piperidines and aceclidine

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA(i)), is most applicable for the analysis of responses at G protein-coupled receptors. RA(i) is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M(2) receptor, including stimulation of phosphoinositide hydrolysis through G(alpha15) in HEK 293T cells, inhibition of cAMP accumulation through G(i) in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through G(s) in CHO cells treated with pertussis toxin. The RA(i) values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy]oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was approximately 10-fold greater at M(2) receptors coupled to G(alpha15) in HEK 293T cells compared with M(2) receptors coupled to G(i) in the same cells or in CHO cells. Our results show that the RA(i) estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Algorithms; Animals; Carbachol; Cell Line; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Diphenylacetic Acids; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Protein alpha Subunits, Gs; Heterotrimeric GTP-Binding Proteins; Humans; Isoproterenol; Oxotremorine; Pertussis Toxin; Phosphatidylinositols; Piperidines; Pirenzepine; Quinuclidines; Receptor, Muscarinic M2; Signal Transduction; Transfection

There are ultrastructural and histochemical differences between the longitudinal (putatively more relevant to outflow facility) and circular (putatively more relevant to accommodation) portions of the primate ciliary muscle. Oxotremorine, a muscarinic agonist putatively somewhat selective for the M2 receptor subtype, binds preferentially to the longitudinal rather than the circular portion. Aceclidine, a putatively non-subtype selective muscarinic agonist, can dissociate accommodative and outflow facility responses in monkeys and humans. We used the muscarinic receptor subtype antagonists 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), 11-([2-(diethylamino)methyl]-1-piperidinyl)acetyl]- 5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116), and pirenzepine to inhibit contractile responses to the muscarinic agonists carbachol, aceclidine and oxotremorine in the longitudinal and circular vectors of the rhesus monkey ciliary muscle in vitro. Oxotremorine generated dose-response curves that were similar in both the circular and longitudinal vectors and intermediate to those previously reported for carbachol and aceclidine. 4-DAMP (M3 selective) was the most potent inhibitor of contractile responses to all three agonists, with IC50 values ranging from 33 to 68 nM for the circular and from 27 to 63 nM for the longitudinal vector, depending on the agonist used to elicit contraction. Pirenzepine (M1 selective) was > or = 25-fold less potent and AF-DX 116 (M2 selective) was > or = 108-fold less potent at inhibiting contractile responses to all three agonists in either vector, indicating that M3 is the predominant receptor subtype mediating ciliary muscle contraction in both vectors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbachol; Ciliary Body; Dose-Response Relationship, Drug; In Vitro Techniques; Macaca mulatta; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Oxotremorine; Piperidines; Pirenzepine; Quinuclidines

Aceclidine can dissociate accommodative and outflow facility responses in monkeys and humans. We sought to determine if different muscarinic receptor subtypes control outflow facility, accommodative and pupillary responses to aceclidine in the living rhesus monkey eye, as a possible basis for this separation. Each eye was cannulated with one branched and one unbranched needle. Baseline measurements (of outflow facility by two-level constant pressure perfusion; refraction by Hartinger coincidence refractometry and pupil diameter by vernier calipers) were recorded after anterior chamber exchange with (one eye) or without (opposite eye), muscarinic receptor subtype antagonist (pirenzepine, AF-DX 116 or 4-DAMP). The eyes were then exchanged a second time with these solutions plus added aceclidine. The response to aceclidine (the differences from baseline) in the presence or absence of antagonist were compared. The M3 muscarinic receptor subtype antagonist 4-DAMP was the most potent inhibitor of all three responses to aceclidine. The dissociation of accommodative, outflow facility and miotic responses to aceclidine in rhesus monkeys does not appear to be due to differences in the muscarinic receptor subtypes that can currently be distinguished pharmacologically.

Topics: Accommodation, Ocular; Animals; Aqueous Humor; Dose-Response Relationship, Drug; Female; Macaca mulatta; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Pupil; Quinuclidines