6103BCBMOL ` 829886
Design and critique a series of experiments to investigate the effects of acetylcholine on
calcium oscillations (2500 words maximum)
Calcium oscillations were first discussed late 1970’s (Glitsch and Pott, 1975, Rapp and Berridge,
1977, Angus, Bobik and Korner, 1977, Dundee, Lilburn, Toner, and Howard, 1978) with an influx of
research in the 1980’s (Lakatta, Capogrossi, Kort, and Stern, 1985, Eisner and Valdeolmillos, 1986,
Holl et al, 1988, Berridge, 1989) and 1990’s (Harootunian, Kao, Paranjape, and Tsien, 1991, Berridge,
1991, Lindströum et al, 1993, Hajnóczky, Robb-Gaspers, Seitz, and Thomas, 1995, Dolmetsch, Xu,
and Lewis, 1998). Dean and Matthews (1968) discussed how sodium and calcium ions were
necessary for insulin release and deduced that the theory of electrical activity in nerve and muscle
cell also applied to pancreatic islet cells. Most oscillatory research was conducted in muscle or
cardiac cells. Palade, Mitchell and Fleischer (1983) investigated spontaneous calcium release from
the sarcoplasmic reticulum in rabbit skeletal muscle cells. They considered that calcium may be
spontaneously released via calcium-induced calcium release or that it may be released
physiologically. The intracellular release of Ca 2+ can result from the inositol 1,4,5-trisphosphate (IP 3)
pathway as shown in figure 1. Calcium oscillations are ubiquitous signals that mediate a multitude of
kinetically distinct processes ranging from extremely fast events such as neurotransmitter release
and skeletal muscle contraction to slower responses including gene transcription and cell
proliferation (reviewed by Cuthbertson and Chay, 1991, Parekh, 2000, Dupont, Combettes, Bird, and
Putney, 2011). Through the regulation of inositol 1,4,5-trisphosphate receptors
Oscillatory spikes are generated through repetitive exchanges of Ca 2+ between the endoplasmic
reticulum (ER) and the cytosol( reviewed by Wacquier, Combettes, Van Nhieu, and Dupont, 2016)
Acetylcholine is a neurotransmitter first discovered by Arthur Ewins and his colleague Henry Dale
(Ewins, 1914, Tansey, 2006), regarding the pancreas it is essential in the production of insulin
(Molina et al, 2014). The muscarinic M3 receptor is the acetylcholine receptor which regulates
calcium influx via the control of monovalent cation channels (Carroll and Peralta, 1998) and has been
shown to play a crucial role in the maintenance of blood glucose homeostasis in mouse models
(Ahrén, 2000, Molina et al, 2014). Acetylcholine is needed for beta cell function as it induces insulin
secretion by increasing the cytoplasmic free Ca 2+ concentration, [Ca2+]i, and enhancing the effects of
Ca2+ on exocytosis in beta cells via protein kinase C (Rodriguez-Diaz et al, 2011) . The three
experiments discussed in this report are to investigate the effects of acetylcholine on calcium
oscillations in pancreatic acinar cells and how they can be altered.
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Figure 1. The pathway in which calcium (Ca 2+) is released from the endoplasmic reticulum via IP 3.
Figure 15-39 taken from Molecular Biology of the Cell (© Garland Science 2008)
Methods and Materials
Substances. Acetylcholine chloride (ACh) HEPES (N-2-hydroxyethylpiperazine-N-ethanesulfonic acid)
(Shipman, 1969) -buffered physiological saline, Collagenase type II, Phorbol 12-myristate 13-acetate
(TPA) would be purchased from Sigma-Aldrich. FURA-2/AM would be purchased from Thermofisher.
Preparation of pancreatic acinar cells. Pancreatic acinar cells were prepared according to the
protocol of Yoon et al, 2017a (used throughout all 3 experiments). Briefly, the dissected tissue was
enzymatically digested for 30 minutes with type II collagenase in HEPES-buffered physiological saline
(physiological solution containing (mM): NaCl 140, KCl 4.7, CaCl2 2, MgCl2 1.1, glucose 10 and N-2-
hydroxyethylpiperazine-N8-2-sulfonic acid (HEPES) 10, pH 7.4.) containing 0.01% trypsin inhibitor
(soybean) and 0.1% bovine serum albumin, followed by incubation at 37°C under gentle agitation,
Pariente et al, 2001 suggests using 60 cycles/min to release the cells into the medium solution
however, according to research carried out by Hosseinizand, Ebrahimi and Abdekhodaie (2016),
60rpm agitation rate causes damaging levels of wall shear stress resulting in lower cell expansion. As
mammalian cells are very shear sensitive, because of mixing cell damage in suspension cultures is
quite common. However, 40rpm was shown to have significantly lower cellular damage and, 20rpm
was related to insufficient mixing and generation of a non-homogenous environment and can cause
concentration gradients in nutrients, metabolites, and cytokines (Choi et al, 2010b, Hosseinizand,
Ebrahimi and Abdekhodaie, 2016). Therefore, the mixture was gently agitated at 40rpm for 10
minutes at 37oC. The procedure was repeated several times (usually 4–6) to ensure all cellular debris
and tissue traces were separated and removed from the solution (Pariente et al, 2001). In cell
culture applications, trypsin inhibitors were used to further inhibit tryptic activity during cell
dissociation, preventing cell damage and death (Sigma-Aldrich), HEPES-buffered saline was used
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