¹Department of Urology (Molecular Pathology), Royal Free and University College Medical School and The Royal Free Hampstead NHS Trust, Pond Street, London, UK

²Department of Molecular Pathology and Clinical Biochemistry, Royal Free and University College Medical School and The Royal Free Hampstead NHS Trust, Pond Street, London, UK

³Department of Biochemistry, Royal Free and University College Medical School and The Royal Free Hampstead NHS Trust, Pond Street, London, UK

Correspondence to: ME Sullivan, Department of Urology, Churchill Hospital, Old Street, Oxford OX3 7LJ, UK. E-mail: sullyme@hotmail.com

New Zealand white rabbit cavernosal smooth muscle strips (n=6) were mounted in organ baths. Relaxations to nitric oxide (10^-7-10^-4 mol/l) were measured and the same procedure was repeated on strips from rabbits 6 months after alloxan-induced diabetes (n=6). Transverse cavernosal sections were obtained from the same penises. Low and high resolution autoradiographs were prepared using [³H]-L-N^G-nitroarginine (an index of nitric oxide binding sites) and analysed densitometrically. Histochemical analysis was performed on adjacent sections using NADPH diaphorase (an index of nitric oxide synthase activity).

Nitric oxide relaxed control rabbit cavernosal smooth muscle strips in a concentration-dependent manner. Diabetic rabbit cavernosal smooth muscle strips were significantly (P<0.03) more sensitive to nitric oxide (mean IC₅₀=3.9 ´ 10^-6 mol/l). Nitric oxide synthase binding sites were localised to the cavernosal endothelium and smooth muscle. Nitric oxide synthase activity was increased in 6 month diabetic cavernosal smooth muscle. These findings suggest impairments in the L-arginine-nitric oxide pathway may play a role in the pathophysiology of diabetic erectile dysfunction.

International Journal of Impotence Research (2002) 14, 523-532. doi:10.1038/sj.ijir.3900935

diabetes mellitus; erectile dysfunction; nitric oxide

Introduction

The mechanism of penile smooth muscle relaxation has not been fully elucidated, but nitric oxide (NO) is increasingly recognised as being of importance in the regulation of cavernosal smooth muscle tone, both in the flaccid state and during erection.^1,2,3 These findings were based on experiments using isolated preparations of human and rabbit corpus cavernosum and eliciting relaxation by electrical field stimulation (EFS). In vivo evidence for the role of the L-arginine/NO pathway in penile erection has also been shown more recently using the rabbit and rat models.^3,4 The source of NO (nerve endings, endothelium or within the smooth muscle) has been a matter of controversy.

Diabetes mellitus (DM) is a major risk factor for erectile dysfunction (ED).⁵ In isolated corpus cavernosum strips from diabetic patients with erectile dysfunction, both neurogenic and endothelium-dependent relaxation has been shown to be impaired.⁶ Similar findings were also apparent in alloxan-induced diabetic rabbit.⁷ Reduced relaxation to EFS in cavernosal tissue taken from impotent diabetic patients has been demonstrated in association with a lack of NO production.⁸ Further support for an impairment in the L-arginine/NO pathway in the diabetic cavernosal bed was provided by studies which showed a significant increase in NO synthase (NOS) binding sites in rat cavernosum two months post induction of DM.⁹ The localisation of NO has been hampered by its short half-life and gaseous nature. These characteristics have forced researchers to study NO production indirectly. As a result, NOS, the enzyme that catalyses the synthesis of NO, has been used for study in different tissues in the body.

We conducted in vitro studies, using isolated rabbit corpus cavernosal tissue strips, to study contractile and relaxant responses to the ₁ agonist phenylephrine (PE) and the known and physiologically relevant vasorelaxants acetylcholine (ACh) and NO, together with EFS. Any changes in these responses brought about by the onset of DM were also assessed.

Autoradiographic and histochemical studies assessing NOS binding sites/activity were also performed on cavernosal sections taken from the same animals.

Materials and methods

Acetylcholine, alloxan, atropine, guanethidine, indomethacin and phenylephrine were all obtained from Sigma (Poole, Dorset, UK). L-arginine, LY85583, L-N^G-nitroarginine, tetrodotoxin and oxadiazoloquinoxalin-1 were provided by Bachem Fine Chemicals (Switzerland). The NO gas was purchased from Lynde gas (Stoke-on-Trent, Staffs, UK). The radiochemical [³H]-L-N^G-nitroarginine was obtained from Amersham Radiochemicals (Amersham International, Aylesbury, Bucks, UK). The histochemical marker, NADPH diaphorase was purchased from Dako Laboratories (High Wycombe, Bucks, UK).

Alloxan administration and blood analysis

Diabetes was induced by a single i.v. (lateral ear vein) injection of alloxan monohydrate 65 mg/kg, in a 5 ml vehicle (0.9% NaCl). Control animals were injected with the vehicle alone. The alloxan-injected rabbits required subcutaneous injections (scruff of neck) of 10 ml of 50% dextrose, at 6 hourly intervals for the first 24 h, to counteract the hypoglycaemia resulting from alloxan-induced pancreatic beta cell necrosis.

Confirmation of the severity of diabetes was obtained by testing for glycosuria (Multistix, Ames division, Miles Laboratories Ltd, Stoke Poges, Berks, UK) and measurement of serum glucose (Hitachi 747 Automatic Autoanalyser, Boehringer Mannheim, Lewes, Sussex, UK). Serum lipids were also determined using standard methodology for the Hitachi 747 Automatic Autoanalyzer (Boehringer Mannheim). All methodologies are monitored by national quality assurance programmes. Rabbits were classified as diabetic if their serum glucose concentrations were 10 mmol/l or greater throughout the study. Alloxan-treated rabbits with plasma glucose concentrations less than 10 mmol/l were considered nondiabetic. These alloxan-treated, nondiabetic rabbits (n=4) were used as a second control group along with vehicle-treated rabbits for the autoradiographic studies.

Functional studies

Tissue preparation and pretreatment: Strips of rabbit corpus cavernosum (4-6 pieces per animal, 2´1.5´6 mm) were obtained from 7 sexually mature, healthy control male New Zealand white rabbits (12 months old). The 'Principles of laboratory animal care' (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws. Rabbits were killed by cervical dislocation and penectomy performed immediately. The penises were placed in chilled Krebs buffer solution (NaCl: 118.1 mmol/l; NaHCO₃: 25 mmol/l; KCl: 4.6 mmol/l; KH₂PO₄: 1.2 mmol/l; CaCl₂: 2.5 mmol/l; MgSO₄: 1.2 mmol/l; and D-glucose: 11.0 mmol/l) and the corpus spongiosum and urethra excised. The corpus cavernosal smooth muscle was then isolated from the enveloping tunica albuginea by careful dissection. The same procedure was performed on 6 age-matched diabetic New Zealand white rabbits (6-8 months duration of DM).

All tissues were studied within 6 h. The tissues were suspended between two small hooks in 1.5 ml water jacketed isolated tissue baths equipped with two parallel platinum electrodes and prepared for experimentation as follows.

The tissue was initially allowed to equilibrate at 2-3 g resting tension for 60-90 min in Krebs buffer solution at 37±1°C. The buffer was constantly aerated with a 95% O₂ and 5% CO₂ gas mixture which maintained the buffer at pH of 7.4±0.1. The tissues were then depolarised with 120 mmol/l KCl. This procedure increases and stabilises subsequent sub-maximal precontractile responses to PE. The strips were then washed and allowed to equilibrate for 30 min and then precontracted by the addition of 10^-5 mol/l concentration of PE. Any tissue which did not achieve a tension of 1 g was discarded. Contractions were measured isometrically with a Grass force displacement transducer (model FT-03, Grass Instruments, Quincy, Massachusetts, USA) and recorded on a Grass polygraph (model 7D, Grass Instruments).

Relaxations to EFS: Following precontraction with 10^-5 mol/l PE, EFS was performed using an S88 Grass stimulator (Grass Instruments). EFS was conducted at 50 V, with sequential frequencies of 2, 4, 8 and 16 Hz in the form of square wave pulses (pulse duration 1 ms) in trains of 10 s and train interval 5-8 min or until the trace returned to the baseline pre-contractile tension. Strips were only used if there was<10% variability in the magnitude of relaxation to EFS.

Following complete relaxation, the strips were preincubated for 60 min in bathing media containing guanethidine (5´10^-6 mol/l), atropine (10^-6 mol/l) and indomethacin (10^-5 mol/l). The purpose of this treatment was to eliminate the adrenergic, cholinergic and prostanoid components, respectively, and to study relaxation responses to the stimulation of nonadrenergic noncholinergic (NANC) nerves. The relaxation to EFS was then studied following precontraction with PE (10^-5 mol/l). Tetrodotoxin (10^-6 mol/l), a neuronal sodium-channel blocker, or L-N^G-nitroarginine (L-NOARG; 2´10^-4 mol/l), an inhibitor of neuronal NO synthesis, were then given 15 min prior to repeating the EFS responses. Tetrodotoxin is used to confirm the neurogenic origin of responses seen to EFS, whilst L-NOARG assesses the role of neuronal NO in the relaxation to EFS.

Endothelium-dependent relaxations: Endothelium- dependent relaxations were determined by precontracting smooth muscle strips with PE (10^-5 mol/l) and then relaxing the tissue by the addition of ACh directly to the organ bath. Concentration-response curves to ACh were obtained using 10^-9 to 10^-4 mol/l, added cumulatively.

NO-induced relaxations: Tissue strips were prepared in exactly the same way as for EFS including incubation with guanethidine, atropine and indomethacin. Following precontraction with PE (10^-5 mol/l) the tissues were relaxed by the addition of NO solutions directly into the organ bath. Concentration response curves were determined in the dose range 10^-8-10^-4 mol/l, with the next dose being added only when the trace had returned to baseline. The NO neutralising agent, LY 85583 (10^-5 mol/l) or the guanylyl cyclase inhibitor, oxadiazoloquinoxalin-1 (ODQ; 10^-6 mol/l) were then given 15 min prior to repeating the responses to NO (10^-7 to 2´10^-5 mol/l).

NO solutions were prepared as follows: double distilled water was boiled for 15 min, allowed to cool to 60°C and pulled under vacuum into specially adapted gas sampling tubes. The water was then flushed with N₂ for 45 min and the tubes sealed with rubber septa. The appropriate amounts of NO was then injected into the gas sampling tubes using gas-tight syringes. Control solutions were prepared by following the above procedure and then bubbling the solutions with O₂ for 1 h. All solutions were prepared freshly on the day and kept on ice for the duration of the experiment.

Receptor studies

Preparation of penile tissue: Following cervical dislocation, the penile tissues were excised and stored immediately at -70°C in air tight cryotubes. The tissues were then mounted in AMES OCT embedding compound (BDH Laboratory Supplies, Poole, UK). Transverse 10 µm sections of rabbit penises were cut in a cryostat (Bright Instrument Co. Ltd., Huntingdon, UK) at approximately -20°C and thaw mounted onto gelatinised microscope slides. The slides were stored at -70°C in air tight containers until use.

Quantitative assessment of [³H]-L-N^G-nitroarginine binding to rabbit corpus cavernosum: Localisation of NOS was carried out essentially as described in previous studies,^10,11 Slide mounted tissues were preincubated in 50 mmol/l Tris-HCl, pH 7.2 for 15 min at 22°C. Consecutive sections were then incubated in Tris buffer containing 3 mmol/l CaCl₂ and 10 nmol/l [³H]-L-N^G-nitroarginine ([³H]-L-NOARG) (Specific activity 55 Ci/mmol) (Amersham International, Amersham, UK) for 60 min at 4°C. This concentration was at the approximate K_D value (10 nM) established from previous saturation studies.¹¹ The degree of non-specific binding was established by incubating alternate sections in the presence of 10 µmol/l unlabelled L-arginine (the non-specific binding for [³H]-L-NOARG was not detectable). After incubation the slides were washed in buffer (4 times for 2 min) to reduce non-specific binding, dipped in glass-distilled water (4°C) and dried in a stream of cold air. Low resolution autoradiography was carried out by exposing sections to Hyperfilm 3H in X-ray cassettes for 12 weeks. After exposure the film was processed in undiluted Kodak D19 (Kodak, Ilford, UK), developed and fixed in Ilford IF-23 (diluted 1:4 with water) (Hypam, Ilford, UK). The autoradiographs were then air dried. Photodensitometric analysis was performed on film images on a VIDAS imaging system (Kontron, Thame, UK) and the degree of specific binding determined from curves generated by ³H microscales that were co-exposed with slide mounted tissue. Binding was expressed in terms of radioligand bound per unit area (ie dpm/mm²). Microscopic localisation (high resolution autoradiography) of binding was performed by post-fixing tissue in paraformaldehyde vapour (2 h at 80°C) and coating slides in nuclear emulsion (LM-1; Amersham International). Slides were then stored in light-proof boxes for 12 weeks at 4°C, after which they were processed in D19 high contrast developer and fixed. Underlying tissue was stained with haemotoxylin and eosin, high resolution autoradiographs were viewed on an Olympus Vanox microscope and selected sections photographed where appropriate.

Histochemical localisation of NOS: NADPH diaphorase histochemistry was used to localise NOS and is derived from NOS activity in neurons.¹² NADPH diaphorase histochemistry relies on the reoxidation of the reduced form of NOS in the presence of the electron acceptor, nitroblue tetrazolium. Sections were incubated in freshly depolymerised 3% paraformaldehyde in phosphate buffered saline for 30 min at 4°C followed by incubation for 60 min at 37°C with 1 mmol/l NADPH, 0.2 mmol/l nitroblue tetrazolium and 0.2% Triton X-100 in 50 mmol/l Tris (pH 7.4).

Adjacent sections were also used for identification of endothelial, smooth muscle and nerve cells using the appropriate immunohistochemical markers, platelet endothelial cell adhesion molecule-1 (PECAM), anti-alpha smooth muscle actin and neurofilament 200, respectively.

The sections were graded by two independent histopathologists blinded to the study.

Statistical analysis

The effects of EFS, ACh and NO are expressed as percentage relaxation of the agonist (PE)-induced tension. The autoradiographic results are expressed as mean±s.e.m. n represents the number of separate animals used. Data was analysed using the Student's t-test. Statistical significance was accepted when P<0.05.

Results

Animal weights and serum glucose and cholesterol concentrations

The starting weights in both the control and diabetic rabbit groups were similar (control: mean±s.e.m.: 3.1±0.1 kg, n=6. Diabetic: 3.2±0.1, n=7). However, after 6 months the diabetic rabbits were significantly (P<0.03) lighter than the control group (control: 4.2±0.1, n=6. Diabetic: 3.7±0.2, n=6).

Serum glucose concentrations (non-fasting) were significantly (P<0.009) elevated in the 6 month diabetic group (32.2±4.8 mmol/l, n=6), when compared to controls (6.4±0.2, n=6).

Serum cholesterol concentrations (non-fasting) were not significantly different between the 6 month control group (1.2±0.3 mmol/l, n=6) and diabetic group (0.9±0.1, n=6). Serum triglycerides (non-fasting) were also not significantly different between control and diabetic groups (results not shown).

Baseline studies

By the end of the equilibration period, 82% of the strips had developed oscillating spontaneous activity at a mean tension of 1.2 g±0.2 (n=13; 52 strips used in total). No significant difference in oscillating spontaneous activity was seen between control and diabetic groups.

Contractions to PE

PE (10^-5 mol/l) caused stable contraction to 4.6 g±0.6 (n=13; 52 strips). No significant difference was seen between control and diabetic rabbit strips in response to PE (10^-5 mol/l).

Relaxations to EFS

EFS of the strips of corpus cavernosum treated with guanethidine, atropine and indomethacin caused frequency-dependent relaxations. Tetrodotoxin (10^-6 mol/l) reduced the electrically elicited relaxation by 92%±4 (Figure 1a). The addition of L-NOARG (2´10^-4 mol/l) significantly reduced the effects of EFS to 18.4±4% of the initial response (Figure 1b).

No significant difference in relaxation between control and diabetic strips to EFS was seen. At higher frequencies (8, 10 and 16 Hz) however, the diabetic response had plateaued (mean per cent relaxation=63.6%±9.6) whilst the response of control strips was continuing to increase in a linear fashion (mean=85.1%±3.4). The responses at the highest frequency (16 Hz) was not however, significant between control and diabetic groups.

ACh-induced relaxations

Relaxations to ACh (10^-9 mol/l-10^-4 mol/l) were not significantly different between control and diabetic corporal strips.

NO-induced relaxations

Relaxations to NO (10^-8-10^-4mol/l) were more potent than to either EFS or ACh (Figure 2a). The relaxations to NO were transient, due to its short half-life and were similar qualitatively to those elicited by EFS (Figure 2a). LY 85583 and ODQ markedly reduced the responses to NO in both control (Figure 2b) and diabetic strips. The relaxations to NO were significantly greater (P<0.03) in diabetic corporal strips (mean IC₅₀=3.4´10^-6 mol/l±0.6) compared to controls (mean IC₅₀=22.9´10^-6 mol/l±14.4) (Figure 2c). The recovery of the cavernosal strips to baseline tension following relaxation to NO was similar in control and diabetic animals. In control experiments, NO solutions which had been bubbled with air prior to use did not induce a relaxation response, indicating that the active component of the solutions was NO and not nitrite or nitrate.

Receptor studies

Autoradiographic localisation of NOS binding sites: There was moderate [³H]-L-NOARG binding to tissue sections, which was markedly reduced when incubated in the presence of L-arginine (non-specific binding was not detectable). [³H]-L-NOARG binding was localised to the corpus cavernosum, urethra and penile blood vessels (Figure 3). Within the corpus cavernosum, there was binding to both the endothelium and the smooth muscle cells.

Densitometric analysis of film images indicated [³H]-L-NOARG binding to the corpus cavernosum was not significantly altered in 6 month diabetic rabbits when compared to controls (receptor binding measured as dpm´1000/mm² and values expressed as mean±s.e.m.: control=0.9±0.2; diabetic= 1.0±0.1; n=6 in each study group).

The cavernosal tissue from alloxan-treated rabbits who had not developed diabetes (and were not included in the study) showed no significant changes in NOS binding sites when compared to age-matched healthy controls. These findings imply alloxan does not have direct toxic effects on corpus cavernosal NOS binding sites.

Histochemical localisation of NOS: NADPH diaphorase activity was demonstrated on cavernosal nerves, blood vessels and cavernosal smooth muscle cells in both control and diabetic tissue (Figure 4). The activity on 6 month diabetic cavernosal smooth muscle cells was ranked greater (two independent histopathologists) than that of age-matched control tissue (Figure 4).

Discussion

The cavernosal smooth muscle relaxant responses to exogenous NO supports a role for this molecule in erectile physiology. The sensitivity of this NO response to LY 85583 and ODQ implies this is a guanylyl cyclase-dependent event. We have recently shown that cGMP is the key mediator of this response.¹³ The inhibitory effects of tetrodotoxin and L-NOARG on EFS responses also support a major role for NO in NANC-induced cavernosal smooth muscle cell relaxation. The increased sensitivity to NO solutions in the diabetic strips implies that the smooth muscle response is not impaired in 6-8 month DM cavernosal tissue. This response may be a positive feedback mechanism secondary to inadequate amounts of NO reaching the end organ, either due to impaired secretion, quenching or inactivation. These findings support studies in impotent diabetic men which have demonstrated that reduced relaxation to EFS is associated with a lack of NO production and not to the inability of the cavernosal smooth muscle to relax.⁸ One explanation for a reduction in NO bioavailability could be a reduction in penile L-arginine content. Reduced plasma and aortic tissue arginine levels have been demonstrated in a rat diabetic model.¹⁴ Furthermore the impaired endothelium-dependent relaxation seen in these diabetic rat aortae was reversible by acute administration of L-arginine. In this context, dietary supplementation of L-arginine has been shown to improve both sexual performance in men with erectile dysfunction¹⁵ and erectile function in aging rats.¹⁶ Other workers have shown that advanced end glycosylation products can quench and inactivate NO in aortic¹⁷ and penile tissue.¹⁸ However, one might expect advanced end glycosylation products to quench both endogenous and exogenous NO equally, thereby impairing the relaxant effects of both, contradicting the results seen in our study and others.⁶ Alternatively, inactivation of NO by the free radical, superoxide, may impair NO bioavailability.

The current study demonstrated no significant alteration in neurogenic and endothelium-dependent relaxations of rabbit cavernosal smooth muscle 6-8 months after the induction of diabetes by alloxan, when compared to controls. This suggests that this diabetic rabbit model has no functional evidence of ED or is compensating for any changes present. The responses to EFS however were plateauing at frequencies of 8-16 Hz in the diabetic strips, whilst they were continuing to rise in control strips at these frequencies and this may be indicative of a developing impairment in NANC-induced corporal relaxation. The responses to NO in this study, in conjunction with the EFS results, support a role for neuronal NO in cavernosal smooth muscle relaxation. These findings might suggest the presence of denervation supersensitivity in the cavernosal NANC pathway, a well recognised observation in other smooth muscle containing tissues with associated neuropathy.¹⁹ This supersensitivity could provide one explanation for the marked erectile responses to low dose intracavernosal agents seen in some men with diabetic ED and in other men with neurogenic ED.²⁰ A loss of antierectile tone is another possible explanation for the enhanced erectile responses to intracavernosal agents.

No significant changes were found in NOS binding sites in the rabbit corpus cavernosum 6 months after the induction of diabetes when compared to age-matched controls. These findings suggest that any decrease in NO production by the rabbit corpus cavernosum in DM cannot be attributable to a decrease in NOS-binding sites. However, in vitro autoradiography, using [³H]-L-NOARG, does not discriminate between the three isoforms of NOS. Subtle changes in the binding sites of one or more NOS isoforms may be difficult to demonstrate. The increased NADPH diaphorase activity on 6 month diabetic cavernosal smooth muscle would provide support for an increase in one or more of the NOS isoforms. Recent studies have shown that induction of inducible NOS (iNOS) can correct ED in a rat model.²¹ An increase in iNOS could be one explanation for the histochemical findings in our study as well as the lack of impaired neurogenic- and endothelium-dependent relaxations seen in the diabetic cavernosal strips. However, Seftel et al have demonstrated iNOS in cavernosal tissue from diabetic men with ED and speculated that upregulation of iNOS and downregulation of eNOS may be a pathophysiological mechanism for advanced glycosylation end product mediated ED.²² Further work is required to clarify the role of iNOS in cavernosal tissue.

These NOS results are unlikely to be due to a toxic effect of alloxan, since the photodensitometric quantification of NOS binding sites in alloxan-treated non-diabetic cavernosal tissue was not significantly different to control rabbit cavernosal tissue.

Hypercholesterolaemia may also adversely influence endothelial function in the corpus cavernosum.²³ However serum cholesterol and triglyceride concentrations in the 6 month diabetic rabbit groups were not significantly different from those in the matched controls. This model therefore has the advantage of the results not being confounded by hypercholesterolaemia and hypertriglyceridaemia and the findings in our study may have important implications for 'early' events in the pathogenesis of diabetic ED.

We recently demonstrated a significant increase in endothelin B (ET_B) receptors on smooth muscle cells of diabetic rabbit cavernosal tissue.²⁴ The ET_B receptor has been shown to effect vasodilatation through the release of NO and prostacyclin. The increase in cavernosal smooth muscle cell ET_B receptors could be one compen-satory mechanism for the apparent reduction in NO bioavailability.

In conclusion, these experiments support a role for NO, ACh and NANC nerve stimulation in cavernosal smooth muscle relaxation. They also demonstrate an increased relaxation response to exogenous NO and increased cavernosal smooth muscle NADPH diaphorase activity in diabetic strips, when compared to controls. This suggests impaired NO bioavailability may play a role in the pathophysiology of diabetic ED.

Acknowledgements

MES is supported by a Novo Nordisk Diabetic Research Grant and MRD by the British Heart Foundation.

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Figure 1 (a) Isometric tension recordings illustrating relaxant effects of EFS in the presence of tetrodotoxin (10^-6 M) or L-NOARG (2´10^-4 M) on PE-contracted strips of control and diabetic rabbit corpus cavernosum. Note the marked inhibition of relaxant response supporting the neurogenic origin of EFS effects and an important role for NO in neurogenically-induced relaxation. Tracings are representative of four strips from two rabbits per group. (b) Dose response relationships of relaxation of control and diabetic corpus cavernosum in response to EFS. Each point represents mean±s.e.m. of 12-18 strips from six rabbits per group.

Figure 2 (a) Isometric tension recordings illustrating relaxant effects of nitric oxide (NO) on PE-contracted strips of control and diabetic rabbit corpus cavernosum. Tracings are representative of 12-18 strips from six rabbits per group. (b) Isometric tension recordings illustrating effects of NO alone and in the presence of LY 85583 (10^-5 mol/l) or ODQ (10^-6 mol/l) on PE-contracted strips of control and diabetic rabbit corpus cavernosum. Tracings are representative of four strips from two rabbits per group. Note the marked inhibition of NO-induced relaxant responses in the presence of LY 85583 or ODQ supporting an important role for guanylyl cyclase in this process. (c) Dose response relationships of relaxation of control and diabetic corpus cavernosum in response to NO. Each point represents mean±s.e.m. of 12-18 strips from six rabbits per group. The IC₅₀ for diabetic strips was significantly (P<0.02) less than control strips.

Figure 3 [³H]-L-NOARG binding to control and diabetic rabbit corpus cavernosum (transverse sections). Left: microautoradiograph generated on nuclear emulsion of [³H]-L-NOARG binding to control six month rabbit cavernosa (dark field illumination, where white grains indicate binding sites). Middle: haematoxylin and Eosin stained six month control rabbit corpus cavernosum (CC). Right: microautoradiograph of binding to six month diabetic rabbit cavernosa (dark field illumination). Scale bar=500 µm.

Figure 4 NADPH diaphorase activity in rabbit corpus cavernosum (CC) and corpus spongiosum (transverse sections). Top left: control rabbit corpus cavernosum (CONTROL CC) showing faint level of staining of smooth muscle cells (SM). Top middle: negative control. Top right: diabetic rabbit corpus cavernosum (DIABETIC CC) showing increased diaphorase activity on smooth muscle cells (SM) when compared to controls. Scale bar=200 µm. Bottom left: NADPH diaphorase activity on diabetic corpus spongiosum blood vessels (BV)´10. Bottom middle: NADPH diaphorase activity in diabetic rabbit corpus cavernosum nerve fibre (N)´10. Bottom right: higher power view of diabetic rabbit corpus cavernosal nerve (N) and smooth muscle cells (SM) demonstrating NADPH diaphorase activity ´20. Scale bar=100 µm.