Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavernosum

Zhi-Shun XU, Qiang FU¹, Sheng-Tian ZHAO, Hai-Nan LIU

Department of Urology, Qilu Hospital, Shandong University, Jinan 250012, China
¹Department of Urology, Shandong Provincial Hospital, Jinan 250021, China

 Asian J Androl  2001 Jun; 3:  139-142

Keywords: nitric oxide synthase;   diabetes mellitus;   impotence; streptozotocin

Abstract

Aim: To study the effect of diabetes mellitus and insulin treatment on rat penile nitric oxide synthase content. Methods: Male Wistar rats were divided at random into two groups: the Control (n=8) and the Diabetic (n=17). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. The diabetic animals were then randomly divided into two subgroups: diabetic rats without insulin treatment (n=7) and diabetic rats with insulin treatment (n=10).  The neuronal nitric oxide synthase (nNOS) in the penile corpus cavernosum were assayed by immumohistochemical staining with specific antibody to nNOS and the nNOS-positive nerve fibers were counted semiquantitatively under a high power microscope. Results: The nNOS- positive nerve fibres in diabetic rats with treatment was higher than that in diabetic rats without treatment (P<0.05) and lower than that in the controls (P<0.01). The nNOS-positive nerve fibres in diabetic rat without treatment were also lower than that in the controls (P<0.01). Conclusion: In streptozotocin-induced diabetic rats, the nNOS content in the penile corpus cavernosum was significantly decreased. Insulin treatment at the dose level employed partially restores the penile nNOS content in these rats.

1 Introduction

The incidence of erectile dysfuntion in diabetes mellitus has been reported to be 35-50%, and in individual papers even as high as 75%, in adult patients with more than 5 years diabetic history^[1-4]. The association of erectile dysfunction and diabetes is also supported by evidences from experimental diabetes in rats: the sexual behavior and reproductive function are adversely affected in experimental diabetic rats, as evaluated by the latency of mounts, the frequency of intromission, the hit rate and the ejaculation rate^[5-7].

2 Materials and methods

2.1 Animals and treatment

Male Wistar rats (180-250 g , 50-70 days, n=30) were obtained from the Shandong University Animal Centre. They were kept in animal house at 222 with 12 hr light/12 hr darkness and free access to animal food pellet and tap water. Rats were randomly divided into two groups: the Diabetic (n=22) and the Control Group (n=8). To each animal of the Diabetic Group, a single intraperitoneal injection of streptozotocin (Sigma, USA, 1% dissolved in 0.1 mol/L, pH 4.5 citric acid-sodium citrate buffer of to a dilution of 1%), at a dose of 60 mg/kg, was given. To the xcontrols, only the vehicle was injected. The fasting serum glucose was determined 24 hr after injection. Streptozotocin-injected rats with serum glucose above 19.5 mmol/L were considered to be diabetic and 19 of the 22 rats fulfilled this requirement. They were further divided at random into two subgroups: the Diabetic non-treated (n=9) and the Diabetic treated (n=10) groups. The serum glucose of the Control group was all within the normal range. Animals of the Diabetic treated group were then given protamine zinc insulin 4u subcutaneously every other day for 12 weeks.

The body weight was determined every week and the serum glucose, every other week in the whole experimental period of 12 weeks.

2.2 nNOS determination  

At the end of the observation period, rats were anaesthetized with intraperitoneal injection of 40 mg/kg sodium pentobarbital. The mid-piece of the penile corpus cavernosum was removed and stored in liquid nitrogen (-70) until analysis  with standardized SP method. Serial 4 m cryosections of corpus cavemosum were performed at 4 m intervals at -18. The sections were then dried in room temperature for 5 minutes and fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer for another 5 minutes. They were stained with polyclonal antibody against nNOS (1:50 dilution) (Santa Cruz, USA), and then detected with biotinylated secondary antibody (Zymed, USA) and streptavididin peroxidase complex procedure. Positive nerve fibers were stained brownish yellow. Semiquantitative evaluation was done by counting the number of positive nerve fibers in 5 visual fields at random under a high power light microscope. 

2.3 Data analysis

3 Results

Table 1. Effect of diabetes and insulin treatment on body weight and serum glucose.

^cP<0.01, compared with Treated; ^fP<0.01, compared with Control.

There were plenty of positive-stained nerve fibers around the trabeculae and the blood vessels in the penile corpus cavernosum of the Control Group (Figure 1). The fibers in the Non-treated Group (Figure 2) were the least among the three groups. Table 2 showed that the number of the positive nerve fibers in the Treated Group (Figure 3) were less than that in the Control Group (P<0.01) and more than that in the Non-treated Group (P<0.05).  

^bP<0.05, compared with Treated; ^fP<0.01, compared with Control.   

Figure 1.   nNOS immunohistochemical staining in penis of Control rats  (400). 
Figure 2.   nNOS immunohistochemical staining in penis of diabetic rats without treatment  (400). 
Figure 3.   nNOS immunohistochemical staining in penis of diabetic rats with treatment  (400).

4 Discussion

Upon electrical stimulation or acetylcholine administration, the relaxation of the corpus cavernosum from diabetic impotence patient in vitro is inadequate as compared with the tissue from the non-diabetics, indicating impairments in both the nerve- and the endothelium-dependent muscular relaxation; on the other hand, there is no significant difference between the diabetic and non-diabetic patients in cavernous muscle relaxation induced by papaverine and the NO donor, sodium nitroprusside, both acting directly on the smooth muscle^[10]. It is be lieved that the impairment lies in the NO synthesis and release, while the cavernous muscle itself is not damaged. This result is consistent with the clinical experience in the management of diabetic impotent patients through intracavernous injection of NO donor, papaverine and prostaglandin E₁^[11].

NO is a nonadrenergic noncholinergic (NANC) mediator for penile erection^[8] and is formed by the conversion of l-arginine to 1-citrulline by the enzyme NOS. NO formed is rapidly diffused to the surrounding musculature to activate guanylatecyclase, thus converting guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). This process results in the depletion of intracellular calcium ions, leading to muscle relaxation. At the penile corpus cavernosum, NO is produced both at the endothelial level and the nitrergic nerves by the endothelial NO synthase (eNOS) and the neuronal NO synthase, respectively^[12]. Although acetylcholine induces endothelium-dependent relaxation of the isolated corpus cavernosum^[13], erection is unlikely to involve the release of acetylcholine from parasympathetic nerves and its subsequent diffusion to the endothelium, since neither atropine nor neostigmine inhibit erection when injected intracavernously^[14].

The principal cause of erectile dysfunction in diabetes is either nervous or vascular damage at the penile corpus cavernosum. However, in diabetic impotence the incidence of nervous involvement, e.g., penile nerve degeneration, is far more frequent than that of vascular impairment^[15]. Furthermore, relaxation of the penile corprs cavernosum by transmural stimulation does not require functional endothelium^[13], thus NO in the penile tissue seems to be largely derived from the nNOS. Vernet et al^[16] found a parallel decrease in the penile reflexes and the NOS activity/content in diabetic rats, suggesting that nerve impairment is the cardinal cause of diabetic impotence. In this study it was indicated that the nNOS nerve fibers in the pernile corpus cavernosum of diabetic rats were markedly reduced. The antibody to nNOS used in the experiment did not crossª²react with eNOS, so that the positive endothelial cells were not stained. Therefore, it seems that a decrease in nNOS content was the main cause of diabetic impotence and the NO from nerves took a greater part in erection. It is interested to note that Ari et al^[17] indicated that the total NOS activity was normal in the cavernosal tissue of streptozotocinª²induced diabetic rats. Although we determined the nNOS content and  the total NOS activity, the discrepancy between these two studies still warrants further investigation.

References

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Correspondence to: Dr Zhi-Shun Xu, Department of Urology, Qilu Hospital, Shandong University, Jinan 250012, China. 
Tel: +86-531-692 1941, Fax: +86-531-692 7544  
E-mail:  xianzhouj@163.net
Received 2001-03-06     Accepted 2001-05-26