Involvement of nitric oxide in the non-adrenergic non-cholinergic neurotransmission of horse deep penile arteries: role of charybdotoxin-sensitive K(+)-channels.
1. The involvement of nitric oxide (NO) and the signal transduction mechanisms mediating neurogenic relaxations were investigated in deep intracavernous penile arteries with an internal lumen diameter of 600-900 microns, isolated from the corpus cavernosum of young horses. 2. The presence of nitric oxide synthase (NOS)-positive nerves was examined in cross and longitudinal sections of isolated penile arteries processed for NADPH-diaphorase (NADPH-d) histochemistry. NADPH-d-positive nerve fibres were observed in the adventitia-media junction of deep penile arteries and in relation to the trabecular smooth muscle. 3. Electrical field stimulation (EFS) evoked frequency-dependent relaxations of both endothelium-intact and denuded arterial preparations treated with guanethidine (10(-5) M) and atropine (10(-7) M), and contracted with 10(-6) M phenylephrine. These EFS-induced relaxations were tetrodotoxin-sensitive indicating their non-adrenergic non-cholinergic (NANC) neurogenic origin. 4. EFS-evoked relaxations were abolished at the lowest frequency (0.5-2 Hz) and attenuated at higher frequencies (4-32 Hz) by the NOS inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-3) M). This inhibitory effect was antagonized by the NO precursor, L-arginine (3 x 10(-3) M). NG-nitro-D-arginine (10(-4) M) did not affect the relaxations to EFS. 5. Incubation with either the NO scavenger, oxyhaemoglobin (10(-5) M), or methylene blue (10(-5) M), an inhibitor of guanylate cyclase activation by NO, caused significant inhibitions of the EFS-evoked relaxations, and while oxyhaemoglobin abolished the relaxations to exogenously added NO (acidified sodium nitrite, 10(-6) - 10(-3) M), there still persisted a relaxation to NO of 24.4 +/- 5.1% (n = 6) in the presence of methylene blue. 6. Glibenclamide (3 x 10(-6) M), an inhibitor of ATP-activated K(+)-channels, did not alter the relaxations to either EFS-stimulation or NO, while the blocker of Ca(2+)-activated K(+)-channels, charybdotoxin (3 x 10(-8) M), caused a significant inhibition of both the electrically-induced relaxations and the relaxations to exogenously added NO. Furthermore, charybdotoxin blocked relaxations induced by the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8 Br-cyclic GMP). 7. These results suggest that relaxations of horse deep penile arteries induced by NANC nerve stimulation involve mainly NO or a NO-like substance from nitrergic nerves. NO would stimulate the accumulation of cyclic GMP followed by increases in the open probability of Ca(2+)-activated K(+)-channels and hyperpolarization leading to relaxation of horse penile arteries.
Simonsen
U
U
Departamento de Fisiologia, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Prieto
D
D
Sánez de Tejada
I
I
García-Sacristán
A
A
D000320:Adrenergic Fibers / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D018999:Charybdotoxin / Q000494:pharmacology*; D002799:Cholinergic Fibers / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D004558:Electric Stimulation; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000294:innervation; D005905:Glyburide / Q000494:pharmacology; D006736:Horses; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology; D009126:Muscle Relaxation / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000294:innervation; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009412:Nerve Fibers / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D019335:Nitroarginine; D010108:Oxyhemoglobins / Q000494:pharmacology; D010413:Penis / Q000098:blood supply* / Q000187:drug effects; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*
D010108:Oxyhemoglobins; D015221:Potassium Channels; D018999:Charybdotoxin; D019335:Nitroarginine; D009569:Nitric Oxide; D001120:Arginine; D009252:NADPH Dehydrogenase; D005905:Glyburide; D008751:Methylene Blue