Woldemussie E, Feldmann B J, Chen J
Department of Biological Sciences, Allergan, Inc., Irvine, CA 92713.
Exp Eye Res. 1993 Apr;56(4):385-92. doi: 10.1006/exer.1993.1052.
Muscarinic receptors present in cultured human iris sphincter and ciliary smooth muscle cells were characterized by both ligand ([3H]QNB binding) and functional (phosphoinositide hydrolysis) studies. Ligand binding studies showed that [3H]QNB represented a single population of binding sites with KD values of 4.02 x 10(-11) M in the ciliary and 5.6 x 10(-11) M in the iris sphincter cells. In competition studies, the selective antagonist, 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP) was the most potent in displacing [3H]QNB with selectivity of 150-350-fold over pirenzepine (M1) and 450-1700-fold over AF-DX 116 (M2). 4-DAMP recognized one site in the iris sphincter cells (Ki = 0.34 nM) but two sites in the ciliary cells (KH = 0.9 nM and KL = 49 nM). 4-DAMP was also the most potent in inhibiting carbachol-induced hydrolysis of inositol phospholipids (PI) in both cell types. However, the IC50 values for PI hydrolysis were several fold lower than those for [3H]QNB binding. Using these selective antagonists, our data supports the presence of functional muscarinic receptors of M3 subtype in human iris sphincter and ciliary cells. It also shows the presence of a second low affinity site in the ciliary smooth muscle cells that is recognized by 4-DAMP.
通过配体（[3H]QNB结合）和功能（磷酸肌醇水解）研究对培养的人虹膜括约肌和睫状平滑肌细胞中的毒蕈碱受体进行了表征。配体结合研究表明，[3H]QNB代表单一的结合位点群体，在睫状肌中的KD值为4.02×10(-11)M，在虹膜括约肌细胞中的KD值为5.6×10(-11)M。在竞争研究中，选择性拮抗剂4-二苯基乙酰氧基-N-甲基哌啶-甲溴化物（4-DAMP）在取代[3H]QNB方面最有效，对哌仑西平（M1）的选择性为150 - 350倍，对AF-DX 116（M2）的选择性为450 - 1700倍。4-DAMP在虹膜括约肌细胞中识别一个位点（Ki = 0.34 nM），但在睫状肌细胞中识别两个位点（KH = 0.9 nM和KL = 49 nM）。4-DAMP在抑制两种细胞类型中卡巴胆碱诱导的肌醇磷脂（PI）水解方面也最有效。然而，PI水解的IC50值比[3H]QNB结合的IC50值低几倍。使用这些选择性拮抗剂，我们的数据支持人虹膜括约肌和睫状肌细胞中存在M3亚型的功能性毒蕈碱受体。它还表明在睫状平滑肌细胞中存在第二个低亲和力位点，该位点可被4-DAMP识别。
