2-thioether 5'-O-(1-thiotriphosphate)adenosine derivatives as new insulin secretagogues acting through P2Y-Receptors.

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.

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 diphosphonate or beta, gamma-methylene ATP, adenosine 5'-alpha, beta-methylene triphosphate or alpha, beta-methylene ATP and adenosine 5'-alpha, beta-methylene diphosphonate or alpha, beta-methylene ADP. 2 beta, gamma-Methylene ATP did not elicit any increase of insulin release: alpha, beta-methylene ATP and alpha, beta-methylene ADP induced a biphasic stimulation of insulin secretion; this effect was dose-related between 1.65 and 165 mumol/l. Relative potency ATP/alpha, beta-methylene ATP was 1.2 and ATP/alpha, beta-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.

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-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.

Evidence for two different P2-purinoceptors on beta 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 than ATP. 3 2-Methylthio ATP also provoked a transient decrease of the flow rate in a concentration-dependent manner but at concentrations (165-825 microM) 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 beta cell of the pancreas. A P2X subtype, mediating vasoconstriction, is present on the vascular bed of the rat pancreas.

Difference in the potentiating effect of adenosine triphosphate and alpha, beta-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 microM 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 alpha, beta-methylene ADP (alpha, beta-MeADP)) at 16.5 microM 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. alpha, beta-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 alpha, beta-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 alpha, beta-MeATP, the ATP potentiating effect was unaffected. 6. It is concluded that ATP and ADP, via activation of beta cell P2 gamma purinoceptors, potentiates the biphasic insulin response to an increase of glucose concentration. On the other hand, alpha, beta-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 beta cell P2 gamma purinoceptors by alpha, beta-MeATP.

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

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.

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

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). 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 X 10(-8) M. A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. Theophylline (50 microM) considerably decreased the peak of glucagon secretion induced by 1.65 microM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. 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.

Evidence for two different types of P2 receptors stimulating insulin secretion from pancreatic B cell.

Adenine nucleotides have been shown to stimulate insulin secretion by acting on P2 receptors of the P2Y type. Since there have been some discrepancies in the insulin response of different analogues of ATP and ADP, we investigated whether two different types of P2 receptors exist on pancreatic B cells. The effects of alpha,beta-methylene ATP, which is more specific for the P2X subtype, were studied in vitro in pancreatic islets and isolated perfused pancreas from rats, in comparison with the potent P2Y receptor agonist ADPbetaS. In isolated islets, incubated with a slightly stimulating glucose concentration (8.3 mM), alpha,beta-me ATP (200 microM) and ADPbetaS (50 microM) similarly stimulated insulin secretion; by contrast, under a non stimulating glucose concentration (3 mM), alpha,beta-me ATP was still effective whereas ADPbetaS was not. In the same way, in islets perifused with 3 mM glucose, alpha,beta-me ATP but not ADPbetaS induced a partial but significant reduction in the peak 86Rb efflux induced by the ATP-dependent potassium channel opener diazoxide. In the isolated pancreas, perfused with a non stimulating glucose concentration (4.2 mM), ADPbetaS and alpha,beta-me ATP (5-50 microM), administered for 10 min, induced an immediate, transient and concentration-dependent increase in the insulin secretion; their relative potency was not significantly different. In contrast, with a slightly stimulating glucose concentration (8.3 mM), ADPbetaS was previously shown to be 100 fold more potent than alpha,beta-me ATP. Furthermore, at 4.2 mM glucose a second administration of alpha,beta-me ATP was ineffective. In the same way, ADPbetaS was also able to desensitize its own insulin response. At 3 mM glucose, alpha,beta-me ATP as well as ADPbetaS (50 microM) induced a transient stimulation of insulin secretion and down regulated the action of each other. These results give evidence that pancreatic B cells, in addition to P2Y receptors, which potentiate glucose-induced insulin secretion, are provided with P2X receptors, which transiently stimulate insulin release at low non-stimulating glucose concentration and slightly affect the potassium conductance of the membrane.

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) (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.

Involvement of K+ channel permeability changes in the L-NAME and indomethacin resistant part of adenosine-5'-O-(2-thiodiphosphate)-induced relaxation of pancreatic vascular bed.

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.

P2y purinoceptor responses of beta 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 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.

Study of the mechanisms involved in adenosine-5'-O-(2-thiodiphosphate) induced relaxation of rat thoracic aorta and pancreatic vascular bed.

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.

Effects of homocysteine on acetylcholine- and adenosine-induced vasodilatation of pancreatic vascular bed in rats.

1. Epidemiological and experimental data have shown that homocysteine may provoke vascular lesions and that moderate homocysteinaemia may constitute an independent risk factor for vascular disease. It is now documented that homocysteine damages human endothelial cells in culture, possibly by producing hydrogen peroxide in an oxygen-dependent reaction. 2. In this study, we have examined the direct effect of this sulphur amino acid on pancreatic vascular resistance. Experiments were performed on the vascular bed of the rat isolated pancreas perfused at constant pressure; thus, any change in pancreatic vascular resistance resulted in a change in the flow rate. D,L-Homocysteine perfused for one hour at three different concentrations (200 microM, 2 mM, 20 mM) did not induce any significant change in the flow rate per se. Homocysteine infusion for 30 min at a concentration of 200 microM or 2 mM abolished the endothelium-dependent vasodilatation induced by acetylcholine (0.05 microM), but did not modify adenosine (1.5 microM)-induced vasodilatation. 3. The effect of D,L-homocysteine (200 microM or 2 mM) cannot be ascribed to a direct antimuscarinic effect since 30 min pretreatment of rat ileum with these concentrations did not significantly change the contractile effect of increasing concentrations of acetylcholine (0.015-15 microM). 4. Preincubation of human umbilical vein endothelial cells with D,L-homocysteine (0.2-5.0 mM) had no significant effect on overall cell number or viability during 18 h of incubation; the endothelial cells exposed to concentrations up to 5 mM exhibited a spindle-shaped, whirled pattern. This pattern was reversed 48 h after the removal of homocysteine. A cytotoxic effect was seen after 18 h incubation in 10 mM D,L-homocysteine. 5. In conclusion, an acute infusion of homocysteine altered acetylcholine endothelium-induced vasodilation, whereas the adenosine vasodilatator effect was insensitive to the deleterious action of homocysteine in vitro.

Attempt to antagonized the stimulatory effect or ATP on insulin secretion.

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.

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.

Dual regulation of pancreatic vascular tone by P2X and P2Y purinoceptor subtypes.

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.

Structural specificity of nucleotides for insulin secretory action from the isolated perfused rat pancreas.

The study concerned the effects of variuos nucleotides on the insulin secretion from the isolated perfused rat pancreas. ATP, the first nucleotide studied, increased the insulin release induced by glucose 1.5 g/l. There was a first immediate peak followed by a second significant and durable increase. The log dose-response curve was linear for concentrations ranging from 0.825 microM to 330 microM. The effects of natural adenine derivatives (ATP, ADP, 5' AMP, cAMP and adenosine) were compared. ATP was the most active compound; ADP had nearly the same activity as ATP (relative potency ATP/ADP = 3.2); 5' AMP, cAMP and adenosine displayed a very weak activity (about 100 fold less active). Adenylimido-diphosphate (AMP-PNP), a non-phosphorylating structural analogue of ATP, clearly stimulated insulin secretion and its effect was concentration-related. It was about 10 fold less active than ATP. The comparison of triphosphorylated derivatives from various purine nucleosides (ATP, GTP, ITP) or pyrimidine nucleosides (CTP and UTP) showed that only the purine derivatives had a strong insulin secretory effect with, in order of decreasing activity: ATP greater than GTP greater than ITP. These results show that certain structural features (purine basis and di- or triphosphate groups) are essential to elicit an insulin secretory effect.

Effect of n-hexacosanol on insulin secretion in the rat.

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.

Inhibition of glucose-induced insulin secretion by a peripheral-type benzodiazepine receptor ligand (PK11195).

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.

Contrasting effects of streptozotocin-induced diabetes on the in vitro relaxant properties of adenosine in rat pancreatic vascular bed and thoracic aorta.

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 microM adenosine for 30 min, or for noradrenaline-contracted aortae, as the relaxant response to adenosine (1 microM-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 Nomega-nitro-L-arginine methyl ester (L-NAME). In all groups of animals, the flow rate of isolated pancreas dropped in the presence of 200 microM L-NAME, but was restored by adenosine to the level observed without L-NAME. L-NAME (10 microM) 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.