Search

In the present work, we have studied adenosine-induced vasodilation in streptozotocin-induced diabetic rats and compared it to that observed in normal age-matched or weight-matched animals. Experiments were performed on a vascular bed, the isolated perfused pancreas, and a large vessel, the thoracic aorta, provided from the same animal. Vasodilator activity was assessed, for isolated pancreas, as the increase in flow induced by the infusion of 2 µM adenosine for 30 min, or for noradrenaline-contracted aortae, as the relaxant response to adenosine (1 µM–1 mM). In both preparations the results obtained with selective adenosine receptors ligands (CPA, CGS 21680 and NECA) agreed with the presence of adenosine receptor of A2a subtype. In normal animals, adenosine vasodilator activity on both preparations diminished with advancing age in the rat, while diabetes was associated with a decreased or increased responsiveness to adenosine in pancreatic vascular bed or aorta, respectively. Further, the involvement of nitric oxide (NO), in relaxant responses, was evaluated by the use of the NO synthase inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME). In all groups of animals, the flow rate of isolated pancreas dropped in the presence of 200 µM l-NAME, but was restored by adenosine to the level observed without l-NAME. l-NAME (10 µM) significantly reduced the dilator response to adenosine in aortic rings from diabetic animals, but not in those from normal rats. These results showed that adenosine vasorelaxant activity is significantly but differentially altered by diabetes according to the origin of the vascular preparation, and suggest that NO is involved in the vasorelaxant activity of adenosine in large vessels of diabetic animals. The potential pathophysiological role of adenosine in the vascular complications of diabetes remains to be determined. [ABSTRACT FROM AUTHOR]

We have recently shown that benzodiazepines with high affinity for peripheral-type receptors such as 4'-chlordiazepam inhibit insulin secretion in vitro. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxami de], a potent and selective ligand for peripheral benzodiazepine binding sites, was also shown to inhibit insulin release from rat pancreatic islets. Both substances have been reported to interact with mitochondrial binding sites. Hence, the present study was performed to investigate the effects of PK 11195 on insulin secretion induced by either a metabolic or a non-metabolic stimulus. In the rat isolated pancreas perfused at a constant pressure with a Krebs-bicarbonate buffer containing a slightly stimulating glucose concentration (8.3 mM), PK 11195 (10(-7)-10(-5) M) induced a progressive and concentration-dependent decrease in insulin secretion. Simultaneously, we recorded the effects on the pancreatic flow rate; in contrast to 4'-chlordiazepam, previously shown to induce vasodilation in the same preparation, PK 11195 was ineffective. The differential effects of these two substances on vascular resistance and insulin secretion may suggest the existence of different subtypes of peripheral benzodiazepine receptors on pancreatic beta-cells and vessels. A metabolic stimulation of insulin secretion was induced by a glucose increment from 4.2 mM to 8.4 mM or by 2 mM alpha-ketoisocaproic acid (KIC), which is directly metabolized in the mitochondria; these stimulations could be reduced by 10(-5) M PK 11195 (P<0.05). In contrast, the drug was ineffective on the insulin secretion induced by 5 mM or 10 mM KCl in the presence of a nonstimulating glucose concentration (4.2 mM). These results suggest that PK 11195 inhibits insulin secretion by interfering with mitochondrial oxidative metabolism.

P2-receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. The aim of our study was to synthesize and evaluate pharmacologically the novel P2Y-R ligands, 2-thioether-5'-O-phosphorothioate adenosine derivatives, as potential insulin secretagogues. An efficient synthesis of these nucleosides and a facile method for separation of the chiral products is described. The enzymatic stability of the compounds towards pig-pancreas NTPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)-adenosine (2-hexylthio-ATP-alpha-S) isomers by NTPDase was 28% that of ATP. The apparent affinity of the compounds to P2Y1-R was determined by measurement of P2Y-receptor-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K0.5 values in the nM range. 2-RS-AMP-S derivatives were full agonists although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nM concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective. A high chemical hydrolytic stability was observed for 2-hexylthio-ATP-alpha-S. Hydrolysis of the phosphoester bond, which was the only detectable degrading reaction under the investigation conditions (pH 7.4, 37 degrees C), was slow, with a half-life of 264 hours. Moreover, even at gastric juice conditions (pH 1.4, 37 degrees C), hydrolysis of the terminal phosphate was the only detectable reaction, with a half-life of 17.5 hours. 2-Hexylthio-ATP-alpha-S isomers are enzymatically and chemically stable. These isomers are highly potent and effective insulin secretagogues, increasing, however, pancreatic vascular resistance.

P2-Receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. Therefore, novel P2Y-R ligands, 2-thioether 5'-O-phosphorothioate adenosine derivatives (2-RS-ATP-alpha-S), were synthesized as potential insulin secretagogues. An efficient synthesis of these nucleotides and a facile method for separation of the chiral products are described. The enzymatic stability of the compounds toward pig pancreas type I ATPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)adenosine (2-hexylthio-ATP-alpha-S) isomers by ATPDase was 28% of that of ATP. Some 2-thioether 5'-(monophosphorothioate)adenosine derivatives (2-RS-AMP-S) exerted an inhibitory effect on ATPDase. The apparent affinity of the compounds to P2Y(1)-R was determined by measurement of P2Y-R-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K(0.5) values in the nanomolar range. 2-RS-AMP-S derivatives were full agonists, although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nanomolar concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective.

The effects of ATP on the pancreatic vascular bed were studied on the isolated rat pancreas perfused at a constant pressure so as any change in the vascular tone induces a modification in the flow rate. This study was performed in two different experimental conditions: 1) In the presence of indomethacin, inhibiting the cyclo-oxygenase and prostacyclin (PGI2) synthesis, ATP (which acts on vasodilatator P2Y receptors and vasoconstrictor P2X and P2U receptors) was used at a concentration (165 microM) which did not modify per se the vascular flow rate. With indomethacin, ATP induced a slight but significant and long lasting decrease in the flow rate. This effect is different from that induced by the stimulation of P2X receptors; it is comparable to that induced by the activation of P2U receptors. 2) In the presence of 2,2'pyridylisatogen tosilate (PIT) used at two different concentrations, the first (5 microM) inhibiting the P2Y effects on insulin secreting B cells and pancreatic vessels, the second (25 microM) inhibiting the P2X effects on pancreatic vessels. The effects of ATP are different according to the concentration of PIT. In both cases, ATP induced only a vasoconstriction. However, the kinetics of the flow rate is totally different: in the presence of 5 microM PIT, an immediate and drastic vasoconstriction was observed, followed by a long lasting vasoconstriction of lesser magnitude, which can be ascribed to P2X and P2U receptor activation, respectively. This hypothesis was confirmed by the results in the presence of PIT at 25 microM. At this concentration this compound completely suppressed the drastic and transient vasoconstriction, so that only a progressive and long lasting vasoconstriction of the P2U type could be observed. From these results, it can be concluded that: 1) PGI2 plays a part in the vasodilatator effects of ATP. 2) At the concentrations used, PIT does not block the vasoconstriction induced by P2U receptors. 3) The effects of ATP on pancreatic vessels is dependent on the balance between its vasodilator effect due to the activation of P2Y receptors and its vasoconstrictor effect which involves two types of receptors: P2X and P2U.

1. We have previously demonstrated that adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS), a potent P2Y-purinoceptor agonist, relaxed pancreatic vasculature not only through prostacyclin (PGI2) and nitric oxide (NO) release from the endothelium but also through other mechanism(s). In this study, we investigated the effects of an inhibitor of the Na+/K+ pump, of ATP-sensitive K+ (K(ATP)) channels and of small (SK(Ca)) or large (BK(Ca)) conductance Ca2+-activated K+ channels. Experiments were performed at basal tone and during the inhibition of NO synthase and cyclo-oxygenase. 2. In control conditions, ADPbetaS (15 microM) induced an initial transient vasoconstriction followed by a progressive and sustained vasodilatation. In the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 200 microM) the transient vasoconstriction was reversed into a one minute vasodilator effect, which was then followed by a progressive and sustained vasodilatation similar to that observed with ADPbetaS alone. The addition of indomethacin (10 microM) did not significantly modify the profile of ADPbetaS-induced vasodilatation. 3. Ouabain (100 microM) decreased basal pancreatic flow rate and did not modify ADPbetaS-induced relaxation. This inhibitor of the Na+/K+ pump increased the pancreatic vasoconstriction induced by L-NAME or by the co-administration of L-NAME and indomethacin. Ouabain did not modify either the L-NAME or the L-NAME/indomethacin resistant part of the ADPbetaS vasodilatation. 4. The K(ATP) inhibitor tolbutamide (185 microM) did not significantly modify basal pancreatic flow rate and ADPbetaS-induced relaxation. This inhibitor which did not change L-NAME-induced vasoconstriction, significantly diminished the L-NAME resistant part of ADPbetaS-induced vasodilatation. Tolbutamide intensified the vasoconstriction induced by the co-administration of L-NAME and indomethacin. In contrast, the L-NAME/indomethacin resistant part of ADPbetaS vasodilatation was not changed by the closure of K(ATP). 5. The SK(Ca) inhibitor apamin (0.1 microM) did not significantly change pancreatic vascular resistance whatever the experimental conditions (in the absence or in presence of L-NAME or L-NAME/indomethacin). In the presence of L-NAME, the closure of SK(Ca) channels changed the one minute vasodilator effect of ADPbetaS into a potent vasoconstriction and thereafter modified only the beginning of the second part of the L-NAME-resistant part of the ADPbetaS-induced vasodilatation. In contrast, the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation remained unchanged in the presence of apamin. 6. Charybdotoxin (0.2 microM), an inhibitor of BK(Ca), increased pancreatic vascular resistance in the presence of L-NAME/indomethacin. In the presence of L-NAME, the closure of BK(Ca) channels reversed the one minute vasodilator effect of ADPbetaS into a potent vasoconstriction and drastically diminished the sustained vasodilatation. In contrast the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation was not modified by the presence of charybdotoxin. Under L-NAME/indomethacin/charybdotoxin/apamin infusions, ADPbetaS evoked a drastic and transient vasoconstriction reaching a maximum at the second minute, which was followed by a sustained increase in the flow rate throughout the ADPbetaS infusion. The maximal vasodilator effect of ADPbetaS observed was not modified by the addition of apamin. 7. The results suggest that the L-NAME-resistant relaxation induced by ADPbetaS in the pancreatic vascular bed involves activation of BK(Ca), K(ATP) and to a lesser extent of SK(Ca) channels, but the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation is insensitive to the closure of K(ATP), SK(Ca) and BK(Ca) channels.

1. The endothelium-dependent relaxation of blood vessels induced by P2Y-purinoceptor activation has often been shown to involve prostacyclin and/or nitric oxide (NO) release. In this work, we have investigated the mechanisms involved in the relaxant effect of the P2Y agonist, adenosine -5'-O-(2-thiodiphosphate) (ADP beta S) using two complementary preparations: rat pancreatic vascular bed and aortic ring. 2. On the pancreatic vascular bed, ADP beta S (1.5 and 15 microM) infused for 30 min induced a concentration-dependent vasodilatation; it was progressive during the first 10 min (first period) and sustained from 10 to 30 min (second period). Indomethacin (10 microM) delayed ADP beta S-induced vasodilatation (1.5 and 15 microM) by about 6 min. N omega-nitro-L-arginine methyl ester (L-NAME) (200 microM) suppressed the relaxation for about 5 min but thereafter ADP beta S at the two concentrations progressively induced an increase in the flow rate. Even the co-administration of L-NAME and indomethacin did not abolish the ADP beta S-induced vasorelaxation. 3. On 5-hydroxy tryptamine (5-HT) precontracted rings mounted in isometric conditions in organ baths, we observed that ADP beta S induced a concentration-dependent relaxation of rings with a functional endothelium; this effect was stable for 25 min. The ADP beta S relaxant effect was strongly inhibited by Reactive Blue 2 (30 microM) and was suppressed by pretreatment of rings with saponin (0.05 mg ml-1 for 30 min), which also abolished the acetylcholine-induced relaxation. 4. ADP beta S-induced relaxation of 5-HT precontracted rings is largely inhibited by indomethacin (100 or 10 microM) or L-NAME (100 microM). 5. We conclude that: the ADP beta S-induced relaxation is endothelium-dependent, mediated by P2Y-purinoceptors, and at least in part linked to NO and prostacyclin release, depending on the preparation used. Furthermore, on the pancreatic vascular bed, (an)other mechanism(s) than prostacyclin and NO releases may be involved in ADP beta S-induced vasodilatation.

n-Hexacosanol, a long-chain saturated fatty alcohol extracted from Hygrophyla erecta Hochr., has been recently shown to exert neurotrophic properties on central neurons and to stimulate phagocytosis in macrophages. The present work was designed to investigate the effects of hexacosanol on stimulated insulin secretion in vivo and in vitro. In anaesthetized rats, hexacosanol (2 mg/kg i.p.) induced a reduction of the insulin response to an intravenous glucose tolerance test (0.3 g/kg) with a consequent increase in hyperglycaemia. In vitro, in the isolated perfused pancreas, hexacosanol at the concentration of 10(-7) M clearly reduced the two phases of glucose-induced insulin secretion. At the higher concentration (10(-5) M), hexacosanol was no longer able to exert an inhibition of glucose-induced insulin release; surprisingly a stimulating effect occurred which was of the same magnitude as in control experiments with Tween alone, at the concentration used to dissolve hexacosanol. In isolated perifused islets, 22 mM glucose-stimulated insulin release was also inhibited by hexacosanol at the concentrations of 10(-9) M and 10(-7) M, but not at 10(-5) M. In contrast, insulin secretion induced by arginine (20 mM) was not affected by the different concentrations of hexacosanol. It is concluded that n-hexacosanol at 10(-9) M and 10(-7) M exerts an inhibitory effect on insulin secretion stimulated by glucose in vivo and in vitro in the rat, suggesting a direct effect on islets of Langerhans.

Purinoceptors have been classified according to their sensitivity to structural analogues of purines. In addition to the well established and widely distributed P2 purinoceptor subtypes (P2X and P2Y), the existence of "pyrimidine" or "nucleotide" receptors was proposed, which are sensitive to the pyrimidine nucleotide, uridine triphosphate (UTP). Recently, this class of receptor has been included in the P2 purinoceptor classification as a P2U subtype. We have previously shown that pancreatic vascular resistance was modulated by the activation of P2Y and P2X purinoceptors, inducing vasodilation and vasoconstriction respectively. In this study, we investigated the effect of pyrimidine nucleotides on pancreatic vessels. The experiments were performed on isolated rat pancreas perfused at a constant pressure, which was selected to obtain a stable flow rate of 2.5 ml/min before drug administration; thus, any change in pancreatic vascular resistance resulted in a change in the flow rate. UTP induced a decrease in the flow rate at all concentrations tested, but the kinetics differed according to the concentration. The lowest concentration used (16.5 microM) induced a delayed, progressive and long lasting vasoconstriction. Ten times and one hundred times higher concentrations (165 and 1,650 microM) induced an immediate, more pronounced and also long lasting vasoconstriction. In contrast, the other pyrimidine nucleotide cytosine triphosphate (16.5 and 165 microM) did not significantly modify the pancreatic flow rate. This study suggests the presence of a P2U purinoceptor on rat pancreatic vessels; this P2U receptor differs from the P2X receptor since the activation of this latter elicits only a transient vasoconstriction.

A possible implication of endogenously released ATP and (or) ADP in insulin response to glucose stimulation was investigated in the isolated rat pancreas. The first phase of insulin response to the same glucose concentration (8.3 mM) was much higher in pancreas previously perfused in the absence of glucose than in pancreas previously perfused with 4.2 mM glucose. A P2 purinoceptor antagonist, 2,2'-pyridylisatogen tosylate, strongly reduced the higher first phase resulting from glucose deprivation; similarly, it reduced exogenous ATP-potentiated insulin response to a glucose increase from 4.2 to 8.3 mM. In contrast, 2,2'-pyridylisatogen tosylate did not modify the first phase of insulin response to 8.3 or 12.5 mM glucose in pancreas previously perfused with 4.2 mM glucose. Our results suggest that endogenous ATP and (or) ADP released in pancreatic islets in the absence of glucose could activate P2 purinoceptors and increase the magnitude of the first phase of insulin response to a glucose stimulation.

1. In vivo effect of a P2y-purinoceptor agonist, adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), on insulin secretion and glycaemia were studied both in rats and dogs. 2. In anaesthetized rats, i.v. administered ADP beta S (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, ADP beta S enhanced insulin release and thus increased the glucose disappearance rate. 3. In anaesthetized fasted dogs, i.v. administered ADP beta S (0.1 mg kg-1) increased pancreaticoduodenal insulin output and slightly decreased blood glucose levels. 4. In conscious fasted dogs, orally administered ADP beta S (0.1 mg kg-1) transiently increased insulinemia and punctually reduced glycaemia. Furthermore, during an oral glucose tolerance test, orally administered ADP beta S at the same dose markedly enhanced insulin secretion and consequently reduced the hyperglycaemia. 5. In conclusion, the P2y-agonist, ADP beta S, is a potent insulin secretagogue in vivo, improves glucose tolerance and is effective after oral administration. Thus, the P2y-purinoceptors of the beta cell may be a target for new antidiabetic drugs.

We have previously shown that the selective peripheral-type benzodiazepine agonist 4'-chlordiazepam inhibited glucose-induced insulin secretion from rat pancreatic islets. The present work was designed to further investigate the mechanism of this effect. Since interactions have been described between benzodiazepines and purinergic modulators such as adenosine, we studied the effect of the adenosine receptor antagonist 8-phenyltheophylline. This substance, which on its own had no effect on insulin release, prevented the inhibiting effect of 4'-chlordiazepam. We then performed experiments in the presence of forskolin, which directly stimulates adenylyl-cyclase, and dibutyryl-cyclic AMP, which enhances the intracellular cAMP content. The inhibiting effect of 4'-chlordiazepam was prevented neither by forskolin nor dibutyryl-cAMP, in contrast to the inhibiting effect of adenosine, which has been previously shown to be prevented by dibutyryl-cAMP. In conclusion, the inhibiting effect of 4'-chlordiazepam on glucose-induced insulin secretion is antagonized by 8-phenyltheophylline; the signalling mechanism of this effect appears to be independent of the adenylyl cyclase/cAMP pathway.

1. To investigate the effect of experimental diabetes on the P2y purinoceptor responses of pancreatic beta-cells and vascular bed, we used adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), 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 microM), 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 ADP beta S (15 microM), 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 ADP beta S stimulating effect occurred independently of the glucose concentration (5, 8.3 and 28 mM) in the diabetic pancreas. On pancreatic vascular resistance, ADP beta S induced a similar vasodilatation in diabetic and age-matched rats. 5. In conclusion, ADP beta S 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.

The effects of ATP on the pancreatic vascular bed of the rat were studied under resting tone. ATP exerted two different effects depending on the concentration used: a slight vasodilatation in the 1.65-49.5 microM range which was statistically significant only at 16.5 microM and a concentration-related vasoconstriction in the 495-4 950 microM range. Theophylline, a P1 purinoceptor antagonist, did not modify the vasodilator effect of ATP. The existence of two P2 purinoceptor subtypes (P2y and P2x) in our preparation may be responsible for the dual effect of ATP. The P2y antagonist 2,2'pyridylisatogen (PIT) used at 5 microM, revealed a vasoconstrictor effect of ATP 165 microM, a concentration without effect per se. Furthermore, the transient vasoconstrictor effect of ATP 495 microM was changed into a long-lasting one in the presence of PIT. On the other hand, the blockade of P2x purinoceptors by the desensitizing agent, alpha,beta-methylene ATP, increased the vasodilator effect of ATP 16.5 microM. In conclusion, two subtypes of P2 purinoceptor do exist on the pancreatic vascular bed: P2y inducing vasodilatation and P2x inducing vasoconstriction. At vascular resting tone, the effect observed with ATP therefore depends on the concentration used and on the balance between P2y/P2x purinoceptors.

1. The effects of a P2 purinoceptor agonist, adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-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-beta-S (4.95-495 nM) evoked a biphasic insulin response in a concentration-dependent manner. A comparison of relative potency between ADP-beta-S and adenosine 5'-triphosphate (ATP) showed that ADP-beta-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-beta-S (165 nM) did not modify the basal insulin secretion. 3. ADP-beta-S, at concentrations effective on insulin secretion and also at higher concentrations (1.65 and 16.5 microM), provoked an increase of the pancreatic flow rate in a concentration-dependent manner. 4. Our results show that ADP-beta-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 beta cells on insulin secretion in vivo.

Our experiments were carried out on the isolated perfused rat pancreas. The effect of ATP on insulin secretion induced by tolbutamide (a hypoglycemic sulfonylurea) was studied in the absence of glucose. The addition of ATP (0.165 mmol/l) to the perfusion medium did not significantly modify the first phase induced by tolbutamide (0.4 mmol/l) but potentiated the second phase.

This work was designed to characterize the adenosine receptor (A1 or A2) involved in glucagon secretion. The most potent adenosine analogues on A1 receptors are the N6 substituted compounds, among them N6-phenylisopropyladenosine (PIA); furthermore L-PIA is 50 to 100 times more potent than D-PIA on the A1 receptor, whereas it is 3 to 5 times more potent on the A2 receptor; thus the A1 receptor shows a much higher stereoselectivity. The effects of L-PIA and D-PIA were studied on glucagon secretion from the isolated perfused rat pancreas. 1) L-PIA at 1.65 microM induced a transient glucagon secretion which was not greater than that induced by the same concentration of adenosine. 2) D-PIA at a 3 fold higher concentration (4.95 microM) elicited a secretion of glucagon comparable to that induced by L-PIA 1.65 microM; thus the involved receptor does not present a high stereoselectivity for L-PIA. These results support the fact that the receptor involved in glucagon secretion is not of the A1 type.

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.

In the presence of a glucose concentration of 1.5 g/1, ATP provokes a biphasic stimulation of insulin secretion from the isolated and perfused rat pancreas. For ATP concentrations ranging from 0.5 mg/1 to 200 mg/1, the increase in insulin secretion presents a linear relation with the logarithm of the concentration. Lowering the temperature from 37.5 degrees C to 28 degrees C provokes a decrease in insulin secretion induced by glucose (1.5 g/1). In response to the stimulation by ATP, the increase in insulin secretion measured during the first phase is weaker at 28 degrees C than at 37.5 degrees C when estimated in ng/min; however, when evaluated in percentage in relation to the baseline value, this increase is more important at the lower temperature.

ATP stimulates glucagon and insulin secretions from the isolated perfused rat pancreas. This effect is modulated by glucose. Glucagon secretion is stimulated by ATP only in the absence or in the presence of a low glucose concentration (0.5 g/1). As to insulin secretion, it is strongly stimulated only in the presence of a glucose concentration of 1.5 g/1.

The study of purinergic receptors in the endocrine pancreas is a recent field of investigations. Until recently, ATP has been considered only as an intracellular fuel by most authors. Nevertheless for some years evidence has been obtained that extracellular ATP and/or ADP could act on the B cell membrane to increase insulin secretion. Furthermore it was shown that adenosine could act on the A cell surface to increase glucagon secretion. In both cases the involvement of purinoceptors could be demonstrated using structural analogues which are metabolically stable and not taken up by cells. Also the effects of activators of purinoceptors on B and A cells were suppressed by antagonists. So, it was possible to establish that the purinoceptors involved in the stimulation of insulin and glucagon secretion were different. The B cell is endowed with P2 purinoceptors (for ATP and/or ADP) and the A cell possess P1 purinoceptors (for adenosine). It is of interest to note that B and A cells, which secrete hormones with opposite roles, anabolic and catabolic respectively, have two different types of purinoceptors involved in stimulatory process. The purinergic modulation of B and A cells appears to be of physiological relevance.

The effect of adenosine (an agonist of the P1 purinoceptors) alone or in presence of a specific blocker of these receptors, theophylline, was studied on the vascular flow of the isolated perfused rat pancreas. Adenosine alone at a low concentration (1.65 microM) induced a rapid increase in vascular flow (+ 17%) which lasted throughout the perfusion of the nucleoside. This effect was suppressed by theophylline (50 microM). Our results show that adenosine provokes a dilator effect on the vessels of the rat pancreas and suggest that this effect is due to an action on purinergic receptors of the P1 type.

An attempt was made to antagonize the stimulatory effect of ATP on insulin secretion from the isolated perfused rat pancreas. The insulin secretory effect of ATP does not seem to be mediated by cholinergic or beta-adrenergic receptors since neither atropine 0.3 micro mol/1 nor propranolol 1 micro mol/1 had any antagonistic action on insulin secretion induced by ATP (16.5 micro mol/1). Theophylline (50 micro mol/1) did not antagonize the insulin secretion evoked by ATP (16.5 micro mol/1). Apamin (10 nmol/1) was also without antagonistic action. 2-2'Pyridylisatogen tosylate (5 micro mol/1) had not effect on insulin secretion induced by glucose )8.33 mmol/1) or acetylcholine (0.5 micro mol/1 and 0.1 micro mol/1) but inhibited the insulin secretory effect of ATP (16.5 micro mol/1). Thus, the antagonism of 2-2'pyridylisatogen for ATP seems selective. We conclude that a purinoceptor of the P2-type is likely to be present on the B-cell of the rat pancreas.

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). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 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, beta, gamma-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 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). 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.

Our experiments were carried out on the isolated perfused rat pancreas. The effect of extracellular ATP (8 microM) on insulin secretion induced by tolbutamide (0.04 mM) was studied in the presence of substimulating glucose concentration 4.2 mM (0.75 g/l). ATP (8 microM), ineffective per se at this concentration, highly potentiated the insulin secretion induced by tolbutamide (0.04 mM).

The stimulating effect of ATP on insulin secretion has been demonstrated by several authors both in vitro and in vivo. The kinetic study carried out on the isolated perfused pancreas of the rat, in the presence of a glucose concentration of 1.5 g/l, showed that the response was biphasic and dose-dependent for ATP concentrations ranging from 10(-6) to 10(-4) M. It can be seen from the results obtained with the natural nucleotides (ATP, ADP, AMP, adenosine, GTP, ITP, UTP, CTP), that there is a structure activity relationship. Some structural features are essential to elicit an insulin secretory effect: a purine base and 2 or 3 phosphate groups. The study with the structural analogues of ATP and ADP modified on the polyphosphate chain, shows the importance of the steric and electronic characteristics of the polyphosphate chain for induction of insulin secretion. The insulin-stimulating action of ATP seems to be selectively antagonized by 2, 2' pyridylisatogen. It is blocked neither by a cholinergic blocker (atropine), nor by a beta-adrenergic blocker (propranolol), nor by an adenosine antagonist (theophylline). In the present state of the experimental work, the results favour a purinergic receptor site to ATP or ADP on the beta-insulin-secretory cells. At least three factors are in favour of a type P2 receptor, according to the classification of purinergic receptors (Burnstock, 1978) : the order of relative potencies of the agonists: ATP greater than or equal to ADP much greater than AMP greater than or equal to adenosine, the lack of antagonist effect of theophylline and the selective antagonism of PIT.