Muscarinic effects on cellular functions in cultured human ciliary muscle cells.
OBJECTIVE
To characterize the pharmacology of the carbachol-induced changes of phospholipase C (PLC) activity and intracellular calcium concentration ([Ca2+]i) in cultured human ciliary muscle cells.


METHODS
Changes in PLC activity of cultured human ciliary muscle cells were determined by production of inositol phosphates. Single-cell dynamic fluorescence ratio imaging was used to determine [Ca2+]i.


RESULTS
Carbachol, oxotremorine-M, aceclidine, and pilocarpine stimulated PLC with mean EC50s of 20, 8, 17, and 2 microM, respectively. The effect of carbachol on PLC was partially suppressed by extracellular Ca2+ depletion. This muscarinic effect was blocked by muscarinic antagonists, such as atropine (apparent pKi = 9.12, nonselective for muscarinic receptor subtypes), pirenzepine (pKi = 6.76, selective for the M1 receptor subtype), 4DAMP (pKi = 9.25, selective for the M1 and M3 subtypes), and fHHSiD (pKi = 7.77, selective for the M3 subtype). In [Ca2+]i experiments, carbachol increased [Ca2+]i transients in human ciliary muscle cells in a dose-dependent manner with a mean EC50 of 7 microM. 4DAMP was approximately 100 times more potent than pirenzepine in the inhibition of the carbachol-induced [Ca2+]i increase. [Ca2+]i oscillations were observed after carbachol stimulation and persisted after extracellular Ca2+ depletion.


CONCLUSIONS
Muscarinic agonists activate PLC and increase [Ca2+]i in cultured human ciliary muscle cells through an M3-like muscarinic receptor subtype.
Matsumoto
S
S
Department of Pharmacology, North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth.
Yorio
T
T
DeSantis
L
L
Pang
I H
IH
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010277:Parasympathomimetics / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000502:physiology*; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D010277:Parasympathomimetics; D011976:Receptors, Muscarinic; D010738:Type C Phospholipases; D002118:Calcium