Endothelial and neuronal-derived nitric oxide mediated relaxation of corpus cavernosal smooth muscle in a rat, in vitro, model of erectile function

(2000) 12, 213±221 ß 2000 Macmillan Publishers Ltd All rights reserved 0955-9930/00 $15.00 www.nature.com/ijir Endothelial and neuronal-derived nitric oxide mediated relaxation of corpus cavernosal smooth muscle in a rat, in vitro, model of erectile function JJ Cartledge 1 *, S Minhas 1, I Eardley 1 and JFB Morrison 2 1 Pyrah Department of Urology, St James's University Hospital, Leeds, UK; and 2 Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates We set out to establish a simple, reproducible, rat in vitro model of erectile function and to use this to demonstrate the functional importance of both neuronal- and endothelial-derived nitric oxide within this animal. Two corpora cavernosal smooth muscle strips were harvested from sexually mature male Wistar rats and mounted in an organ bath for measurement of isometric tension. Following contraction with noradrenaline the strips were relaxed by the addition of either acetylcholine or sodium nitroprusside. Electrical eld stimulation was performed in the presence of atropine and guanethidine. Relaxation responses were repeated in the presence of methylene blue, L-arginine, L-NNA and haemoglobin L-arginine. Methylene blue abolishes the relaxation to acetylcholine and EFS; L-NNA and haemoglobin cause a signi cant impairment in the relaxation response. L-arginine reverses the effect of haemoglobin. In conclusion, the inhibitory, relaxant stimulus of rat corpora cavernosa is due to both neuronal nitric oxide and endothelial-derived nitric oxide released in response to cholinergic stimulation. (2000) 12, 213±221. Keywords: penile erection; nitric oxide; enos; nnos; rat; haemoglobin; Introduction Penile erection is a dynamic process mediated by relaxation of smooth muscle within the trabeculae of the corpora cavernosa and the arterial vessels of the penis. 1 A carefully controlled balance exists between relaxant, pro-erectile, factors and contractile, anti-erectile factors, which will determine the functional state of the penis. 2 It is generally accepted that nitric oxide (NO) is the principal agent responsible for smooth muscle relaxation in the penis. 3±6 NO, generated from L-arginine by the enzyme nitric oxide synthase (NOS), may be released from endothelium in response to cholinergic stimulation 7 or directly from nonadrenergic noncholinergic (NANC) nerve terminals. 4 Following its release, NO stimulates the intracellular guanylate cyclase second messenger system to bring about smooth muscle relaxation. 8 *Correspondence: J Cartledge, Specialist Registrar in Urology, Pyrah Department of Urology, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK. E-mail: j.cartledge@ukgateway.net Received 20 October 1999; accepted 9 May 2000 Whether neuronal- or endothelial-derived NO is most important in penile smooth muscle relaxation is unclear, and may vary depending on species. Immunoreactivity and functional studies have clearly shown the presence and importance of NOS in nerve bres innervating the corpora cavernosa in man, 9±11 dogs, 12 rabbits 13,14 and rats. 15 ± 17 Tissue from mice bred genetically de cient in neuronal NOS (nnos) over-expresses endothelial NOS (enos) and is still able to relax normally in response to acetylcholine. 18 Other groups of mice, genetically enos negative are able to breed and reproduce normally, although functional data on corpora cavernosal tissue from these animals is not available to con rm normal erectile function. 19 The rat is a widely used laboratory animal that is inexpensive to breed and house. There is very limited data on the use of the rat as an in vitro model of erectile function. Initial descriptions of two different corpora cavernosal in vitro models have not been followed up by the authors that developed them, 20,21 although other groups have adapted their techniques. 22 ± 25 There is controversy over the role of enos in the rat and whether it is a good model of human corpora cavernosal function. Certainly nnos is important in the rat since it has both been identi ed immunologically, 15 ± 17 and

214 con rmed to be functionally relevant. 26 The role of enos, however, is unclear in the rat. Dail et al were unable to demonstrate the presence of enos in the sinusoidal endothelium of the corpus cavernosa immunologically, 16 although it was seen in the endothelium of arteries within the cavernosa. There is only limited functional data to support its presence. We set up this study with two de nite aims, rstly to develop a simple, reproducible, in vitro model of rat corpus cavernosal function. Secondly, we intended to clarify the role of NO in the rat penis and demonstrate if corpora cavernosal smooth muscle relaxation in this animal is due only to neuronal-derived NO or whether there is in fact a contribution from endothelial-derived NO. Methods Animals Sexually mature male Wistar albino rats were used in all experiments. All animals were maintained in a 12 h light=dark cycle with ambient temperature regulation at 20 ± 22 C. Tissue preparation Strips of corpora cavernosa were used in all experiments. Animals were killed by cervical dislocation and the penes rapidly dissected and placed in Krebs solution at 4 C. To excise the penis the rat was placed supine and the skin incised over the dorsal surface of the penis. Penile skin was stripped and the incision continued cranial to the abdomen, and caudal to the anus. Skin was re ected laterally to expose the insertion of the abdominal muscles onto the symphisis pubis. Tissue caudal to the symphisis pubis was excised to expose the midline dorsal venous complex and the lateral crura of the penis. Incision of the vessels, crura and ventral urethra allowed removal of the whole penis. The whole penis was pinned out in a Petri dish under Krebs solution chilled to 4 Cand bubbled with 95% O 2 =5% CO 2, which maintained ph 7.4. With the aid of a 12 6 binocular dissecting microscope, loose connective tissue and adventitia were removed. All skeletal muscle was removed from the proximal crura to expose the corpus spongiosum surrounding the urethra. The corpus spongiosum was re ected off the underlying tunica albuginea in a proximal to distal direction before division at the base of the glans penis. The penis was then turned over to expose the dorsal venous complex, which was also excised. Sharp incision of the thick tunica albuginea of the crus penis exposed the underlying corpora cavernosa. A strip of corpus cavernosum was dissected by separating it from the thick, midline, tunica albuginea, leaving the lateral, thin, tunica intact and adherent to the corpus. This process was repeated on the contralateral side to produce two identical strips, approximately 3 6 3 6 12 mm. One end of the corpus was tied to a loose length of 4 ± 0 silk (Davis & Geck, USA), the second attached to a glass rod, fashioned to form a hook at each end, using 4 ± 0 silk. In vitro preparation For all in vitro experiments, the strips of corpora cavernosa were attached between a xed point by the loose silk suture and an isometric force transducer by a hooked glass rod. The strip was suspended in an organ bath between parallel platinum electrodes which allowed it to be immersed in Krebs solution gassed with 95% O 2 =5% CO 2, ph 7.4. A temperature of 37 C was maintained in the organ bath by circulating water through a surrounding jacket. Water was also circulated in a closed system to ensure that stock Krebs solution was warmed to 37 C. To maintain oxygenation stock, Krebs solution was also gassed with 95% O 2 =5% CO 2. Krebs solution within the organ bath was exchanged completely every 15 min during periods of equilibration. Whenever washout of the organ bath was performed, this procedure drew from the stock reservoir of warmed, aerated Krebs solution. The signal from the force transducer was passed trough a bridge ampli er (Fylde 492BBs, Fylde Electronic Laboratories Ltd, Preston, UK) and a 50 Hz lter to a pen recorder (Lectromed MX216, Lectromed (UK) Ltd, Letchworth, UK). At the beginning of each experiment the bridge ampli er was balanced and the pen recorder was calibrated so that active tension between 0 and 500 mg could be recorded following the application of optimal resting tension. Optimal resting tension Once tissue strips were mounted in vitro a xed resting tension had to be applied prior to measurement of any active tension generated in response to the application of stimuli. Initially strips were contracted by the addition of 10 76 M noradrenaline (NAd) following the application of 125 ± 175 mg resting tension. This value was chosen from published data. 20,21 In our experience, application of this range of resting tension resulted in inconsistent contractions.

Optimal resting tension, for the strips prepared by the dissection techniques outlined above; was determined as follows. Strips were mounted in the organ bath as described and allowed to equilibrate for 90 min at a resting tension within the range 125 mg to 2 g before application of 1 6 10 76 M NAd. The magnitude of contraction (active tension) was recorded for each incremental resting tension. Between contractions at different resting tension the strips were allowed to relax following washing in at least three changes of Krebs solution. The next incremental resting tension was applied and, prior to contraction with 1 6 10 76 M NAd, strips were allowed to equilibrate for 30 min. The peak of the best t curve, 1.6 g, the mean of 14 experiments, was considered optimal resting tension and was applied to tissue strips in all subsequent experiments. EC50 of noradrenaline To calculate the EC50 for NAd tissue strips were mounted in the organ bath, as described, and allowed to equilibrate for 90 min at optimal resting tension. Noradrenaline was added incrementally, at concentrations between 1 6 10 79 and 1 6 10 73 M, and the active tension generated at each dose recorded. The maximum active tension generated for each strip was considered 100% contraction, the percentage response to each dose of NAd was calculated and the mean results from 10 experiments plotted (Figure 1). The dose of NAd responsible for causing 50% of maximal contraction was calculated from the curve at 8.5 6 10 77 M, which for all further experiments was approximated to 1 6 10 76 M NAd. Figure 1 EC50 of Noradrenaline. Active tension (y) is calculated as the percentage of maximal contraction to a range of doses of NAd (x). The mean s.e.m. response from 10 experiments is shown. The drop line indicates the dose of NAd required to cause 50% maximum contraction. Endothelial nitric oxide Corpora cavernosal muscle strips from 12-week-old male Wistar rats were mounted in vitro as described above for measurement of isometric tension. After equilibration at optimal resting tension of 1.6 g for 90 min, strips were contracted by the addition of 1 6 10 76 M NAd. Following the establishment of a stable contractile response the strips were washed in at least three exchanges of Krebs solution and allowed to re-equilibrate at optimal resting tension for 30 mins. Lo-nitro-L-arginine (L-NNA; 1 6 10 74 M) or L- arginine (1 6 10 74 M) was added to the organ bath and after 15 min incubation; contraction was induced by the addition of 1 6 10 76 M NAd. After a steady-state contraction was achieved, acetylcholine (ACh) was added incrementally in the range 1 6 10 79 to 1 6 10 73 M. At least 3 min were allowed between doses of ACh to establish a stable response at each concentration. In other experiments, after a steady contractile response to 1 6 10 76 M NAd was established, 100 ml human haemoglobin with and without L-arginine (1 6 10 74 M) were added to the organ bath before the construction of relaxation responses to ACh. Neuronal nitric oxide Following 90 min of equilibration at optimal resting tension, corpora cavernosa tissue strips were contracted with 1 6 10 76 M NAd. When a stable contraction was achieved, strips were washed in at least three exchanges of Krebs solution and allowed to re-equilibrate for 30 min at optimal resting tension prior to contraction with 1 6 10 76 M NAd; guanethidine (5 6 10 76 M) and atropine (1 6 10 76 M) were then added to the organ bath. After at least 15 min, electrical eld stimulation (EFS) was performed as follows. A train of square wave pulses (band width 0.5 ms, intensity 20 V) were generated at a range of frequencies using a dual channel electrical stimulator (Palmer 8173, Searle Instruments, Harlow, UK). The impulse was applied across parallel platinum electrodes initially at a frequency of 2 Hz for 10 s. Strips were allowed to recover before application of the next train of impulses. This process was performed for each of the following pulse frequencies: 2, 5, 10, 20 and 50 Hz. After measurement of one series of responses to EFS, L-NNA (1 6 10 74 M), L-arginine (1 6 10 74 M) or haemoglobin (100 ml) was added to the organ bath and, after 15 min incubation, the cycle of EFS was repeated. In some experimental strips tetrodotoxin (1 6 10 76 M) was added to the organ bath between the rst and second cycle of EFS. 215

216 In addition to the responses recorded secondary to the addition of ACh and EFS, baseline studies were also performed using sodium nitroprusside (SNP). Following equilibration at optimal resting tension and contraction with 1 6 10 76 M NAd, incremental doses of SNP were added to the organ bath in the dose range 1 6 10 79 to 1 6 10 73 M. In other experiments methylene blue (1 6 10 74 M) was added to the organ bath prior to the construction of relaxation responses secondary to ACh or EFS. The response of tissue strips under all experimental conditions was calculated in the same way. Relaxation response ˆ maximum contraction relaxation to dose applied maximum contraction 100 A response to any agent which relaxes tissue to below the applied optimal resting tension will result in a greater than 100% relaxation response. Chemicals and solutions The constituents for the Krebs solution (NaCl, 118 mm; KCl, 4.5 mm; KH 2 PO 4, 1 mm; NaHCO 3, 25 mm; glucose, 6 mm; CaCl, 2.5 mm; MgSO 4, 1 mm) were purchased from Merck, Lutterworth, Leicestershire, UK. All other chemicals were purchased from Sigma-Aldrich, Poole, Dorset, UK. Noradrenaline bitartrate (Arterenol) was mixed in distilled water with 1 mm ascorbic acid to act as preservative. Lo- Nitro-L-arginine (L-NNA) was initially dissolved in 1 M HCl before further dilution in distilled water. Indomethacin was dissolved in NaHCO 2. Acetylcholine chloride, guanethidine sulphate, atropine sulphate, L-arginine hydrochloride, human haemoglobin and methylene blue were all mixed in distilled water. Statistical analysis Comparison of the response of a single tissue sample under different conditions to a range of doses of ACh, SNP or EFS was performed using a two-way analysis of variance (ANOVA). All statistical analysis was performed on a personal computer using SigmaStat 1 2.0 (Jandel Corporation, USA). Graphical representation of results was compiled using SigmaPlot 1 5.0 (SPSS Inc., USA). A signi cance level was applied at P < 0.05 for all analyses Results Sexually mature male Wistar albino rats were used in all experiments. A summary of the essential characteristics of these animals is given in Table 1. In all instances the number of tissue strips used to provide data is given. The number of strips for each experiment is given in Tables 2 and 3. Harvesting of corpora cavernosa tissue strips from young, 12-week-old rats yielded strips with a mean weight of 57.2 mg that developed a mean active contractile force of 339.4 mg in response to 1 6 10 76 M NAd. Once mounted in the organ bath at optimal resting tension, following contraction with 1 6 10 76 M NAd, strips relaxed in response to the addition of ACh (Figure 2), SNP (Figure 3) or EFS Table 1 12 weeks Weight Animal 341.4 14.5 g Tissue strip 57.2 1.5 mg Serum Sodium 141.8 0.9 mmol=l Potassium 11.8 0.1 mmol=l Urea 7.8 0.1 mmol=l Creatinine 75 2.2 mmol=l Triglyceride 1.95 0.3 mmol=l Cholesterol 1.6 0.03 mmol=l HbA 1c 4.0 0.01 Table 2 The response of tissue strips to ACh ACh (mol) Control Control L-arginine Control L-NNA Haemoglobin Hb L-arginine 10 79 7 2.0 1.0% 0 7 0.16 0.16% 7 3.29 3.29% 7 2.5 2.5% 10 78 7 10.7 2.6% 7 4.8 3.2% 7 1.4 1.0% 7 6.21 3.38% 7 16.2 8.7% 10 77 7 27.3 4.6% 7 19.3 9.2% 7 12.4 4.0% 7 23.5 5.46% 7 30.6 14% 10 76 7 48.9 4.6% 7 43.3 11.2% 7 27.1 7.6% 7 24.9 9.93% 7 37.6 14% 10 75 7 65.3 5.2% 7 76.7 12.0% 7 37.6 9.7% 7 38.5 4.34% 7 65.5 12.8% 10 74 7 95.5 7.0% 7 98.0 15.8% 7 56.1 9.6% 7 49.7 6.92% 7 91.9 13.9% 10 73 7 115 5.6% 7 104.7 15.5% 7 75.5 13.1% 7 57.6 4.12% 7 112.4 11.8% Overall 7 52.11% 7 49.5% 7 30.%* 7 24.35%* 7 40.72% n ˆ 24 n ˆ 8 n ˆ 8 n ˆ 7 n ˆ 8 *Signi cant difference between the overall response of that tissue strip and control tissue strips, P < 0.05.

Table 3 The response of tissue strips to EFS 217 EFS (Hz) Control Control L-arginine Control L-NNA Haemoglobin Hb L-arginine 2 7 4.74 2.01% 7 1.9 1.9% 0 7 1.43 1.43% 0 5 7 22.19 4.84% 7 24.7 9.2% 0 7 4.76 2.14% 7 10.9 3.2% 10 7 39.46 3.92% 7 40.2 9.7% 7 1.6 1.1% 7 9.05 2.67% 7 38.9 7.1% 20 7 48.2 3.85% 7 49.3 11.7% 7 1.8 1.2% 7 14.13 3.26% 7 51.2 9.1% 50 7 47.14 1.93% 7 48.9 11.5% 7 4.7 2.0% 7 17.79 4.73% 7 52.6 9.2% Overall 7 32.4% 7 33.0% 7 1.6%* 7 9.43%* 7 30.72% n ˆ 7 n ˆ 8 n ˆ 8 n ˆ 7 n ˆ 7 *Signi cant difference between the overall response of that tissue strip and control tissue strips, P < 0.05. (Figure 4). All results are represented as the mean standard error of the mean for the indicated number of tissue strips. Endothelial nitric oxide The overall mean relaxation response of control tissue strips to ACh was recorded at 52.11%. Incubation of control tissue strips with the substrate for NO synthesis, L-arginine, did not signi cantly alter this response, with an overall mean relaxation response of 49.45%. If the control strips were preincubated with an inhibitor of NOS, a signi cant reduction in the relaxant response to ACh is seen (Figure 2). In the presence of L-NNA the overall mean relaxation response is recorded as 30%, an overall reduction of 42.5% (P < 0.05). Haemoglobin (100 ml) caused an overall 49% reduction in mean relaxation response over control (P < 0.05) which was completely reversed by the addition of 1 6 10 74 M L-arginine (Figure 5). In the presence of 100 mm methylene blue, no relaxation response to the application of ACh was recorded. The response to individual doses of ACh are given in Table 2. Neuronal nitric oxide Figure 2 The mean s.e.m. relaxation of control corpus cavernosal tissue strips to ACh is unaffected by the addition of L- arginine. The addition of L-NNA causes a signi cant impairment in the relaxant repsonse, *P < 0.05. n ˆ 8. Incubation of control tissue strips with L-arginine prior to construction of relaxation responses to EFS had no signi cant effect. The overall mean relaxation response in the presence of L-arginine was 32.99% and 32.34% for control tissue alone. The addition of L-NNA to the organ bath led to a signi cant reduction in the relaxant response of Figure 3 Mean s.e.m. relaxation responses of control strips to SNP. Strips from 12 week control animals relaxed in a dose dependent manner to the application of exogenous SNP. n ˆ 7. Figure 4 The mean s.e.m. relaxation responses of control corpus cavernosal tissue strips to EFS is unaffected by the addition of L-arginine. The addition of L-NNA causes a signi cant impairment in the relaxant response, *P < 0.05. n ˆ 8.

218 Discussion Figure 5 The effect of L-arginine in addition to haemoglobin on ACh mediated relaxation. The addition to L-arginine to control strips incubated with haemoglobin reverses the impaired relaxation seen following the incubation of control strips with haemoglobin alone. This effect reached statistical signi cance, *P < 0.05. n ˆ 8. 95% overall (Figure 4). In the presence of L-NNA the mean relaxation response to EFS was recorded as 1.61%. Haemoglobin (100 ml) caused a 70.8% reduction in the mean overall relaxation response of tissue strips to EFS (P < 0.05), which was completely reversed by the addition of 1 6 10 74 M L- arginine (Figure 6). The responses of tissue strips to individual frequencies of EFS are given in Table 3. The addition of methylene blue and tetrodotoxin to the organ bath abolished the relaxant response at all frequencies of EFS. Figure 6 The effect of L-arginine in addition to haemoglobin on EFS mediated relaxation. The addition of L-arginine to control strips incubated with haemoglobin reverses the EFS induced impaired relaxation seen following the incubation of control strips with haemoglobin alone. This effect was statistically signi cant, *P < 0.05. n ˆ 8. It is known that the vasodilator mediator, NO, can be produced by the sinusoidal endothelium as well as the penile nerves. It is generally accepted that in man NO, derived from both these sources, plays a role in the relaxation of cavernosal smooth muscle that is necessary for penile erection, although the relative contribution of each source of NO is unclear. 3±6 A similar pattern of dual NO origin is known to be relevant in other species. In the rat the role of endothelial NO is controversial, and this has hampered the acceptance of the rat as a good in vitro model of erectile function. We believe that the data we present clearly demonstrates the functional importance of endothelial-derived NO in the rat and con rms the additional role of neuronal-derived NO, clarifying the pharmacology of rat corpus cavernosal smooth muscle and supporting the use of the rat as a model of erectile function. Tissue from rat corpora cavernosa mounted in vitro contracted with NAd relaxes in a dosedependent manner to the application of incremental doses of SNP (Figure 3). This con rms that, at least in part, rat corpus cavernosal tissue relaxes in response to NO; SNP, and other organic nitrates, exert their physiological effect by a non-enzymatic production of NO and activation of guanylate cyclase. 27 It is clear that neuronal-derived NO is important for relaxation of corpus cavernosal smooth muscle in man. 2,4,9,10 Hedlund et al presented both immunological and functional data from experiments performed on cavernosal tissue derived from Sprague ± Dawley rats that suggest that a similar pattern is seen in rats. 26 We have shown that corpus cavernosal tissue derived from Wistar rats, contracted by NAd in the presence of guanethidine and atropine, relaxes in a frequency-dependent manner to EFS (Figure 4). Maximal relaxation of the corpora cavernosal tissue strips was noted at a stimulation frequency of 20 Hz. Following contraction with 1 6 10 76 M NAd, a relaxation response of 50% was recorded at this frequency, equivalent to the response described by other groups in the rat 22 and comparable to the response seen in man and rabbit. 4 Since guanethidine and atropine block the adrenergic and cholinergic nerve-mediated effects of electrical stimulation, the relaxation response of cavernosal smooth muscle induced by EFS must be due to NANC mechanisms. The addition of an inhibitor of NO synthesis, L-NNA, caused a signi cant inhibition in the relaxation response recorded secondary to EFS, which suggests that NO is partly responsible for the NANC effect in this model (Figure 4). This is supported by the observation that human haemoglobin signi cantly impairs the EFSgenerated relaxation response, and that this effect may be reversed by the addition of the precursor of

NO, L-arginine (Figure 6). NO readily binds to ferrous haem, both within guanylate cyclase and human haemoglobin, 28,29 an observation that was used to support early evidence con rming that the endothelial-derived relaxing factor was NO. 7,30 Additionally, we report that methylene blue, which inhibits soluble guanylate cyclase, 14 the second messenger in smooth muscle relaxation activated by NO, blocks the relaxant response normally recorded secondary to EFS. We can therefore be con dent that neuronal derived NO is involved in the relaxation of rat corpus cavernosa, as it is in other species. The role of endothelial-derived NO in the rat is more controversial, and this has probably contributed to a reluctance in accepting the rat as a model of corpora cavernosal function. The sinusoidal endothelium is an important factor in penile erection. It is clear from functional studies performed on human and rabbit tissue that disruption of the endothelium, either by rubbing or perfusion of detergent, blocked the relaxant response secondary to ACh, 4 highlighting the fact that ACh relaxes penile smooth muscle by generating an endothelialderived relaxant factor in the same way that is does in vascular tissue. 7 Furthermore, the mating and in vitro studies performed by Burnett et al on mice genetically de cient in nnos revealed that, not only do these animals breed normally, but that isolated corpora cavernosal smooth muscle function is maintained. Immunohistochemical localization of enos in these animals revealed a compensatory over-expression of enos in nnos negative mice compared to control animals. 18 With good evidence to support both the presence of, and functional importance of endothelial NO in mice, rabbits and humans, it is dif cult to accept that it does not exist in the rat. Acetylcholine contributes to the relaxation of cavernosal smooth muscle directly by inhibition of contractile adrenergic inputs 31 and indirectly through its action on the endothelium, liberating NO. 32 We have demonstrated that the addition of acetylcholine to the in vitro experimental set-up caused a dose-dependent relaxation of rat corpora cavernosa tissue strips that had been pre-contracted with 1 6 10 76 M NAd (Figure 2). This response was signi cantly impaired by the addition of L-NNA, suggesting that acetylcholine is acting through the generation of NO (Figure 2). Further support for this observation comes from our demonstration that ACh-mediated cavernosal smooth muscle relaxation was signi cantly impaired by a NO scavenger, haemoglobin, and that this effect was reversed by the administration of L-arginine (Figure 5). In addition, the soluble guanylate cyclase inhibitor, methylene blue, blocked the relaxation response to ACh. From this data it is clear that ACh causes a relaxation of rat corpus cavernosal smooth muscle via the generation of NO. In response to exogenous cholinergic stimulation we describe a greater relaxation response of cavernosal tissue than some authors. 24,26 Indeed, other groups have not shown an ACh-mediated relaxation response in similar studies. 21,22 This may be explained by differences in experimental technique, in particular the preparation of tissue. The technique originally described by Italiano et al 21 and adopted by Way and Reid 22 mounts the penis as a single specimen, albeit with excision of part of the tunica albuginea to allow drugs to penetrate the tissue. Furthermore, both these groups excised the penis at the base and excluded the crura of the cavernosa from the sample. Keegan et al 24 demonstrated approximately 50% relaxation to ACh. Their dissection also excluded the crus penis, but did mount a single corpus cavernosal strip with the midline septum still attached and tunica albuginea excised to allow drug penetration. The dissection technique we developed ensured that the whole penis was excised to allow cavernosal strips to include the crus penis. In addition, we detached the corpus cavernosum from the thick midline septum leaving only the thin lateral tunica covering cavernosal tissue. We believe this has three advantages. Firstly, the cavernosal tissue specimen is covered by the minimum of tissue, allowing maximum penetration of drugs under experimental conditions. Secondly, excision of the thick septum allows examination of the effect of agents on cavernosa alone, which is comparable to tissue specimens harvested from human and rabbit. Finally, during the dissection, leaving the midline septum behind anchors the specimen rmly, allowing the cavernosa to be excised from it. Minimal handling of the cavernosa is then possible; indeed the entire length of cavernosal tissue may be excised simply by holding a silk tie, attached to the crus, that will subsequently be used to mount the specimen in vitro. It is likely that the effects of the midline septum do not contribute mechanically to the increase in relaxation that we have recorded, since the relaxation response to EFS we measured is equivalent to that in other groups. 24,26 Minimal handling of cavernosal tissue is vital though, if the action of the endothelium is to be examined. Kim et al 4 were able to abolish endothelium-mediated relaxation in strips of corpus cavernosum by rubbing them between nger and thumb to disrupt the endothelium. Since the dissection technique we adopted allowed minimal tissue handling, this may contribute to the greater relaxation response we recorded to ACh. The cavernosal tissue strips that we mounted in vitro included the crus penis; the tissue examined by other groups did not. 22,24 In addition to allowing a larger specimen, and reduced handling of cavernosal tissue, this may also be functionally relevant. Within the crura of the corpora cavernosa of the rat the trabeculae consist mainly of smooth muscle cells whereas distally 219

220 connective tissue predominates, with a reduced smooth muscle distribution. 33 Doubt over the presence of enos in the rat comes from the immunohistochemical studies of Dail et al. 16 Using corpora cavernosa derived from Sprague ± Dawley rats, they were unable to demonstrate the presence of enos in the sinusoidal endothelium of the corpus cavernosa, although the presence of this isoenzyme was con rmed in the vascular endothelium lining all arteries and veins of the penile crura. 16 It is possible that the sectioning technique used by these authors cutting at a thickness of 16 mm and immuno-stained once mounted upon a slide, may have restricted penetration of the reagents throughout the section. 34 Staining for enos within the endothelium in nnos-negative mice was successful on tissue sections cut at 6 mm. 16 Cutting rat cavernosal tissue at a thickness of 8 mm, though, did not allow demonstration of sinusoidal enos, but did again con rm its presence in penile vasculature. In more recent studies, by using reverse transcriptase-polymerase chain reaction and immuno-histochemistry, a clear demonstration of the presence of enos within cavernosal smooth muscle of the rat has been made. 35 Using autoradigraphic techniques Sullivan et al 36 were able to demonstrate evidence of speci c binding sites for NOS located within the endothelium lining the cavernosal lacunar spaces, which suggests, but does not con rm the presence of endothelial NOS. In conclusion, we have con rmed the ndings of other groups 20 ± 26 that have demonstrated an adrenergic contractile response of rat corpus cavernosa. In addition, we support the evidence which demonstrates that inhibition of penile smooth muscle contraction in the rat is secondary, at least in part, to the action of neuronal NO. 22,24 Furthermore, although there have been doubts raised over the histochemical con rmation of enos in the rat, we have clari ed the functional importance of endothelium-derived NO in cavernosal smooth muscle relaxation in this species. We believe that this model provides a reliable, reproducible technique for the study of penile erection. Acknowledgement This study was supported by a British Urological Foundation=Wyeth (UK) research scholarship. References 1 Krane RJ, Goldstein I, Saenz de Tejada I. Impotence. New Engl J Med 1989; 321: 1648 ± 1659. 2 Andersson K-E, Wagner G. Physiology of erection. Physiol Rev 1995; 75: 191 ± 236. 3 Ignarro LJ et al. Nitric oxide and cyclic GMP formation upon electrical eld stimulation cause relaxation of corpus cavernosum smooth muscle. 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