In young animals the HT A
In young animals, the 5-HT2A melanin antagonist ketanserin, at relatively low concentrations, shifted 5-HT concentration-response curves rightward, suggesting a role for 5-HT2A receptors in mediating 5-HT-induced contractions of the ureter. The slopes of the ketanserin Schild plots for the antagonism of frequency and AUC responses were not significantly different from unity, suggesting that 5-HT2A is the sole receptor subtype responsible for these responses. Furthermore, in these tissues from younger animals the affinity estimate for ketanserin was comparable to that reported at the 5-HT2A receptor in the literature (8.7) (Bonhaus et al., 1995). Also, in tissues from younger animals there was a slight rightward shift of the 5-HT concentration-response curve in the presence of methiothepin, generally a non-selective 5-HT antagonist but it's highest affinity being at the 5-HT2A receptor subtype (Hoyer et al., 1994). This again suggests the involvement of the 5-HT2A receptor subtype. This conclusion is further supported by the lack of effect of all of the other 5-HT receptor subtype selective antagonists. Based on the affinity values published in the literature, for each antagonist a concentration was chosen that would produce a significant blockade of the specific receptor subtype, without an action at the other receptor subtypes. None of these antagonists had any effect on concentration-response curves to 5-HT, supporting the results obtained with ketanserin and again suggesting that 5HT2A is the sole receptor subtype involved in mediating the responses to 5-HT in tissues from younger animals. In ureteral tissues from older animals, again ketanserin had a relatively high affinity, although slightly lower than that obtained in the younger animals. For the frequency responses to 5-HT, the slope of the Schild plot for ketanserin was significantly greater than unity, indicating additional actions of ketanserin in ureters from the older animals. For this reason the affinity estimate of 7.7 obtained from the intercept of the Schild plot for frequency responses is most likely not reflective of the true affinity value for ketanserin. The affinity estimate obtained from AUC responses to 5-HT in tissues from the older animals was also lower (8.0) than values reported in the literature and lower than that obtained in tissues from younger animals. In addition, methiothepin (10nM) did not produce a rightward shift of the 5-HT response curves in tissues from the older animals. Since methiothepin is generally a non-selective 5-HT receptor antagonist (Hoyer et al., 1994), this finding suggests the possibility of methiothepin having an additional action on the 5-HT-induced frequency of phasic contractions in the ureter of older animals, which could be masking the effects of it's 5-HT2A receptor inhibition. It suggests that whilst in the younger animals the 5-HT-induced contractile response is due solely to stimulation of the 5-HT2A receptor subtype, in the older animals the 5-HT2A receptor is responsible for the contractile effect of 5-HT and stimulation of another receptor by 5-HT could be causing a relaxatory effect. When considering which other receptor may be involved in the 5-HT response in older tissues, the additional action of methiothepin is unlikely to be via 5-HT2B or 5-HT2C receptors, since affinity estimates for ketanserin at 5-HT2A receptors following α-methyl-5-HT stimulation of ureters from older animals were comparable to affinity values (8.7) found in the literature (Bonhaus et al., 1995). This is further supported by the lack of inhibition of 5-HT contractile response by the 5-HT2C selective antagonist, RS-10221. The 5-HT4, 5-HT6 and 5-HT7 receptor subtypes were considered, since these receptors are known to mediate relaxatory effects, acting via adenylyl cyclase inhibition (Nichols and Nichols, 2008). However, in the present study the lack of effect of the 5-HT4, 6 and 7 selective antagonists, as well as the 5-HT3 selective antagonist, on 5-HT contractile responses in the ureters from older animals suggests that none of these receptors are likely to be involved and cannot explain the additional action of methiothepin.