then applied for a second time in the continued presence of α,β-methylene ATP but was without effect. When ATP was applied after wash out of both drugs, its response was restored. Mean data from five similar experiments is presented in Figure 5B. This shows that the response to ATP was reduced from a control value of −755 ± 235 pA to −78 ± 52 pA in the presence of α,β-methylene ATP (P < 0.05, N = 5). The ATP response returned to −748 ± 177 pA, following wash out of α,β-methylene ATP, which was not significantly different from control (P > 0.05).

Next, the effect of NF449, a powerful selective inhibitor of P2X1 receptors [21] was examined (Figure 6). Again, ATP (20 μM) evoked a large inward current when applied alone but after several minutes of exposure to NF449 (5 μM), repetition of the ATP application failed to evoke a current (Figure 6A). After wash out of NF449, ATP again evoked a robust response, similar to the control. In six cells, NF449 reversibly reduced the mean current from −419 ± 109 pA to −51 ± 13 pA (P < 0.05, N = 6) and the response returned to −351 ± 119 pA following wash out of NF449, which was not significantly different from control (P > 0.05).

The above results suggested that ATP could evoke inward currents in corpus cavernosum myocytes by acting on P2X receptors. As P2X receptors are known to be ligand-gated cation channels, it was of interest to identify the main ion species responsible for the inward current. As rabbit corpus cavernosum smooth muscle cells (CCSMC) have previously been shown to express an IClCa, and this can be activated as a result of intracellular Ca2+-release in response to stimulation by agonists such as phenylephrine or caffeine [18,19], it was important to distinguish between IClCa and any potential cation current (Icat) mediated by P2X receptors. Figure 7A shows a comparison of the effect of ATP and caffeine under the same conditions as all of experiments described so far (holding potential = −60 mV, ECl = 0 mV, extracellular [Na+] = 130 mM). Under these conditions we would expect both IClCa and Icat to be inward. Indeed, under these conditions, the effects of ATP and caffeine were similar (Figure 7A). However, when ECl was adjusted to −40 mV by reducing intracellular [Cl] (using solution 4, see Methods) and the holding potential changed so that it was now positive to ECl (holding potential = −10 mV), the caffeine-evoked current shifted to the outward direction, consistent with Cl ions as the charge carrier (Figure 7B). Under these altered conditions, the ATP-evoked current remained inward, demonstrating that it was predominantly carried by a different ionic species to that evoked by caffeine (Figure 7B). Figure 7C demonstrates that the main ionic species involved in the ATP-response was Na+, as when the external [Na+] was reduced from 130 to 13 mM; this current also shifted to the outward direction (Figure 7B, C, from same cell). Figure 7D summarizes the results of four cells treated as in Figure 7B, C, where the conditions were initially set so as to discriminate between the ATP- and caffeine-evoked currents (ECl = −40, holding potential = −10 mV, [Na]o = 130 mM), followed by reduction of [Na]o to 13 mM. Again, reduction in [Na]o resulted in a reversal of the ATP-evoked current, consistent with Na+ acting as the main charge carrier.

Calcium Imaging

We also examined the effect of ATP on cytosolic Ca2+ and cell length in freshly isolated CCSMC. Figure 8A shows a record, derived from a movie of a cell, where spontaneous Ca2+ waves are displayed as a distance (y) vs. time (t) plot (on this time scale, each wave appears as an almost vertical bright line). The lower panel of Figure 8A shows a plot of the changes in cytosolic [Ca2+], averaged over the whole cell area, with respect to time. ATP (20 μM) initially increased basal Ca2+ (measured as F/F0, see Methods) from the basal value of 1.0–3.29. This was followed by several oscillations, before the Ca2+ dipped below the previous basal level following wash out of ATP. In the upper panel of Figure 8A, it is also clear that the cell contracted during application of ATP (as evidenced by the decrease in the width of the bright area). Figure 8B shows a summary of the effect of ATP on cytosolic Ca2+ in six cells, where F/F0 increased (from unity) to 2.44 ± 0.36 (P < 0.05). Figure 8C shows the effect of ATP on mean cell length, which decreased from 40.97 ± 4.87 μm to 35 ± 5.11 μm (P < 0.05, N = 6).

Discussion

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

In the present study, we have provided the first functional evidence that ATP induces an inward cationic current in corpus cavernosum myocytes. Ion substitution experiments showed that this is distinct from the inward current mediated by IClCa and therefore different from the spontaneous inward currents generated by these cells [17,18] and from the previously reported inward currents generated by phenylephrine [19]. As P2X receptors are known to be ligand-gated cation channels [6], it was highly likely that the cation current described here was mediated by P2X receptors. This was confirmed by the fact that desensitization to α,β-methylene ATP abolished the ATP response. The fact that NF449 reduced the ATP response suggests that P2X1 receptors contributed to the response, although other receptor subtypes cannot be excluded. As the inward current resulting from stimulation of this pathway would be expected to depolarize the smooth muscle cells and open voltage-gated Ca2+ channels [22], this mechanism would be expected to result in contraction of the corpus cavernosum and push the balance toward penile detumescence.

Interestingly, despite its usual relaxant effect [11], ATP and closely related purinergic agonists have also been reported to contract the corpus cavernosum in cats, horses, rabbits, and humans [9,12,13]. Also, several older studies reported that ATP caused contraction of the closely related retractor penis muscle in several species including bull, dog, cat, pig, and ram [9,10]. These variable effects of ATP may be explained by the complexity associated with the action of ATP and its metabolites. For example, ATP may itself act as an agonist, or it may be broken down by the action of ectonucleotidases into a series of metabolites, including ADP and adenosine [2,11]. Purinergic receptors are classified as either P1 receptors, further subdivided into A1, A2A, A2B, and A3, where the main ligand is adenosine [11] and P2 receptors, further subdivided into P2X (P2X1–5,7) [6] and P2Y (P2Y1,2,4,6,11,12,13,14) [7], where ATP, ADP, uridine triphosphate, and uridine diphosphate may be the preferred agonists, depending on receptor subtype. The picture is complicated further by overlapping specificity of the currently available agonists and antagonists that act on these receptors. In addition, the receptors may be expressed on a variety of cell types within the tissue, including smooth muscle cells, endothelial cells, interstitial cells of Cajal, and on the nerve terminals, increasing the potential to influence the state of contraction of the corpus cavernosum in many different ways. Finally, species differences in the pharmacological profiles and distribution of the receptors may also add to the variability of the responses [23–25].

Thus, the relaxations commonly observed in response to exogenous ATP may not be due to direct effects of ATP acting on cavernosum smooth muscle cells but by its actions, or actions of its metabolites, on other cell types. A large part of the relaxant effect appears to be mediated by adenosine acting on either A2A or A2B receptors [11], or of ATP or ADP acting on via P2Y receptors [26,27]. The relaxations appear to be partly mediated by release of nitric oxide (or other mediators) from the endothelium [12,27–30], although NO-independent relaxations have also been reported [26,29,31,32]. Finally, adenosine has also been shown to act on presynaptic A1 receptors on adrenergic neurons to reduce output of norepinephrine, thus facilitating erection [33].

In contrast, the contractile effect of ATP appears to be mediated by P2X receptors in humans and rats [12,16]. In humans, although P2Y agonists produced endothelium-dependent relaxations, robust contractions occurred in response to stimulation with the P2X agonist, α,β-methylene ATP [12]. Several recent studies suggest that such antierectile effects of P2X1 stimulation may be important in pathological conditions or age-related changes that affect erectile function [15,16]. Gur et al. [16] used immunohistochemistry to show that P2X1 receptor expression on corpus cavernosum smooth muscles cells greatly increased in diabetic rats compared with normal controls. This appeared to have a functional consequence, as relaxations in response to nerve stimulation were reduced in diabetic animals but these were restored to control levels by PPADS, a P2X antagonist, suggesting that excessive P2X receptor stimulation contributed to the reduced erectile function seen in diabetic animals [16]. Another study, using Western blotting, reported that an increase in P2X1 expression in the corpora cavernosa of diabetic rats was negatively correlated to erectile capacity [15].

So far, there have been few studies that have investigated the distribution of purinoceptor subtypes in corpus cavernosum. In situ hybridization was used to demonstrate the presence of mRNA encoding P2Y1 receptors in the endothelial cells surrounding the sinusoids in rat corpus cavernosum [34]. Interestingly, it was reported that the trabecular smooth muscle cells did not express these receptors [34]. However, this was in contrast to the study by Gur et al. [16], who did appear to find P2Y1 receptors on both smooth muscle cells and endothelium of rat corpus cavernosum. Furthermore, these authors also found evidence of P2X1 receptors on both cell types. Expression of P2X1 receptors, as well as P2X2, on the corpus cavernosum smooth muscle cells was also reported in an earlier study [14].

Our results show that MRS2500, a selective P2Y1 antagonist had little or no effect on the response to ATP; however, 2-MeSADP, a P2Y1,12,13 agonist, evoked large inward currents that were blocked by MRS2500, seeming to confirm the finding of Gur et al. [16] that P2Y1 receptors are present on the smooth muscle cells. In the light of this finding and, given that ATP has been widely reported as a full agonist at P2Y1 receptors (see Palmer et al. [35]), it may seem strange that almost none of the ATP-induced effects in the present study were attributable to stimulation of P2Y1 receptors. However, carefully controlled studies have shown that when highly purified ATP is used, and breakdown to ADP is prevented, that ATP is only a partial agonist at best at the P2Y1 receptor, and it may even act as a competitive inhibitor in the micromolar range [35–37]. The endogenous agonist for the P2Y1 receptor is ADP, whereas 2-MeSADP stimulates the receptor with a higher potency. Using P2Y1 receptor-expressing 1321N1 cells, it was shown that stimulation with ATP could only produce a physiological effect if there was a high receptor reserve, whereas ADP could produce a response even when the number of functional receptors was low [35]. Thus, in the present study, a low level of expression of P2Y1 receptors on corpus cavernosum myocytes would explain the fact that 2-MeSADP, but not ATP, evoked a response susceptible to blockade with MRS2500.

It is highly likely that the P2Y-mediated current evoked by 2-MeSADP is mediated by the IClCa, as P2Y1 receptors are coupled to Gq/11 G proteins that activate phospholipase C, leading to production of inositol trisphosphate (IP3). It has previously been shown that phenylephrine, which stimulates the same pathway, via Gq/11-coupled α-adrenoceptors, activates the IClCa, by IP3-mediated Ca2+ release [17,19]. Direct stimulation of the smooth muscle cells by this pathway would be expected to be excitatory and antierectile, causing contraction. As P2Y1 receptors are also expressed on the endothelium, causing release of NO and relaxation (see above), the overall physiological effect of ADP might depend on where in the tissue it was being released or produced. It is interesting to note that a P2Y agonist caused relaxation intact but caused contraction in endothelium-denuded, human corpus cavernosum [12]. However, this contraction amounted to only to 17% of that evoked by α,β-methylene ATP, consistent with the idea that the expression levels of P2Y receptors on the smooth muscle cells are much less than those of P2X receptors.

Conclusions

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

In conclusion, the main findings presented here are that in rabbit corpus cavernosum myocytes, ATP activates a P2X-receptor mediated cation current, as well as increasing cytosolic Ca2+. These effects are pro-contractile but at present it is not clear whether they have a physiological role in contributing to penile detumescence under normal circumstances. However, the mechanism described here is likely to explain the antierectile effects of P2X receptors that have been reported in pathological conditions such as diabetes [15,16]. Thus, taken together with immunohistochemical data in these studies, we believe that we have provided further evidence for a previously neglected mechanism that may be detrimental to erection under certain pathophysiological conditions, thus raising the possibility that P2X receptors are drug targets for treating erectile dysfunction.

Further studies are required to establish the extent of these effects in animal models of disease and to evaluate their role in human patients with erectile dysfunction. For example, it will be interesting to determine if there are enhanced responses to ATP in corpus cavernosum smooth muscle cells derived from animal models for diabetes and hypertension.

Acknowledgments

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

The authors gratefully acknowledge grant funding from Science Foundation Ireland (07/RFP/BIMF377) and Enterprise Ireland (ARE20080001 and CE20080020) and technical assistance from Mrs. Billie McIlveen.

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

Statement of Authorship

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

Category 1

  • (a)
    Conception and Design
    Keith D. Thornbury; Gerard P. Sergeant; Mark A. Hollywood; Noel G. McHale; Claire Doyle
  • (b)
    Acquisition of Data
    Claire Doyle; Gerard P. Sergeant
  • (c)
    Analysis and Interpretation of Data
    Keith D. Thornbury; Gerard P. Sergeant; Claire Doyle

Category 2

  • (a)
    Drafting the Article
    Keith D. Thornbury
  • (b)
    Revising It for Intellectual Content
    Keith D. Thornbury; Gerard P. Sergeant; Mark A. Hollywood; Noel G. McHale

Category 3

  • (a)
    Final Approval of the Completed Article
    Keith D. Thornbury; Gerard P. Sergeant; Mark A. Hollywood; Noel G. McHale

References

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