Human Pancreatic Islets Express the Purinergic P2Y₁₁and P2Y₁₂Receptors

Abstract

P2Y 12 receptors as future antidiabetic targets in human pancreatic islets and to determine their cellular localization: 1. An increase in cAMP is responsible for the glucose-dependant insulinotropic effect of GLP-1, the most promising antidiabetic drug presently in development; 2. P2Y agonists induce a glucose-dependent increase in insulin secretion in human pancreatic islets [11] ; and 3. The cAMP / PKA pathway intervenes in ATP S insulin secretory effect in rat islets [12] . Materials and Methods & Isolation of human isletsHuman pancreata were obtained from healthy brain-dead donors in the context of a human islet transplantation protocol, as previously reported [13] . After perfusion and extraction, organs were maintained at 4 ° C for 2 -6 hours in a solution developed by the University of Wisconsin. Human islets were then isolated after digestion according to a modifi cation of Ricordi ' s automatic method. Islets were then collected by hand-picking under a stereomicroscope and frozen at − 80 ° C. RNA isolation and RT-PCRTotal RNAs were isolated by TRIzol Reagent (Invitrogen, Carlsbad, CA) from human islets and treated with DNAse I. Then, fi rst strand cDNA was generated from 3 g RNA by using Superscript II RNAse H -Reverse Transcriptase (Invitrogen), oligo(dT) (Promega, Madison, WI), and random primers (Promega). Subsequently, PCR was carried out using the following conditions: 95 ° C for 3 minutes, 95 ° C for 30 seconds, 56 ° C for 45 seconds, 72 ° C for 30 seconds (45 cycles), and fi nally 72 ° C for 5 minutes. The sequences of the primers used for P2Y 11 were 5 Ј -ACTGGTGGTTGAGTTC-CTGG-3 Ј and 5 Ј -TCAGGTGGGAGAAGCTGAGT-3 Ј

Purinergic receptors expressed in human skeletal muscle fibres

“…1). Previous research using the P2Y 11 Alomone antibody has shown that the molecular weight of the band varies within the interval 34-45 kDa, depending on the tissue or cell under study [25][26][27][28]. P2Y 4 also gave rise to a single band (∼25 kDa).…”

“…The following primary antibodies were used, either alone or two together (rabbit+mouse): Rabbit polyclonal anti-P2X 1 (APR-001, Alomone Labs, Israel; 1:500; previously used in the following studies [6,8,[13][14][15][16][17][18][19]); mouse polyclonal anti-P2X 4 (H00005025-B01P; Abnova, Taiwan; 1:500); rabbit polyclonal anti-P2Y 1 (APR-009, Alomone Labs; 1:500; previously used in the following studies [18,[20][21][22]); polyclonal anti-P2Y 2 (APR-010, Alomone Labs; 1:500; previously used in the following studies [6,8,18,[22][23][24]); rabbit polyclonal anti-P2Y 4 (P6497, Sigma-Aldrich, Denmark; 1:250); rabbit polyclonal anti-P2Y 11 (APR-015, Alomone Labs; 1:500; previously used in the following studies [18,[25][26][27][28][29][30]); rabbit polyclonal anti-P2Y 12 (HPA013796, Sigma-Aldrich; 1:30; previously used in the following study [31]). The P2X 1 antibody was highly specific and directed against the epitope corresponding to amino acid residues 382-399 of rat P2X 1 (human 15/18 residues identical); the P2Y 11 antibody was corresponding to amino acid residues 357-373 of human P2Y 11 ; western blots for anti-P2X 1 and anti-P2Y 11 are shown on the Alomone homepage.…”

A critical look at the function of the P2Y11 receptor

“…When used for western blot, the #APR-015 antibody resulted in bands of 33-60 kDa, all of which were stated to be monomeric P2Y 11 . In one study, western blot of placental tissue protein extracts resulted in bands of 50, 60, 100, 150, and 200 kDa, which were interpreted as being the result of multimeric P2Y 11 receptor assembly [32]. Multimerization of G protein-coupled receptors including purinergic receptors has been widely observed [33], and, indeed, P2Y 11 is known to form a heterodimer with P2Y 1 , as described below [34].…”

“…Hence, it is not clear what caused the bands seen in the human placental tissue. The 50-kDa band from placental control tissue believed to be P2Y 11 was compared with homogenate from pancreatic islets with a smaller band size of 45 kDa that were also claimed to be P2Y 11 [32]. In most studies, specificity of the signal from the #APR-015 antibody was tested by blocking with the control peptide antigen [11,[35][36][37].…”

Purinergic signalling in endocrine organs

“…Other studies showed clear stimulation of insulin secretion via these receptors at glucose concentrations >8 mM [17,405]. A further two receptors were identified, P2Y 11 and P2Y 12 , in human pancreatic islets and their involvement in stimulation of insulin secretion was postulated [333]. In the hamster β-cell line HIT-T15, P2Y 11 receptors stimulated insulin secretion while P2X7 receptors inhibited it; the net effect depending on the glucose concentration [292].…”