J. Chapal

Purinergic receptors on insulin-secreting cells.

Summary— The insulin secreting B cell is fitted with the two types of purinergic receptors: P2 (for ATP and/or ADP) and P1 (for adenosine). The activation of P2 purinoceptors by ATP or ADP evokes a biphasic stimulation of insulin secretion from isolated perfused rat pancreas; this stimulation is dose‐dependent between 10−6 and 10−4 M. Non hydrolysable structural analogues are also effective, and the relative potency of various agonists (2‐methylthio ATP ≫ ATP = ADP = α, β‐methylene ATP ≫ AMP) gave evidence for a P2y purinoceptor subtype. Proposed mechanisms include both an increased Ca2+ uptake and an increased intracellular Ca2+ mobilization via the hydrolysis of polyphosphoinositides. ATP (or ADP) potentiates physiological insulin‐secreting agents (glucose and acetylcholine) and P2 purinoceptors could play a physiological role in the stimulation of insulin secretion. The activation of P1 purinoceptors (adenosine receptors) decreases insulin secretion. Using structural analogues of adenosine, the receptor was characterized as an A1 subtype; it is coupled to a pertussis toxin sensitive G protein and it inhibits adenylate cyclase. It is of physiological relevance that the B cell has the two types of purinoceptors with opposite effects. Recently, a metabolically stable structural analogue of ADP, adenosine‐5′‐0‐(2‐thiodiphosphate) or ADPßS, has been described as a potent secretory agent, effective at nanomolar concentrations on isolated perfused rat pancreas. In vivo, this substance is able to increase insulin secretion and to improve glucose tolerance after IV administration in rats and oral administration in dogs. Furthermore in streptozotocin‐induced diabetes, ADPßS retains its insulin secreting effects. These results suggest that P2y purinoceptors could be a new target for antidiabetic drugs.

Evidence for two different P2‐purinoceptors on β cell and pancreatic vascular bed

1 The effects of a 2‐substituted analogue of adenosine 5′‐triphosphate (ATP), 2‐methylthioadenosine triphosphate (2‐methylthio ATP) have been studied on insulin secretion and flow rate of the isolated pancreas of the rat, perfused in the presence of glucose (8.3 mm). 2 2‐Methylthio ATP (16.5–1650 nm) increased insulin secretion in a biphasic and concentration‐dependent manner; the kinetics were comparable to those previously obtained with ATP. A comparison of relative potency between ATP and 2‐methylthio ATP showed that 2‐methylthio ATP was 45 times more potent that ATP. 3 2‐Methylthio ATP also provoked a transient decrease of the flow rate in a concentration‐dependent manner but at concentrations (165–825 μm) about 1000 fold higher than those needed to increase insulin secretion. A comparison of relative potency between the natural derivative and 2‐methylthio ATP showed that 2‐methylthio ATP was only twice as potent as ATP. 4 These and other previous results (with phosphate‐modified analogues of ATP) provide evidence for two different types of P2‐purinoceptors on endocrine cell and vessel cells of the pancreas. A P2Y subtype, mediating an increase of insulin secretion, is present on the β cell of the pancreas. A P2X subtype, mediating vasoconstriction, is present on the vascular bed of the rat pancreas.

Effects of phosphate-modified adenine nucleotide analogues on insulin secretion from perfused rat pancreas.

1 The effects of three methylene analogues of adenosine 5′‐triphosphate (ATP) or 5′‐diphosphate (ADP) have been studied on insulin secretion from the isolated perfused pancreas of the rat: 5′‐adenylmethylene diposphonate or β, γ‐methylene ATP, adenosine 5′‐α, β‐methylene triphosphate or α, β‐methylene ATP and adenosine 5′‐α, β‐methylene diphosphonate or α, β‐methylene ADP. 2 β.γ‐Methylene ATP did not elicit any increase of insulin release; α, β‐methylene ATP and α, β‐methylene ADP induced a biphasic stimulation of insulin secretion; this effect was dose‐related between 1.65 and 165 μmol/1. Relative potency ATP/α, β‐methylene ATP was 1.2 and ATP/α, β‐methylene ADP was 0.31. 3 Our results point to the importance of the steric and electronic characteristics of the polyphosphate chain of the analogues of ATP and ADP in inducing an insulin secretory effect. They support the hypothesis of a purine receptor for ATP and ADP.

Stimulation of insulin secretion and improvement of glucose tolerance in rat and dog by the P2y‐purinoceptor agonist, adenosine‐5′‐O‐(2‐thiodiphosphate)

1 In vivo effect of a P2y‐purinoceptor agonist, adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), on insulin secretion and glycaemia were studied both in rats and dogs. 2 In anaesthetized rats, i.v. administered ADPβS (0.2 mg kg−1) produced an insulin response dependent on the nutritional state of the animals, since we observed only a transient increase in overnight‐fasted rats and a sustained insulin secretion followed by a reduction in plasma glucose levels in fed rats. During an i.v. glucose tolerance test, ADPβS enhanced insulin release and thus increased the glucose disappearance rate. 3 In anaesthetized fasted dogs, i.v. administered ADPβS (0.1 mg kg−1) increased pancreaticoduodenal insulin output and slightly decreased blood glucose levels. 4 In conscious fasted dogs, orally administered ADPβS (0.1 mg kg−1) transiently increased insulinemia and punctually reduced glycaemia. Furthermore, during an oral glucose tolerance test, orally administered ADPβS at the same dose markedly enhanced insulin secretion and consequently reduced the hyperglycaemia. 5 In conclusion, the P2y‐agonist, ADPβS, is a potent insulin secretagogue in vivo, improves glucose tolerance and is effective after oral administration. Thus, the P2y‐purinoceptors of the β cell may be a target for new antidiabetic drugs.

Evidence for an A2-subtype adenosine receptor on pancreatic glucagon secreting cells.

1 The effects of a 5′‐substituted analogue of adenosine, 5′‐N‐ethylcarboxamidoadenosine (NECA) have been studied on glucagon secretion in vitro, using the isolated pancreas of the rat perfused in the presence of glucose (2.8 mM). 2 NECA provoked a peak of glucagon secretion, the kinetics of which were comparable to those previously obtained with adenosine. The effect was concentration‐dependent and appeared at nanomolar concentrations. The EC50 was approximately 4 × 10−8 M. 3 A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. 4 Theophylline (50 μM) considerably decreased the peak of glucagon secretion induced by 1.65 μM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. 5 These and other previous results (low stereoselectivity of N6‐phenylisopropyladenosine) provide evidence for an adenosine receptor of the A2‐subtype being involved in glucagon secretion.

Adenosine-5'-O-(2-thiodiphosphate) is a potent agonist at P2 purinoceptors mediating insulin secretion from perfused rat pancreas

1 The effects of a P2 purinoceptor agonist, adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐S) have been studied on insulin secretion and flow rate of the isolated perfused pancreas of the rat. 2 In the presence of a moderately stimulating glucose concentration (8.3 mm), ADP‐/7‐S (4.95–495 nm) evoked a biphasic insulin response in a concentration‐dependent manner. A comparison of relative potency between ADP‐β‐S and adenosine 5′‐triphosphate (ATP) showed that ADP‐β‐S was 100 times more potent than ATP. On the other hand, in the presence of a non stimulatory glucose concentration (4.2 mm), ADP‐β‐S (165 nm) did not modify the basal insulin secretion. 3 ADP‐β‐S, at concentrations effective on insulin secretion and also at higher concentrations (1.65 and 16.5 μm), provoked an increase of the pancreatic flow rate in a concentration‐dependent manner. 4 Our results show that ADP‐β‐S is a potent insulin secretory P2 purinoceptor agonist. As it is resistant to hydrolysis it might be useful in studying the effect of activation of the P2 purinoceptor of β cells on insulin secretion in vivo.

Evidence for purinergic receptors on vascular smooth muscle in rat pancreas.

The changes in flow rate in the isolated pancreas of the rat in response to natural purine nucleotides and phosphate-modified analogues were recorded. ATP and ADP transiently decreased the flow rate in a dose-dependent manner but only at very high concentrations (greater than 165 microM). In contrast, alpha,beta-methylene ATP, the most active of the drugs used, decreased the flow rate of the preparation at very low concentrations: 0.495 microM induced about 50% of the maximum effect. beta, gamma-Methylene ATP and AMP-PNP had an intermediary potency. Pyrophosphate had no effect. AMP and adenosine had no vasoconstrictor effect even at 1650 microM. The vasoconstrictor effect of alpha,beta-methylene ATP was not antagonized by phenoxybenzamine (6 microM) or by apamin (0.01 microM). In contrast PIT, a P2-purinergic receptor antagonist, partially or totally blocked the alpha,beta-methylene ATP effect depending on the concentrations used. From our results it can be concluded that purinergic receptors of P2-type are present on the vascular smooth muscle of the pancreatic bed in the rat.

Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro.

1 The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). 2 Adenosine induced a transient increase of glucagon secretion. This effect was concentration‐dependent in the range of 0.165 to 165 μM. ATP also induced an increase, but the effect was no greater at 165 μM than at 16.5 μM. 3 2‐Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, β,γ‐methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 μM); in contrast α,β‐methylene ATP and α,β‐methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. 4 Theophylline (50 μM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2‐chloroadenosine, ATP and β,γ‐methylene ATP (1.65 μM). 5 An inhibitor of 5′ nucleotidase, α,β‐methylene ADP (16.5 μM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 μM). 6 These results support the hypothesis of adenosine receptors (P1‐purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine.

P2y purinoceptor responses of β cells and vascular bed are preserved in diabetic rat pancreas

1 To investigate the effect of experimental diabetes on the P2y purinoceptor responses of pancreatic β‐cells and vascular bed, we used adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), a potent and stable P2y agonist. This work was performed in the isolated perfused pancreas of the rat. 2 Diabetes was induced by streptozotocin (66 mg kg−1, i.p.). Five weeks after the induction of diabetes, on the day of pancreas isolation, the animals displayed marked hyperglycaemia (37.6 ± 2.7 mm). Age‐matched rats were used as controls. 3 Insulin response to a glucose stimulation from 5 to 10 mm was completely lost and stimulation of insulin release by the sulphonylurea, tolbutamide (185 μm), was drastically impaired in the diabetic pancreas (maximum responses were 1.5 ± 0.4 and 7.0 ± 1.4 ng min−1 for diabetic and age‐matched rats respectively). 4 In contrast, in the diabetic pancreas ADPβS (15 μm), infused in the presence of glucose 5 mm, elicited an immediate and significant insulin release similar to that observed in the age‐matched pancreas (maximum responses were 7.6 ± 1.5 and 6.7 ± 1.3 ng min−1 respectively). This ADPβS stimulating effect occurred independently of the glucose concentration (5, 8.3 and 28 mm) in the diabetic pancreas. On pancreatic vascular resistance, ADPβS induced a similar vasodilatation in diabetic and age‐matched rats. 5 In conclusion, ADPβS retains its insulin stimulatory and vasodilator effects in experimental diabetes; P2y purinoceptors could therefore be considered as a new target for the development of antidiabetic drugs.

Difference in the potentiating effect of adenosine triphosphate and α,β-methylene ATP on the biphasic insulin response to glucose

1 The effects of exogenous adenine nucleotides and structural analogues on the biphasic insulin response to an increase of glucose concentration in the physiological range (from 4.2 to 8.3 mm) were studied in the isolated perfused rat pancreas. Purinoceptor agonists were added either simultaneously or 15 min before increasing glucose. 2 ATP and ADP at 16.5 μm were ineffective per se in the presence of the non stimulatory glucose concentration (4.2 mm) but markedly potentiated the biphasic insulin response to glucose rise in both experimental protocols. 3 Two more stable analogues of ATP and ADP (adenylylimidodiphosphate and α,β‐methylene ADP (α,β‐MeADP)) at 16.5 μm behaved like the natural compounds: they were ineffective at a glucose concentration of 4.2 mm and potentiated both phases of insulin response to glucose rise. 4 α,β‐MeATP added simultaneously with the high glucose concentration, markedly potentiated the first phase of insulin response to glucose rise but did not potentiate the second one. When α,β‐MeATP infusion began 15 min before glucose rise, the biphasic response to glucose was not potentiated, in contrast to what occurred with ATP. 5 In the presence of α,β‐MeATP, the ATP potentiating effect was unaffected. 6 It is concluded that ATP and ADP, via activation of β cell P2y purinoceptors, potentiates the biphasic insulin response to an increase of glucose concentration. On the other hand, α,β‐MeATP did not behave like natural and other structural analogues of ATP and ADP: this difference appears not to be the consequence of desensitization of β cell P2y purinoceptors by α,β‐MeATP.