1 The antagonistic actions of parafluorohexahydrosiladiphenidol (pFHHSiD) at muscarinic receptors has been studied in cardiac muscle, smooth muscle and cell culture preparations. In this paper, the classification scheme of Doods et al. (1987) is employed. This scheme is based upon differential affinities of muscarinic antagonists. pFHHSiD exhibited high pA2 values at M3 receptors mediating contractions of guinea-pig ileum and oesophageal muscularis mucosae (7.8 and 8.2 respectively) whereas low values were determined at M2 receptors mediating negative inotropic responses in guinea-pig atria (6.0). Intermediate pA2 values were determined at M1 receptors mediating contractions of the canine femoral and saphenous veins. 2 The pA2 values of pFHHSiD at receptors mediating endothelial-dependent relaxation of rat aortic rings, rabbit jugular vein and canine femoral artery (7.6-7.9) were similar to those determined on the ileum. However, the pA2 values of pFHHSiD at receptors mediating contractions of the guinea-pig trachea (7.1), which has been previously shown to possess M3 receptors, were different from those determined in the ileum. 3 The similarity in pA2 values of pFHHSiD between the M3 receptors in guinea-pig ileum and the receptors mediating endothelial-dependent relaxations provide further evidence for the role of M3 receptors in this vascular response. Taken together, pA2 values for pFHHSiD range from 7.1 to 8.2, depending upon the M3 preparation used. The selectivity of the compound therefore for the M3 versus the M2 muscarinic receptor ranged from 13 to 163 fold. 4 At muscarinic receptors mediating stimulation of phosphatidylinositol hydrolysis, pFHHSiD paradoxically displayed a high affinity for the M1 receptor in the SH-SY5Y cell line (pA2 = 7.9) as well as for the M3 receptor in the human astrocytoma (1321 NI cell line (pA2 = 7.6). The value at the M1 receptor in SH-SY5Y cells was greater than was observed at M1 receptors mediating contractions of both the canine saphenous and femoral veins (7.1). 5 pFHHSiD, therefore, clearly delineated M3 from M2 muscarinic receptors, whilst the separation between M1 and M3 receptors was variable. The reason for the anomalous affinity estimates in some functional studies remains unclear. These data indicate that the pA2 values for pFHHSiD appear to be tissue-dependent since the M3 selectivity varies according to the preparations studied. As a result the utility of pFHHSiD in muscarinic receptor classification is limited.

Previous studies have demonstrated species‐specific differences in 5‐hydroxytryptamine3 (5‐HT3) receptors, but unequivocal evidence of 5‐HT3 receptor subtypes, within a species, has not yet been obtained. The purpose of the current study was to test for heterogeneity in 5‐HT3 receptors in murine tissues. 5‐HT3 receptors in membranes derived from brain cerebral cortex of CD‐1, C57BI/6, and Swiss Webster mice and ileum of CD‐1 mice were labeled with the 5‐HT3 receptor antagonist [3H]RS‐42358–197. Structurally diverse competing ligands were then used to characterize the binding site. [3H]RS‐42358‐197 bound with similar affinity in each of the cortical tissues (mean KD= 0.14 nM; range, 0.06–0.32 nM) but bound with lower affinity in ileal tissue (2.5 nM). The density of sites labeled with [3H]RS‐42358–197 ranged from 10.4 fmol/mg of protein in Swiss Webster mouse cortex to 44.2 fmol/mg of protein in Sprague‐Dawley rat cortex. Displacing ligands produced a pharmacologic profile of the [3H]RS‐42358–197 binding site consistent with it being a 5‐HT3 receptor: (R)‐YM060 > (S)‐zacopride > (R)‐zaco‐pride > MDL 72222 > 2‐methyl‐5‐HT. However, 10‐fold differences in the affinity of certain ligands were found when comparing 5‐HT3 binding sites in membranes from cerebral cortex of the different strains of mice and when comparing 5‐HT3 binding sites in brain and ileal membranes prepared from the CD‐1 mouse strain. Ligands demonstrating selectivity included RS‐42358–197, (R)‐za‐copride, 1‐(m‐chlorophenyl) biguanide, (R)‐YM060, and MDL 72222. These studies demonstrate tissue‐and strain‐dependent differences in murine 5‐HT3 binding sites. This suggests that 5‐HT3 receptors exist as multiple subtypes within species and that subtype‐selective 5‐HT3 ligands may be identified.

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1 Muscarinic receptors present in the human astrocytoma cell line 1321 N1 were characterized in radioligand binding studies and in functional studies of carbachol-stimulated phosphatidylinositol (PI) turnover. 2 In radioligand binding studies the muscarinic receptor in intact cells could be labelled usingIn the intact cells these receptors displayed low pirenzepine affinity (pKi = 6.83) indicating that they were not of the M1 subtype. Furthermore, the 1321 Ni muscarinic receptors displayed low affinity for the two M2-cardiac selective ligands methoctramine (pKi = 5.82) and AF-DX 116 (pKi = 6.29). This pharmacology was consistent with the 1321 NI cells containing a single population of muscarinic receptors that displayed a similar pharmacology to the M2-receptor present in exocrine gland tissue. 3 The M2-gland nature of the receptors was further indicated in the functional studies where antagonist affinities were determined from their ability to antagonize carbachol-stimulated PI turnover in 1321 Ni cells. pA2 values for pirenzepine (7.31), methoctramine (6.10) and AF-DX 116 (6.52) were similar to those determined in the binding studies. 4 From these studies we conclude that 1321 N1 astrocytoma cells contain an M2-gland muscarinic receptor which mediates muscarinic receptor-mediated stimulation of PI turnover in these cells.

1 The action of hexamethonium has been studied at a range of muscarinic receptors in vitro by use of both functional and radioligand binding studies. 2 In functional studies, hexamethonium exhibited little or no significant (P < 0.05) antagonism of contractile responses to carbachol at muscarinic receptors in the guinea-pig ileum, oesophageal muscularis mucosae, urinary bladder and trachea. However, antagonism was observed at muscarinic receptors in the guinea-pig left atria mediating negative inotropic responses and the calculated pKB value was 3.80. Hexamethonium also antagonized contractile responses to carbachol in the canine saphenous vein. The pKB value at these receptors was 3.75. 3 In the presence of 3.2 mm hexamethonium, the pA2 value for methoctramine at atrial muscarinic receptors was reduced by approximately 10 fold (control pA2 value was 7.81 + 0.05; pA2 value in hexamethonium was 6.73 + 0.04). In contrast at tracheal muscarinic receptors, the pA2 values for methoctramine were unaffected in the presence of 3.2mm hexamethonium (control pA2 = 5.58 + 0.07; pA2 value in hexamethonium was 5.63 + 0.12). All values quoted are mean + s.e.mean, n = 8. 4 In competition radioligand binding studies, hexamethonium exhibited a higher affinity for cardiac M2 receptors (pKi = 3.68) than for cerebrocortical M1 receptors (pKi = 3.28) or for submaxillary gland M3 receptors (pKi = 2.61). At M2 receptors hexamethonium at concentrations of 0.1-10mm, increased the half life of the dissociation rate of [3H]-N-methylscopolamine 1.6-4.3 fold. This was observed at M3 receptors only at 10 mM, when the half life was increased 1.7 fold. 5 We conclude that hexamethonium, in addition to its well characterized nicotinic antagonist properties, can act as a weak muscarinic antagonist and differentiates between cardiac M2 receptors and glandular/smooth muscle M3 receptors. However, hexamethonium differentiates less clearly between M1 and M2 receptors. The selectivity between M2 and M3 receptors observed in the present study with hexamethonium is comparable to other M2 selective antagonists such as AF-DX 116 and himbacine. 6 Caution should be exercised with regard to the inclusion of hexamethonium in functionsal studies of M2 muscarinic receptor subtypes at concentrations of 0.1 mm and above.