NANC transmitters in the female pig urethra--localization and modulation of release via alpha 2-adrenoceptors and potassium channels

1. To investigate further the release, localization and identity of a non-nitrergic mediator of smooth muscle relaxation in the female pig urethra, we studied the effects of drugs acting at alpha 2-adrenoceptors or K+ channels, the effects of capsaicin and chemical sympathectomy, and the actions of several transmitter candidates. 2. Electrical field stimulation (EFS; frequencies above 12 Hz) of spontaneously contracted smooth muscle strips from the female pig urethra evoked long-lasting, frequency-dependent relaxations in the presence of prazosin, scopolamine, and NG-nitro-L-arginine. Treatment with the selective alpha 2-adrenoceptor agonist UK-14 304 markedly reduced the relaxations evoked by EFS at all frequencies tested (16-30 Hz). The inhibitory effect of UK-14 304 was completely antagonized by the alpha 2-adrenoceptor antagonist rauwolscine. The muscarinic M1 receptor antagonist, pirenzepine, or exogenously administered carbachol, did not have any effects on the electrically evoked relaxations. 3. Inhibition of high conductance Ca2+ activated K+ channels by iberiotoxin or charybdotoxin significantly enhanced the relaxations evoked by EFS at all frequencies. However, inhibition of voltage-sensitive K+ channels with 4-aminopyridine or dendrotoxin-1, treatment with the ATP-sensitive K+ channel blocker, glibenclamide, or treatment with the high and low conductance Ca2+ activated K+ channel blockers, tetraethylammonium chloride and apamin, had no effect on the relaxations evoked by EFS. 4. Electrically evoked relaxations were not affected by adrenergic denervation with 6-hydroxydopamine (6-OHDA) at any frequency. However, treatment with 6-OHDA abolished prazosin-sensitive electrically induced contractions, and a long-lasting relaxation was revealed. Treatment with capsaicin, believed to damage selectively a subpopulation of primary afferent fibres, did not affect basal tone or relaxations evoked by EFS. 5. Exogenously applied vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP)-27, PACAP-38, adenosine, ATP and 5-hydroxy-tryptamine caused relaxations of the urethral preparations, whereas prostaglandin E2 and calcitonin gene-related peptide had no effects. VIP 10-28, alpha, beta-methylene-ATP, reactive blue-2, suramin or indomethacin did not reduce the electrically-evoked relaxations at any frequency. However, the relaxations were slightly reduced by trypsin or alpha-chymotrypsin. 6. The present results suggest that the release of the unknown mediator in the female pig urethra can be modulated via alpha 2-adrenoceptors and high conductance Ca2+ activated K+ channels. Furthermore, the mediator does not appear to be localized to or released from adrenergic or capsaicin-sensitive sensory nerve-endings. The identity of the transmitter remains to be established