Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and betaHC9 cells

Abstract

The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is an intracellular Ca 2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP 3 R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP 3 R mRNA to -actin mRNA were 0·08 0·02, 0·08 0·03 and 0·25 0·04 respectively. Types I, II and III InsP 3 R mRNA were also expressed in rat (RINm5F) and mouse ( HC9) pancreatic -cell lines, and rat cerebellum. Type III InsP 3 R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In HC9 cells, types II and III InsP 3 R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP 3 R mRNA in cerebellum. Culture of HC9 cells for 5 days at 2·8 and 25 mM glucose, or RINm5F cells for 7 days at 5·5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP 3 R mRNA in the cells at the higher glucose concentrations. During short-term (0·5-2 h) incubations, HC9 cell type III InsP 3 R mRNA levels increased in response to glucose in a timeand concentration-dependent manner. Actinomycin D inhibited the glucose response. -Ketoisocaproic acid also stimulated HC9 cell type III InsP 3 R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3-Omethylglucose were without effect. The different levels of expression of mRNA for three InsP 3 R isoforms in islets and insulinoma cells, and the influence of glucose and -ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP 3 R subtypes may be unique with each playing a distinct role in -cell signal transduction and insulin secretion.

Proteasomal Activation Mediates Down-Regulation of Inositol 1,4,5-Trisphosphate Receptor and Calcium Mobilization in Rat Pancreatic Islets*

“…Among the CCh-responsive muscarinic receptor subtypes, only m1, m3, and m5 stimulate IP3 production, and m1 and m3 (but not m2) receptor subtypes have been shown to down-regulate IP3Rs (17). In the rat pancreatic islet and insulinoma cell lines, IP3R isoforms I, II, and III have been identified and characterized for regulation in response to short-term and long-term glucose stimulation (3,4). The binding of IP3 to IP3Rs activates the receptor ion channel; and Ca 2ϩ stored in the endoplasmic reticulum, and perhaps in other subcellular organelles, enters the cytoplasm.…”

“…(Endocrinology 142: 1744 -1751, 2001) T HE INOSITOL 1,4,5-TRISPHOSPHATE (IP3) receptor (IP3R) is an integral ligand-activated Ca 2ϩ -selective channel found in many tissues (1,2), including the pancreatic islet ␤-cell (3). The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca 2ϩ channel-blocking agent nimodipine.…”

Adaptive changes in insulin secretion by islets from neonatal rats raised on a high-carbohydrate formula

“…Total RNA was extracted from rat pancreatic islets using TRIzol. First strand cDNA was obtained by RT from 0.5 g of total RNA by use of a random hexamer and SuperScript II ribonuclease H reverse transcriptase kit as described previously (28). Islet cDNA was amplified with primers from a highly conserved sequence among adenylyl cyclase isoforms (types I-VIII), as reported previously (13): sense 5Ј-GCTCTAGACCATCGGTAGCAC-CTACATGGC-3Ј and antisense 5Ј-GCGACATTCACTGCAT-TICCCCAGATGTCATA-3Ј.…”

Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes

“…They strongly express the three IP 3 R isoforms (I-III), although to different levels depending on the species [124][125][126][127][128][129]. Hence, it is not surprising that well established PLC-activating agents, such as ACh, elicit robust IP 3 ICR in ␤-cells [71,130].…”