Activation and potentiation of the NO/cGMP pathway by NG-hydroxyl-L-arginine in rabbit corpus cavernosum under normoxic and hypoxic conditions and ageing

Abstract

1 When nitric oxide synthase (NOS) produces NO from N(G)-hydroxy-L-arginine (OH-arginine) instead of L-arginine, the total requirement of molecular oxygen and NADPH to form NO is reduced. The aim of this work was to evaluate the effects of OH-arginine on the contractility of rabbit corpus cavernosum (RCC) and to compare the capacities of L-arginine and OH-arginine to enhance NO-mediated responses under normoxic and hypoxic conditions and in ageing, as models of defective NO production. 2 OH-arginine, but not L-arginine, was able to relax phenylephrine-contracted rabbit trabecular smooth muscle. OH-arginine-induced relaxation was inhibited by the NOS-inhibitor, L-NNA (300 microM), and by the guanylyl cyclase inhibitor, ODQ (20 microM), while it was not affected by the cytochrome P450 oxygenase inhibitor, miconazole (0.1 mM). Administration of OH-arginine, but not L-arginine, produced a significant increment of cGMP accumulation in RCC tissue. 3 Relaxation elicited by OH-arginine (300 microM) was still observed at low oxygen tension. The increase of cGMP levels induced by ACh (30 microM) in RCC was significantly enhanced by addition of OH-arginine (300 microM) in normoxic conditions, as well as under hypoxia, while L-arginine did not alter the effects of ACh on cGMP accumulation. 4 Endothelium-dependent and nitrergic nerve-mediated relaxations were both significantly reduced in RCC from aged animals (>20-months-old) when compared with young adult rabbits (5-months-old). Treatment with OH-arginine (300 microM) significantly potentiated endothelium-dependent and neurogenic relaxation in corpus cavernosum from aged rabbits, while L-arginine (300 microM) did not have significant effects. 5 Results show that OH-arginine promotes NO-mediated relaxation of RCC and potentiates the NO-mediated responses induced by stimulation of endogenous NO generation in hypoxic and aged tissues. We propose that the use of OH-arginine could be of interest in the treatment of erectile dysfunction, at least in those secondary to defective NO production.

Figure 1

Schematic representation of the metabolic pathway of endothelial or neuronal production of nitric oxide (NO), which stimulates guanylyl cyclase (GC) promoting cGMP generation and relaxation of trabecular smooth muscle. The sites of action of the NO synthase (NOS) inhibitor (N^G-nitro-L-arginine (L-NNA), the cytochrome P450 oxygenase (CYP) inhibitor, miconazole, and the GC inhibitor, ODQ, are shown.

Figure 2

Relaxant effects of L-arginine and N^G-hydroxy-L-arginine (OH-arginine) (1 μM to 1 mM) in rabbit corpus cavernosum strips contracted with phenylephrine. Data are expressed as mean±s.e.mean of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of strips used, each strip from a different animal. ^*Indicates P<0.05 vs L-arginine.

Figure 3

Effects of cytochrome P450 oxidase inhibition with miconazole (0.1 mM) (A) and NO synthase and guanylate cyclase inhibition with N^G-nitro-L-arginine (L-NNA; 0.1 mM) and ODQ (0.02 mM), respectively (B) on relaxations induced by N^G-hydroxy-L-arginine (OH-arginine) in rabbit corpus cavernosum strips contracted with phenylephrine. Data are expressed as mean±s.e.mean of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of strips used, each strip from a different animal. ^***Indicates P<0.005 vs control.

Figure 4

Effects of L-arginine and N^G-hydroxy-L-arginine (OH-arginine) on cGMP accumulation in rabbit corpus cavernosum. Data are expressed as mean±s.e.mean of pmoles of cGMP per mg of tissue protein. n indicates the number of strips used, each strip from a different animal. ^**Indicates P<0.01 vs control.

Figure 5

Relaxant effects of L-arginine (300 μM) and N^G-hydroxy-L-arginine (OH-arginine, 300 μM) in rabbit corpus cavernosum strips contracted with phenylephrine under different oxygen concentrations (20, 5, 2 and 1%). Data are expressed as mean±s.e.mean of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of strips used, each strip from a different animal. ^*Indiates P<0.05 vs L-arginine.

Figure 6

Effects of L-arginine and N^G-hydroxy-L-arginine (OH-arginine) on the increase of cGMP levels after exposure of rabbit corpus cavernosum to acetylcholine (ACh) under normoxic (A) or hypoxic (B) conditions. Data are expressed as mean±s.e.mean of pmoles of cGMP per mg of tissue protein. n indicates the number of strips used, each strip from a different animal. ^*Indicates P<0.05, ^**P<0.01 vs ACh alone.

Figure 7

Effects of ageing on endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM to 10 μM) (A) and neurogenic relaxation induced by transmural electrical stimulation (TES; 0.5 to 12 Hz) (B) in rabbit corpus cavernosum strips contracted with phenylephrine. Control group consisted of young adult rabbits (5-months-old), while aged rabbits were above 20-months-old. Data are expressed as mean±s.e.mean of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of strips used, each strip from a different animal. ^*** Indicates P<0.005 vs control.

Figure 8

Effects of L-arginine (300 μM) (A) and N^G-hydroxy-L-arginine (OH-arginine; 300 μM) (B) on endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM to 10 μM) in aged rabbit corpus cavernosum strips contracted with phenylephrine. Data are expressed as mean±s.e.mean of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of strips used, each strip from a different animal. ^***Indicates P<0.005 vs control.

Figure 9

Effects of L-arginine (300 μM) (A) and N^G-hydroxy-L-arginine (OH-arginine; 300 μM) (B) on neurogenic relaxation induced by transmural electrical stimulation (TES; 0.5 to 12 Hz) in aged rabbit corpus cavernosum strips contracted with phenylephrine and treated with guanethidine (5 μM) and atropine (0.1 μM). Data are expressed as mean±s.e.mean of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of strips used, each strip from a different animal. ^***Indicates P<0.005 vs control.