The Role of Calcium in Signal Transduction: Kinetics and Regulation of Intracellular Calcium During the Photomechanical Response of the Albino Rat Sphincter Pupillae Smooth Muscle
Krivoshik, Andrew Peter
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https://hdl.handle.net/2142/85417
Description
Title
The Role of Calcium in Signal Transduction: Kinetics and Regulation of Intracellular Calcium During the Photomechanical Response of the Albino Rat Sphincter Pupillae Smooth Muscle
Author(s)
Krivoshik, Andrew Peter
Issue Date
2001
Doctoral Committee Chair(s)
Lloyd Barr
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Animal Physiology
Language
eng
Abstract
Intracellular calcium regulates multiple processes in all cells, including contraction in myocytes. In smooth muscle the primary signal transduction cascade for agonist activation is receptor → G-protein → phospholipase C → inositol trisphosphate → intracellular calcium → calcium-calmodulin complexes → myosin light chain kinase → phosphorylated myosin → force. In the isolated vertebrate sphincter pupillae, the myosin phosphorylation cascade can be activated very precisely by controlled light stimuli, which result in equivalently precise photomechanical responses (PMR). This precision makes the PMR ideal for testing quantitative models of regulation of smooth muscle myosin phosphorylation. The PMR is initiated by absorption of light by a member of the opsin family in the cell membrane of the sphincter pupillae myocytes. In frog sphincter pupillae the PMR and the retinal pretectal pupillary reflex are equally strong; however, in mammals, the PMR is much weaker than the contraction elicited by muscarinic receptor activation. Nonetheless, forces developed during mammalian PMRs, 1--50 micronewtons, were easily measured, even in albino preparations. In order to test a quantitative PMR model based on the myosin phosphorylation cascade, force and [Ca2+] i were concurrently measured on a fast time scale following stimulation by light flashes of varying duration and intensity. The calcium kinetics are more complex than those predicted by the PMR model in that force decouples from intracellular calcium and falls while calcium is continuing to rise. While for some other mammalian smooth muscles alternative activating pathways have been proposed, it can be conclude from our kinetic data that an inhibitory component must be added to the myosin phosphorylation cascade. The presence and regulation of certain components of the signal transduction cascade were also tested, including (1) [Ca 2+]i and force are attenuated by cAMP but not cGMP mediators, (2) [Ca2+]i and force are attenuated by heparin, an IP3 antagonist, (3) force development but not [Ca 2+]i release is attenuated by calmidazolium, a calmodulin antagonist.
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