Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells
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There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells.
We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca2+ ([Ca2+]i) and insulin secretion in human beta cells.
Extracellular application of ATP evoked small inward currents (3.4 ± 0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4 ± 2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca2+]i by stimulating Ca2+ influx and evoking Ca2+ release via InsP3-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca2+ release was sufficient to trigger exocytosis in cells voltage-clamped at −70 mV. All effects of ATP were mimicked by the P2Y(1/12/13) agonist ADP and the P2Y1 agonist MRS-2365, whereas the P2X(1/3) agonist α,β-methyleneadenosine-5-triphosphate only had a small effect. The P2Y1 antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca2+]i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca2+]i and exocytosis without affecting Ca2+ influx.
These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y1 receptors, stimulating electrical activity and coupling Ca2+ influx to Ca2+ release from ER stores.
KeywordsATP Calcium Electrophysiology Exocytosis Insulin Islets of Langerhans Purinergic Secretion
Intracellular Ca2+ concentration
P2X2–green fluorescent protein fusion protein
In addition to serving as an energy carrier and intracellular signal, ATP has an important role as an extracellular signal and neurotransmitter. After its release from cells by exocytosis or via non-vesicular pathways, ATP activates two types of purinergic P2 receptors in the plasma membrane. P2X receptors are ligand-gated non-selective cation channels, while P2Y receptors are G-protein coupled. In humans, the P2X and P2Y families comprise 7 and 11 isoforms, respectively [1, 2].
ATP is present at millimolar concentrations in insulin granules [3, 4] and is released from beta cells upon glucose stimulation [5, 6, 7]. There is evidence for the expression of both P2X and P2Y receptors in rat and mouse beta cells, suggesting that ATP acts as an autocrine signal in islets, although it is debatable whether purinergic signalling stimulates or inhibits insulin secretion [8, 9]. Overall, the few studies that have been conducted in human islets suggest a stimulatory role for ATP [10, 11, 12]. However, controversy exists regarding the receptor subtypes and signal transduction pathways involved. While one study proposed that ATP acts principally via P2X3 receptors, membrane depolarisation and increasing the intracellular Ca2+ concentration ([Ca2+]i) , a more recent study suggested a prominent role for P2Y1 receptors and activation of protein kinase C . Involvement of P2X7 has also been proposed . An ATP-evoked, P2X-mediated membrane current in human beta cells has been suggested , although the effect of ATP on glucose-induced electrical activity has not been investigated.
We sought to characterise the effects of extracellular ATP on membrane currents and membrane potential in human beta cells. We found that the effects of ATP were mimicked by the P2Y agonist ADP and demonstrated that autocrine activation of P2Y1 receptors plays a significant role in the regulation of electrical activity, [Ca2+]i and insulin secretion in human beta cells. Autocrine signalling via P2Y1 represents a novel link between Ca2+ influx and Ca2+ release from intracellular stores.
MRS-2279, MRS-2365, MRS-2500, α,β-methyleneadenosine-5-triphosphate (α,β-meATP), 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), heparin, thapsigargin and bafilomycin A1 were from R&D Systems (Minneapolis, MN, USA). Fura-2AM and Fura-2 Na+-salt were from Life Technologies (Burlington, ON, Canada). Nucleotides and other chemicals were obtained from Sigma-Aldrich (Oakville, ON, Canada).
Islet isolation, culture and transfection
Human islets were from the Clinical Islet Laboratory at the University of Alberta or the Alberta Diabetes Institute IsletCore [15, 16]. The study was approved by the local Human Research Ethics Board. Islets were dispersed in Ca2+-free buffer and then plated onto plastic or glass-bottom Petri-dishes (In Vitro Scientific, Sunnyville, CA, USA) and incubated in RPMI-1640 medium containing 7.5 mmol/l glucose for at least 24 h before experiments. For measuring ATP release, cells were infected with an adenovirus encoding a P2X2–green fluorescent protein fusion protein (AdP2X2-GFP) for 24–48 h . All experiments, except for the assessment of insulin secretion, were carried out using dispersed beta cells.
Paraffin-embedded tissue sections were heated in 10 mmol/l Na+-citrate (pH 6) for 10 min. Sections were blocked using 20% goat serum and incubated with anti-P2Y1 (1:50 dilution; P6487; Sigma-Aldrich) and anti-insulin antibodies for 1 h, followed by fluorescently labelled secondary antibodies. Images were captured using a Zeiss Apotome inverted microscope (Carl Zeiss Canada, Toronto, ON, Canada). Identification of beta cells by immunocytochemistry after patch-clamp and Ca2+ imaging was as described previously .
Cells were pre-incubated with Fura-2AM (1 μmol/l) for 15 min. Glass-bottom Petri dishes were mounted onto an inverted microscope (Zeiss Axioobserver, Carl Zeiss Canada Ltd.) equipped with an ICCD-camera and a rapid-switching light source (Oligochrome; Till Photonics, Grafelfing, Germany). Fluorophore, excited at 340 and 380 nm (intensity ratio 10:4) and emission detected at 510 nm, was imaged at 0.5 Hz using Life Acquisition software (Till Photonics). Beta cells were identified by immunostaining and fluorescence ratios were calculated using ImageJ (v1.46r; http://imagej.nih.gov/ij/).
Fifteen size-matched islets (in triplicates) were pre-incubated in 0.5 ml KRB buffer containing 1 mmol/l glucose and 0.1% BSA for 1 h, followed by a 1 h test incubation in KRB with the indicated glucose concentrations and test substances. The supernatant fraction was removed and the insulin concentration was determined using the MSD human insulin kit (Meso-Scale Discovery, Rockville, MD, USA).
Patch-clamp was performed using an EPC-10 amplifier and Patchmaster software (Heka Electronics, Lambrecht, Germany). Patch-pipettes were pulled from borosilicate glass (resistance 3–8 MΩ; Sutter Instruments, Novato, CA, USA). Solutions for whole-cell and perforated-patch recording are detailed in the electronic supplementary materials (ESM) Methods. Cells were continuously superfused (~1 ml/min) with extracellular solution at ~32°C. Rapid application of ATP was performed using a Fast-Step system (Warner Instruments, Hamden, CT, USA). Beta cells were identified by immunostaining or based on cell size (12.5 ± 0.3 pF; n = 189) .
Data are presented as means ± SEM. The n values represent the number of cells, unless indicated otherwise. Statistical significance was evaluated using Student’s t test, or by multiple-comparison ANOVA and Bonferroni post test when comparing multiple groups.
Membrane currents evoked by purinergic receptor agonists
In perforated-patch whole-cell recordings, ATP-evoked an inward current in all beta cells (1.2–8.6 pA) and averaged 3.4 ± 0.7 pA (Fig. 1b). Similar responses were obtained with the P2Y(1/12/13) agonist ADP (4.2 ± 0.6 pA; 0.4–11.1 pA) and the P2Y1 agonist MRS-2365 (2.4 ± 0.3 pA), but not the P2X(1/3) agonist α,β-meATP (Fig. 1b). The ATP- or ADP-evoked current was inhibited 78 ± 13% by the P2Y1 antagonist MRS-2279 (2–3 μmol/l, p < 0.05, n = 4). The ADP-evoked current was inward during voltage ramps from −110 to −60 mV (Fig. 1c, d) and was not inhibited by tolbutamide (4.2 ± 1.1 pA, n = 4; Fig. 1e). The rapid upstroke in Fig. 1e following application of tolbutamide and ADP is likely to be an artefact. Instead, the current was attenuated (by 90 ± 25%; p < 0.05, n = 5) when Na+ was replaced by the membrane-impermeable cation N-methyl-d-glucamine (NMDG+) (Fig. 1f–h).
Effect of purinergic agonists on the membrane potential
Effect of purinergic agonists on [Ca2+]i
Effect of ATP on exocytosis
Exocytosis was elicited by voltage-clamp depolarisations from −70 to 0 mV, which triggers Ca2+ influx through voltage-gated Ca2+ channels. Exocytosis was potentiated 64% and 32% by extracellular application of ATP and ADP, respectively (Fig. 4c, d). We next monitored the membrane capacitance in cells clamped at −70 mV to prevent opening of voltage-gated Ca2+ channels, and in the presence of 5 mmol/l glucose. ATP application alone was sufficient to evoke a clear exocytotic response under these conditions (Fig. 4e). This response was strongly inhibited in cells pretreated with thapsigargin (Fig. 4f).
Expression of P2Y receptors in human beta cells
Exocytotic release of ATP
Autocrine activation of P2Y1 receptors potentiates glucose-induced [Ca2+]i signals, electrical activity and insulin secretion
P2Y1 receptors couple Ca2+ influx to Ca2+ release from stores
This study provides evidence that ATP acts as a positive autocrine feedback signal in human beta cells, by amplifying glucose-induced [Ca2+]i responses. Several findings support a central role for P2Y1 in this: (1) The effects of ATP were mimicked by ADP (at five- to tenfold lower concentrations), which selectively activates P2Y(1/12/13) (only P2Y1 was detected in human islets) ; (2) the selective P2Y1 agonist MRS-2365 increased electrical activity and [Ca2+]i, while the P2X(1/3) agonist α,β-meATP did not; (3) P2Y1 inhibition blocked the ATP-evoked membrane depolarisation; (4) MRS-2500 reduced insulin secretion to a similar extent as the non-specific P2 antagonist suramin . While in agreement with the findings of a recent study , our findings vary from those of another study suggesting a dominant role for P2X3 . However, the latter study employed pyridoxalphosphate-6-azophenyl-2′,5′-disulfonic acid (iso-PPADS) and oxidised ATP at concentrations that also strongly inhibit P2Y1 [21, 22]. The more selective P2X(1/3) blocker TNP-ATP  does not affect [Ca2+]i (Fig. 7a). While others have also suggested a role for P2X7 , human P2X7 has a very low affinity for ATP (half maximal effective concentration [ED50] 0.78 mmol/l) and is insensitive to ADP and AMP , making a role for P2X7 unlikely here.
P2Y1 has a ~20-fold lower affinity for ATP than P2X3 [1, 2]. However, it has been reported that insulin granules contain similar concentrations of ATP and ADP , suggesting that both nucleotides play an important role. We demonstrated that human beta cells secrete ATP in response to increased [Ca2+]i, and the magnitude of ATP-release events was increased by glucose. This could result from intragranular ATP accumulation via granule-resident vesicular nucleotide transporter . It should be noted, however, that in intact rodent islets extracellular ATP plays an important role in synchronising the electrical and Ca2+ responses among beta cells within and between islets through the induction of Ca2+ release from InsP3-sensitive stores [26, 27, 28], in addition to stimulating exocytosis.
Some studies conducted in rodents, particularly mice, have found that ATP inhibits insulin secretion  and that insulin secretion in islets from mice lacking P2Y1 is elevated . This was attributed to direct inhibitory effects of ATP on exocytosis  or voltage-gated Ca2+ currents . It has been reported that adenosine, acting on P1 receptors, inhibits insulin secretion from INS-1 cells , but this was not confirmed in human islets . We show here that ATP stimulates depolarisation-evoked exocytosis without affecting Ca2+ currents in human beta cells. We found no evidence for a negative role of ATP in insulin secretion from human islets, consistent with the potentiation of insulin secretion from human islets following block of extracellular ATP degradation [11, 33].
ATP increased [Ca2+]i in a biphasic manner, with an initial peak reflecting Ca2+ release from stores and a plateau reflecting Ca2+ influx. Similar to rat beta cells , Ca2+ was released via heparin-sensitive InsP3 receptors, but was from thapsigargin-sensitive (ER) rather than bafilomycin-sensitive (acidic) compartments. Our findings differ from those of Jacques-Silva et al, who concluded that Ca2+ stores contribute little to the ATP-evoked Ca2+ signal in human beta cells , but this may be explained by experimental differences: the Ca2+ response under Ca2+-free conditions is transient and will appear small (as AUC) when compared with prolonged agonist application in the presence of extracellular Ca2+. Although a recent study suggests that autocrine activation of P2Y1 stimulates diacylglycerol production in rodent and human beta cells , we were unable to determine a role for phospholipase C as the inhibitor U-73122 (5–10 μmol/l) also suppressed KCl-evoked Ca2+ responses (data not shown).
In mice, P2Y1 receptors depolarise beta cells via inhibition of KATP channels . In contrast, the ADP-evoked membrane current in human beta cells did not reverse at the K+ equilibrium potential and was insensitive to tolbutamide. Instead, the current was abolished by removal or replacement of Na+, indicating a Na+- or non-selective cation conductance similar to P2Y1-activated currents in neurons [35, 36, 37]. This effect was insensitive to thapsigargin (arguing against a store-operated channel) and to Gd3+, a blocker of the Na+ leak channel NALCN . While the molecular identity of the P2Y1-activated leak channel remains unclear, candidates include members of the transient receptor potential channel family .
We now show that blocking P2Y1 receptors also inhibits electrical activity, [Ca2+]i signalling and insulin secretion in human beta cells in the absence of exogenous ATP (Fig. 7). While P2Y1 blockade inhibits spontaneous [Ca2+]i transients in mouse beta cells , this was in the presence of a Ca2+ channel blocker and thus not likely caused by electrical activity and Ca2+ influx. We obtained similar results with two different, selective P2Y1 antagonists [41, 42] that lacked non-specific effects on a number of human beta cell ion channels (ESM Fig. 1).
In cells stimulated with action potential-like depolarisations, P2Y1 blockade reduces [Ca2+]i and exocytosis without affecting Ca2+ currents (Fig. 8). Thus the MRS-2279-sensitive component of the [Ca2+]i increase reflects Ca2+ release from stores triggered by autocrine activation of P2Y1 receptors. This, in combination with activation of diacylglycerol and protein kinase C (DAG/PKC) , potentiates exocytosis. The Ca2+ signal required for insulin secretion is largely generated by Ca2+ influx through voltage-gated Ca2+ channels [43, 44]; our results are compatible with this because membrane depolarisation and Ca2+ influx are necessary to evoke ATP release and initiate the feedback loop. The secondary acceleration of depolarisation-evoked exocytosis, which we suggest reflects ER Ca2+ release, is observed in human [17, 45] but not mouse  beta cells.
Our data indicate that the contribution of Ca2+ release to the glucose-induced Ca2+ signal may have been underestimated. We present some limited data suggesting that release of Ca2+ from thapsigargin-sensitive stores during ATP stimulation is absent in a donor with type 2 diabetes; this could contribute to impaired secretion. There is evidence that ER stress is involved in the pathogenesis of type 2 diabetes  and is associated with reduced SERCA2b [48, 49], the main ER Ca2+ pump in beta cells . The resulting lowering of ER Ca2+ levels may not only promote apoptosis but also impair beta cell stimulus–secretion coupling.
All authors wish to thank the senior author of this work, the late Professor Matthias Braun, for providing guidance and vision in this study and over many years. The authors also thank P. Rorsman (University of Oxford, Oxford, UK) and Q. Zhang (University of Oxford) for their helpful comments, which contributed to the completion of the manuscript, and A. Spigelman (University of Alberta, Edmonton, AB, Canada) for assistance with human insulin assays. Human islets were provided by the University of Alberta Clinical Islet Isolation Laboratory directed by J. Shapiro and the Alberta Diabetes Institute IsletCore programme. We thank J. Lyon (University of Alberta) and T. Kin (University of Alberta) for their work in human islet isolation, and the Human Organ Procurement and Exchange (HOPE) and Trillium Gift of Life Network (TGLN) programmes for efforts in obtaining human pancreases for research.
This work was funded by an operating grant from the Canadian Institutes of Health Research (MOP-106435). Human islet isolation was funded in part by the Alberta Diabetes Foundation and the University of Alberta. SK and RY-D hold studentships from the Alberta Diabetes Foundation. PEM is supported by an AI-HS Scholarship and holds the Canada Research Chair in Islet Biology.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
SK, RY-D, ED, XW, AB, SC and MB researched data. MB designed the study and wrote the manuscript. All authors reviewed/edited the manuscript and contributed to the discussion. PEM analysed data, reviewed/edited the manuscript and takes full responsibility for the work as a whole, including the study design, access to data and the decision to submit and publish the manuscript. All authors, with the exception of MB, approved the final version of the paper.