The subject of this thesis was an investigation of the mechanism of action of smooth muscle relaxants and included two projects. I. the Nature of the NANC Neurotransmitter in the Bovine Retractor Penis and the Rat Anococcygeus Muscles (1) The relaxant action of endothelium-derived relaxing factor (EDRF), the smooth muscle inhibitory factor (IF) isolated from the bovine retractor penis (BRP) muscle, nitric oxide (NO) and sodium nitroprusside (NaNP) were examined on four isolated smooth muscle preparations; the rabbit aortic strip, the BRP muscle, the rat anococcygeus muscle and the guinea-pig trachea. EDRF (released by acetylcholine), the IF and NO produced powerful relaxation of the rabbit aortic strip and the BRP muscle but had little or no effect on the rat anococcygeus muscle or the guinea-pig trachea. The same rank order of potency of these stimuli in all four muscles suggests all might owe their effects to NO. NaNP, however, produced complete relaxation of the rabbit aortic strip, the rat anococcygeus and the BRP muscles at low concentrations but the guinea-pig trachea was much less sensitive. This suggests an action in addition to the release of NO. (2) Haemoglobin added to solutions of the IF, NO or EDRF released from a donor aorta, completely abolished their relaxant properties. Haemoglobin added to the bath solution also greatly reduced effects of NANC nerve stimulation in the BRP and the rat anococcygeus muscles and that of EDRF released in situ, consistent with all acting by virtue of NO. (3) Suspensions of erythrocytes with an intracellular haemoglobin concentration equivalent to that in (2) were as effective as free haemoglobin in abolishing the relaxant effect of NO or EDRF released from the donor aorta. Erythrocytes were less effective in abolishing the response to EDRF released in situ. Erythrocytes had no effect on the inhibitory response induced by the IF or NANC nerve stimulation in the BRP or the rat anococcygeus muscles. (4) The superoxide anion generators, pyrogallol and hydroquinone, both significantly reduced the response to NANC nerve stimulation in the rat anococcygeus muscle but had no effect in the BRP muscle. The inhibition of the NANC response in the rat anococcygeus muscle by pyrogallol was completely reversed by superoxidedismutase. Pyrogallol also inhibited the response to NO in the rat anococcygeus muscle but not that to 3-isobutyl-1-methyl-xanthine (IBMX) confirming this is a selective action. (5) These results suggest the neurotransmitter in the rat anococcygeus muscle may be NO or a substance releasing NO. The transmitter in the BRP muscle which is unaffected by superoxide anions or the environment in which it acts may be different. In addition, EDRF, the IF and the NANC transmitter in the BRP and the rat anococcygeus muscles may rely on NO for activity, but the IF, like the NANC transmitter, may be a less diffusible substance than NO itself. II. The Effects of Cromakalim on Spasmogen- or Stretch-induced Contraction of a Variety of Smooth Muscle Preparations (1) The relaxant effect of cromakalim has been examined on four isolated preparations, the rabbit aortic strip, the BRP muscle, the guinea-pig trachea and the guinea-pig taenia coli contracted by either histamine, carbachol, noradrenaline or KCl. Even though the contribution of external calcium entering through voltage-operated channel (VOC) in these tissues varied, the expected greater effectiveness of cranakalim on tissues using this pathway was not observed. (2) Desensitization was observed in the guinea-pig trachea when the tissue was exposed to cranakalim 30 min before the spasmogen was added. This did not happen when cromakalim was added at the peak of contraction. Such desensitization was not found in the rabbit aortic strip. (3) Verapamil significantly reduced the relaxant effect of cromakalim in the guinea-pig trachea. In contrast, cromakalim potentiated the relaxant effects of isoprenaline and NaNP in the guinea-pig trachea. (4) The relaxant effects of cromakalim on the BRP muscle and the guinea-pig taenia coli were unaffected by haemoglobin (3.3 muM) or apamin (0.5 muM) in concentrations which block completely the relaxant effect of respectively NANC nerve stimulation in these tissues respectively. Methylene blue (30 muM) greatly reduced the response to both cromakalim and NANC nerve stimulation in the BRP suggesting a non-specific action. (5) Cromakalim in concentrations causing maximal relaxation did not increase the tissue level of either cyclic AMP or cyclic GMP in the BRP muscle, although the appropriate synthetic enzymes were present and could be stimulated by forskolin or NaNP. (6) In the BRP muscle, isoprenaline (30 muM) caused maximal relaxation but did not raise the level of cyclic AMP, even though lower concentrations of 2 muM did raise the level of this nucleotide in the rabbit uterus.
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