The Role of phospholipase-C β1 in intracellular calcium release in smooth muscle contraction Neel Gohil.

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The Role of phospholipase-C β1 in intracellular calcium release in smooth muscle contraction Neel Gohil.

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The Role of phospholipase-C β1 in intracellular calcium release in smooth muscle contraction Neel Gohil

What is smooth muscle contraction (SMC)? When myosin filaments move towards the dense bodies on their sides resulting in dense bodies coming closer to each other

Role of intracellular calcium in SMC? Calcium is bound to calmodulin Myosin light chain kinase is activated Myosin in thick filaments is phosphorylated Leads to SMC

Role of phospholipase C- β1 (PLC-β1) Once agonist acetylcholine (ACh) is bound to its muscarinic receptor, it activates the G-protein which interacts with PLC-β1 Cleave the bond between phosphatidylinositol 4,5-bisphosphate (PIP2) and lipid bilayer How does PLC-β1 effect intracellular calcium release?

Gene Knockout cell line PLC-β1 knockout KO mice will be obtained online with the null mutation on a C57Bl6 background strain http://www.findmice.org/summary?gaccid=MGI:97613. Smooth muscle tissues (myocytes) will be isolated enzymatically as done in (Tokita 2015)

Fluo-4 dye

Fluo-4AM dye The dye is now able to enter the cell due to no ionization

Experiment Procedure Fluo-4 AM solution will be mixed with a buffer solution Myocytes will be placed in a chamber of a slide Fluo-4 solution will be added to the slide and incubated Non-specific esterase

Experiment procedure (micro-fluorometer) Myocytes will be placed in micro-fluorometer ACh will be added on top of the myocytes Laser pointed at 485nm images analyzed using ImageJ software on an inverted LCD microscope

Experiment procedure (Fmin) Triton X-100 (detergent) will be added to lyse myocytes BAPTA will be added to bind to calcium Fluorescence will be imaged using inverted microscope with LCD monitor

Experiment procedure (Fmax) Calcium in the form of CaCl2 will be added to the solution Saturation of BAPTA Increase in fluorescence

Discussion It is anticipated that there will be an increase in Ca2+ release in myocytes from wild type mice in response to ACh and that there will be no Ca2+ release in myocytes from PLC-β1 KO mice. This is because there will be no PIP2 hydrolysis, and IP3 release in PLC-β1 KO mice. Understanding the role of these enzymes will help us understand the regulation of smooth muscle contraction and how to address problems related to lack of or unregulated contraction of the smooth muscle.

Questions?