To determine whether chronic renal failure (CRF) reduces nitrergic relaxant responses in a rabbit model.
To determine whether chronic renal failure (CRF) reduces nitrergic relaxant responses in a rabbit model.
Ten rabbits underwent surgery to induce uraemia (CRF rabbits) and a further 10 a sham operation (controls). Corpus cavernosal tissue was prepared and used in organ-chamber experiments, with relaxation assessed against a background of pre-contraction with phenylephrine. At the plateau of contraction, relaxation responses to cumulative concentrations of carbachol or sodium nitroprusside (SNP), to test endothelium-dependent and -independent relaxations, respectively, were assessed. Before electrical-field stimulation (EFS), the tissue was treated with an adrenergic nerve blocker and a muscarinic receptor blocker to eliminate the adrenergic and cholinergic components, and to determine the relaxation responses to the stimulation of nonadrenergic, noncholinergic (NANC) nerves. The relaxation responses in corporal strips obtained from CRF rabbits were compared with those from controls.
When tissues were contracted with KCl, tensions were similar in all groups. The impairment in concentration-dependent relaxation with carbachol was significant in CRF rabbits, but SNP- and papaverine-induced concentration-dependent relaxation responses were no different among the groups. EFS-induced frequency-dependent relaxations were significantly lower in CRF rabbits than in controls.
CRF inhibits the NANC-mediated relaxation of rabbit corpus cavernosum smooth muscle. Changes in NANC-mediated and carbachol-induced (endothelium-dependent) relaxation of corporal smooth muscle in the rabbit are probably caused by uraemia and subsequently, hyperthyroidism, hyperparathyroidism or low testosterone levels in CRF. These results also suggest that if vasoactive agents are to be used for treating erectile dysfunction in uraemic patients, direct-acting vasodilators and phosphodiesterase inhibitors will be useful.
Erectile dysfunction (ED) is defined as the persistent inability to achieve and/or maintain an erection sufficient for satisfactory sexual activity . It is a common condition in middle-aged and older men, and frequently occurs in association with various illnesses, e.g. cardiovascular disease, hepatic and renal failure . The incidence of ED among patients with renal failure is high, at 20–100% in men with chronic renal failure (CRF), as discussed previously .
The initiation and maintenance of penile erection requires the integration of thoracolumbar and sacral afferent signals, intact autonomic nerves to the penis, normal erectile tissue and adequate blood supply to the penis. The relaxation of penile corporal smooth muscle allows the expansion of lacunar spaces and compression of subtunical venules, with entrapment of blood in the corporal endothelium [4,5]. Over the last decade there have been significant advances in the understanding of the physiology of the penile erection. Neurogenically mediated relaxation responses that persist after adrenergic and cholinergic blockade are thought to be mediated by the nonadrenergic-noncholinergic (NANC) mechanism [5,6]; however, endothelium-dependent cholinergic neurotransmission may also mediate penile erection . Recent studies have shown that nitric oxide (NO) generation in response to NANC transmission, is the main event leading to corporal smooth muscle relaxation, through the activation of soluble guanylyl cyclase [8,9]. Dysfunction of the NO signalling pathway has been implicated as a factor in the effects of endothelial damage in patients with CRF .
To date, there is no published in vitro study investigating that the alteration of nitrergic system in the corpus cavernosum in CRF. In the present study we investigated the changes in nitrergic relaxation responses in corporal strips obtained from rabbits with CRF and compared them with control rabbits.
The details of the animals (20 male New Zealand white rabbits, 2.5–3 kg) and the experimental system were reported previously [3,11]; briefly, the rabbits were divided into two equal groups; 10 underwent surgery to induce uraemia (by partial excision of the left kidney and 2 weeks later, right nephrectomy; CRF rabbits) and the remaining 10 a sham operation (controls). After 4 weeks the rabbits were killed, their penises removed and strips of cavernosal tissue prepared for organ-bath experiments to determine their contraction and relaxation responses to various agents.
Relaxant responses to carbachol (10−8−10−4 mol/L) were elicited in endothelium-intact corpus cavernosal strips that were contracted with submaximal concentrations of phenylephrine (10 µmol/L). These relaxations were compared with those of strips obtained from CRF rabbits.
Relaxant responses to sodium nitroprusside (SNP, 10-8−10-4 mol/L) and papaverine (10−8−3 × 10−4 mol/L) were produced in strips pre-contracted with phenylephrine (10 µmol/L) in control and CRF rabbits.
For electrical-field stimulation (EFS), the strips were stimulated for 10 s with two parallel platinum electrodes at sequential frequencies of 2, 4, 8, 16, 32 and 64 Hz as square-wave pulses of 50 V (0.8 ms) delivered by a current amplifier and a stimulator. The strips were allowed to return to the baseline pre-contractile tension between the tests at each frequency. Before EFS, the tissue was treated with an adrenergic nerve blocker, guanethidine (10 µmol/L) and a muscarinic receptor blocker, atropine (1 µmol/L) for 30 min to eliminate the adrenergic and cholinergic components, and to determine the relaxation responses to the stimulation of NANC nerves. The complete blockade of electrically stimulated relaxation of corporal smooth muscle with the addition of an autonomic ganglion blocker, hexometonyum (100 µmol/L) is consistent with the hypothesis that electrically elicited relaxation is mediated by a NANC neuronal pathway . The relaxation responses elucidated by EFS were abolished with 1 µmol/L tetrodotoxin, suggesting that the relaxant stimulation was neurogenic. Relaxation was elicited with EFS determined after submaximal contraction with phenylephrine. The technical procedure used for obtaining the responses to EFS were described previously . One strip from each rabbit was contracted with phenylephrine (10 µmol/L) and the second strip maintained unstimulated. Relaxant responses to EFS were compared with those obtained from CRF rabbits.
The following drugs were used in the experiments: atropine sulphate, SNP, acetylcholine chloride, guanethidine, hexometonyum, l-arginine N-nitro-L-arginine methyl ester (L-NAME), carbachol, papaverine, phenylephrine hydrochloride (Sigma Chemical Co, St Louis, MO). All drugs were dissolved in distilled water and freshly prepared on the day of the experiments.
Experimental values are expressed as the mean (sem); the relaxant effects of agonists were expressed as a percentage of the pre-contraction to phenylephrine. To evaluate the effects of an agonist, the maximum response (Emax), the concentration for a half-maximal response (EC50) and pD2 values were calculated from the concentration-response curve obtained in each experiment, as predicted from the Scatchard equation for drug–receptor interaction, where: response/concentration = 1/EC50 × response + Emax/EC50
The pD2 value was expressed as the negative logarithm of the EC50. Groups were compared using general linear models of analysis, with independent paired t-tests, and P < 0.05 considered to indicate statistical significance.
The blood and hormone values of both groups before surgery and after 4 weeks were reported previously . When tissues were contracted with 124 mmol/L KCl the tensions were similar in all groups, with respective mean contractions of 2143 (261) and 2286 (260) mg in controls and CRF rabbits. In corpus cavernosal strips pre-contracted with phenylephrine at submaximal concentration, cumulative concentrations of carbachol (0.01–100 µmol/L) caused concentration-dependent relaxation (Fig. 1a). The impairment of relaxation in CRF rabbits was statistically significant compared with the control (P < 0.05). In corpus cavernosal strips pre-contracted with phenylephrine, SNP (0.01–30 µmol/L) induced concentration-dependent relaxation (Fig. 1b). Papaverine induced concentration-dependent relaxation (Fig. 1c), but there were no significant differences between the groups and no significant differences in Emax and pD2 (Table 1). The application of L-NAME (30 µmol/L), a potent inhibitor of NO synthesis, impaired the relaxation responses elicited by EFS and led to an increase in basal tension. This impairment in relaxation response was restored by l-arginine (300 µmol/L) and it was concluded that the relaxation responses elicited by EFS were nitrergically mediated. There were significant differences between control and CRF rabbits in EFS-induced frequency-dependent relaxations (Fig. 1d). The Emax and pD2 values for each drug in control and CRF groups are also shown in Table 1.
|Mean (sd) variable||Controls||CRF|
|Body weight, kg||2.60 (0.04)||2.13 (0.01)*|
|Emax||80.2 (6.1)||12.86 (5.40)*|
|PD2||6.82 (0.056)||6.92 (0.079)|
|Emax||98.0 (4.8)||93.0 (4.3)|
|PD2||5.86 (0.063)||5.84 (0.072)|
|Emax||98.8 (2.2)||98.0 (1.73)|
|PD2||4.64 (0.063)||4.72 (0.069)|
|EFS Emax||76.2 (5.1)||30.5 (4.2)*|
As discussed previously , a 4-week period of uraemia after 83% nephrectomy (CRF rabbits) is a suitable model to investigate the effects of uraemic status; serum urea and creatinine were significantly higher in CRF than in control rabbits, and the T3, T4, and parathyroid hormone levels significantly greater and testosterone levels significantly lower in CRF rabbits.
There was an impairment of the endothelium-dependent mechanism of corporal smooth muscle relaxation in cavernosal tissue strips from CRF rabbits. Endothelium-dependent relaxation of corporal tissue to carbachol and relaxation responses to EFS were also significantly lower.
Recent studies suggest that NO mediates both neurogenic and endothelium-dependent relaxation of trabecular smooth muscle [9,13,14]. NO synthase synthesizes NO by converting l-arginine to l-citrulline. Soluble NO freely diffuses into corpus cavernosum smooth muscle cells and activates soluble guanylate cyclase, which than converts GTP to cGMP in the tissue cells. Intracellular increases in cGMP levels have been shown to be directly related the process of smooth muscle cell relaxation and tumescence .
NO is widely accepted to be important in the relaxation of corporal smooth muscle and vasculature. The endothelium and/or the nerves innervating the corpus cavernosum may be the source of NO and probably more than one isoform of NO synthase may be involved. NO is present in cavernosal nerves and their terminal endings in the corpora cavernosa, and in the branches of the dorsal penile nerves and neural plexus in the adventitia of the deep cavernosal arteries [15,16]. Dail et al. showed that all smooth muscle regions of the penis of rat were richly innervated by nerves containing neuronal NO synthase and that the endothelium of the vessels stained for both endothelial NO synthase and NADPH diaphorase. In a recent study, in the rats with experimental CRF, it was concluded that CRF is associated with oxidative stress which promotes NO inactivation by reactive oxygen species leading to functional NO deficiency, hypertension and widespread accumulation of protein nitration products. In the same study, CRF did not change the plasma l-arginine level, whereas antioxidant therapy ameliorated the CRF-induced hypertension, improved vascular tissue NO production, lowered tissue nitrotyrosine burden, and reversed down-regulations of NO synthase isoforms .
That CRF impaired EFS-induced neurogenic relaxation at all frequencies in trabecular smooth muscle suggest a possible common pathophysiological mechanism by which changes in the NO/cGMP pathway and other possible pathways of CRF may impair the relaxation of trabecular smooth muscle or diminish its sensitivity to NO. However, these possibilities are unlikely, as the corporal strips relaxed in response to SNP, which is metabolized by the smooth muscle to NO . The normal responses to SNP in the CRF rabbits therefore indicate a normal cGMP-dependent relaxation of corporal smooth muscle from CRF rabbits. Thus it is possible that CRF impairs the synthesis or availability of NO in corpus cavernosum tissue. In addition, there were no differences in the KCl-induced contractile responses between control and CRF rabbits. Thus, the contractile mechanisms were intact in the cavernosal smooth muscle.
The relaxation of human corporal smooth muscle in response to acetylcholine requires the presence of an intact endothelium . Another finding of the present study was that endothelium-dependent cavernosal relaxation in response to carbachol was decreased in the CRF rabbits. Corporal relaxation responses to SNP were identical in the controls and CRF rabbits. Because this inorganic nitrate donates NO directly to smooth muscle , this finding indicates that the smooth muscle response to NO is not altered by CRF. In addition to these findings, the relaxation responses to papaverine were similar between the controls and CRF rabbits, suggesting that the decrease in the endothelium-dependent relaxation response to carbachol in CRF rabbits probably occurs at the level of the endothelium and not the smooth muscle cells, and is most likely a result of endothelial cell responses to carbachol receptor-mediated activation.
The thyroid factors were significantly increased and testosterone levels significantly decreased in CRF rabbits. It is known that testosterone and its metabolite 5α−dihydrotestosterone stimulate neuronal NO synthase gene expression and increase the amount of NO produced by corpus cavernosum and penile arteries during erection [20,21]. Hyperthyroidism can cause secondary hypogonadism and increased sex hormone-binding globulin may contribute to lowering free testosterone levels . Özdemirci et al. suggested that hyperthyroidism impairs both neurogenic and endothelium-dependent relaxation of corporal smooth muscle and appears to be related to the alteration of the NO/cGMP pathway, or hyperthyroidism might impair the relaxation of corporal smooth muscle or diminish its sensitivity to NO.
In conclusion, the present results suggest that CRF inhibits the NANC-mediated and carbachol-induced relaxation of the rabbit corpus cavernosum smooth muscle. Changes in the NANC-mediated relaxation of corporal smooth muscle in the rabbit are probably a results of uraemia and hyperthyroidism, hyperparathyroidism or low testosterone levels in CRF. Further evaluation is required to clarify which of the possible mechanisms contributes to ED in CRF.
chronic renal failure
l-arginine N-nitro-L-arginine methyl ester.