Identification Of Ip₃R-Ⅲ In Secretory Granules Of Rat Pancreatic Islet β Cells By Immunoelectron Microscopy

IntroductionThe lack of insulin secretion/insulin resistance are the main factors ofdiabetes. After some substances such as glucose, glucagon, lactose or ribosestimulus β cell, insulin is secreted to transport to the target tissue （such asmuscle, liver and adipose tissue）through blood circulation，then plays thephysiological effect by combining with insulin receptor. The main effect ofinsulin is to decrease blood glucose, at the same time to promote glycogen, fatand protein synthesis.Previous studies have proven，the cracking efflux insulin secretion byinsulin secretory granule of pancreatic β cells is directly triggered by theincreased intracellular Ca²⁺concentration. Insulin secretion is triggered by a rapid rise of calcium ions concentration due to mobilization from intracellularstores and/or influx of extracellular Ca²⁺. There are several Ca²⁺stores in βcells[1]， such as IP3-sensitive calcium store，NAADP-sensitive calcium store，ryanodine-sensitive calcium store and mitochondrial calcium store. IP3-sensitivecalcium store was first discovered in mouse pancreatic cells，later studiesconfirmed that it also existed in a variety of cells. IP3R is a kind of ligand-gatedCa²⁺release channel protein which exists in the endoplasmic reticulum，sarcoplasmic reticulum and nuclear membrance. Secretory granules is a kind ofcalcium stores in β cells, which has the highest calcium content. After granulemembrane fusions with plasma membrane, exocytosis process is occurred. Theefflux of Ca²⁺from granules accompanies with insulin contents in order tomaintain the dynamic equilibrium of Ca²⁺which exists outer and inner ofplasma membrane. This process may be achieved by the activation of IP3R insecretory granule membrane. The mechanism that secretory granules participateand regulate the process of insulin secretion is not clear. This respect of report isvery few, and the existance of IP3R-Ⅲ is still controversial[3]. In this study，weimproved Blondel’s experimental design[2]， the IP3R-Ⅲ type of insulinsecretory granules in the rat pancreatic islet β cell was localized by usingcolloidal gold immune electron microscopy. The results showed that IP3-IP3RⅢmay be involved in the regulation of insulin secretion in β cell.ObjectiveThe IP₃R-Ⅲ type of insulin secretory granules in the rat pancreatic islet βcell was localized by using colloidal gold immune electron microscopy. Thisstudy will further understand the mechanism of insulin secretory granules in the process of insulin secretion.Methods1. The pancreatic choledoch of male SD rat retrogradely primed Ⅴtypecollagenase digestion, purified islets in histopaque-1077.2. The islets were identified with dithizone solution.3. Islet specimens were fixed and prepared for electron microscopy.4. Immunohistochemical staining for IP₃R-Ⅲ type on insulin secretorygranules of SD rat pancreatic islet β cells. Primary antibody was rabbitanti-rat IP₃R-Ⅲ antibodies, secondary antibody for5nm colloidalgold-labeled goat anti-rabbit IgG. The primary antibody of control group wasTBS（pH7.4）， the same as other steps.5. After the staining of uranyl acetate and lead citrate, the samples wereobserved with JEM-2000EX transmission electron microscope.Results1. The islet cells which were stained by dithizone, were opaque entity, round oroval. Their diameter were100-300μm. The islet structural integrity of theboundaries clear, dark down.2. The results showed that immunogold particles were detected in the granularcores and at the periphery of the secretory granules. A few immunogoldparticles were also detected in the endoplasmic reticulum. In the controlgroup photos， there were no immunogold paiticles marked in the insulinsecretory granules.Conclusions 1. It can be used to identify IP₃R-Ⅲ in secreory granules of pancreatic islet βcells by using immunoelectron microscopy.2. The IP₃R-Ⅲ exists in insulin secretory granules indeed.