Evidence for the possible involvement of the P2Y(6) receptor in Ca (2+) mobilization and insulin secretion in mouse pancreatic islets

Abstract

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca(2+)](i)) and insulin release in mouse pancreatic beta-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca(2+)](i) rise via cytoplasmic Ca(2+) mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 microM) transiently increased [Ca(2+)](i) in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y(1) receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y(6) receptor agonist UDP (200 microM) transiently increased [Ca(2+)](i) and reduced insulin secretion at high glucose, whereas the P2Y(2/4) receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca(2+)](i) transients induced by 2-MeSADP and UDP were antagonized by suramin (100 microM), U73122 (2 microM, PLC inhibitor), and 2-APB (10 or 30 microM, IP(3) receptor antagonist), but neither by staurosporine (1 microM, PKC inhibitor) nor depletion of extracellular Ca(2+). The effect of 2-MeSADP on [Ca(2+)](i) was also significantly inhibited by MRS2500, a P2Y(1) receptor antagonist. These results suggested that P2Y(1) and P2Y(6) receptor subtypes are involved in Ca(2+) mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca(2+)](i) and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y(1) and P2Y(6), but not P2Y(2) and P2Y(4) receptors.

Fig. 1

Effect of a high concentration of glucose and acetylcholine (ACh) on [Ca²⁺]_i in a single mouse islet in culture. Glucose at 16.7 mM (G16.7, a) and ACh at 100 μM (b) were added to the mouse islet in the presence of 5.5 mM glucose (G5.5) at the time indicated by the bar. The change in [Ca²⁺]_i was monitored for 30 min using excitation at 340 and 380 nm and emission at 510 nm, and the bar indicates exposure to the indicated nucleotide. Each experiment was repeated 3 times

Fig. 2

Effect of ATP, 2-MeSADP, and UDP on [Ca²⁺]_i in a single mouse islet and beta-TC6 cells in culture. ATP at 100 μM (a, d), 2-MeSADP at 100 μM (b, e), and UDP at 200 μM (c, f) were added to the mouse single islet (a–c) and beta-TC6 cells (d–f) in the presence of 5.5 mM glucose at the time indicated by the bar. In beta-TC6 cells, [Ca²⁺]_i rise occurring in each islet-like cluster composed of a few or several cells (9–10 clusters) is shown with overlapping different colors. The change in [Ca²⁺]_i was monitored for 20 min using excitation at 340 and 380 nm and emission at 510 nm, and the bar indicates exposure to the indicated nucleotide. Each experiment was repeated 7–15 times

Fig. 3

The effect of purinergic compounds on [Ca²⁺]_i in a mouse single islet in culture and beta-TC6 cells. The following compounds were tested: ATP (100 μM), 2-MeSADP (100 μM), ADP (100 μM), and UDP (200 μM). The effect of the nucleotides on [Ca²⁺]_i is shown as % of the peak response induced by ATP (100 μM) in the mouse single islet in culture (a) and beta-TC6 cells (b) in the presence of 5.5 mM glucose. Each bar represents the mean ± SE (n = 7–15 in a and n = 24–54 in b)

Fig. 4

Effect of the PLC inhibitor U73122 and the IP₃ receptor antagonist 2-APB on 2-MeSADP- or UDP-induced [Ca²⁺]_i rise in a single mouse islet in culture. U73122 at 2 μM (a, c) and 2-APB at 30 μM (b) or 10 μM (d) were added to the islet 20 min prior to the addition of the nucleotides. The change in [Ca²⁺]_i was monitored for 20 min using excitation at both 340 and 380 nm and emission at 510 nm, and the bar indicates exposure to the indicated nucleotide or the inhibitor. Each experiment was repeated 3–4 times

Fig. 5

Effect of the PLC blocker U73122 and the IP₃ receptor antagonist 2-APB on 2-MeSADP- or UDP-induced [Ca²⁺]_i rise in beta-TC6 cells. Cells were incubated in the presence of either 2 μM U73122 (a, c) or 30 μM 2-APB (b, d) for 20 min prior to addition of the nucleotides. The results are expressed as a change in ratio (calculated by subtraction of a baseline value from a peak height value). Each bar represents the mean ± SE (n = 16–32). *P < 0.05 in comparison to control in the absence of nucleotides

Fig. 6

Effect of 2-MeSADP and UDP on insulin secretion in mouse islets in culture. The islets were incubated in Krebs/HEPES buffer (pH 7.4) containing 5.5 mM (G5.5, a) or 16.7 mM glucose (G16.7, b) in the presence of 100 μM 2-MeSADP or 200 μM UDP for 60 min at 37°C. Each bar represents the mean ± SE (n = 8 in a and n = 10 in b). *P < 0.05 in comparison to control in the absence of nucleotides

Fig. 7

RT-PCR analysis of P2Y receptor subtype mRNAs in mouse islets and beta-TC6 cells. PCR reactions were performed by 40 cycles in the islets (a) and by 30 cycles in beta-TC6 cells (b) with specific primers for each P2Y receptor subtype. The expected PCR product lengths for P2Y₁, P2Y₂, P2Y₄, P2Y₆, and β-actin were 410, 440, 499, 452, and 778 bp, respectively. Reverse transcription was performed with (+) or without (−) a reverse transcriptase to assess genomic DNA contamination. M shows a 100-bp DNA ladder